CLINICAL IMMUNOLOGY AND EMMUNOPATHOLOGY &t,299-314 (1982)

Localization of Neutrophil Cationic Proteins and Loss of Anionic Charges in Glomeruli of Patients with Systemic

Lupus Erythematosus Glomerulonephritisl

GIOVANNI CAMUW, CIRO TETTA, GIUSEPPE SEGOLONI, RENATO CODA,* AND ANTONIO VERCELLONE

Lahorutorio di Immunopatologia, Cattedra di Nejiiologia Medica, Divisione di Nefrologia e Dialisi. Ospedale Maggiore S.G. Butt&a, and *Zstituto di Anatomia ed Istologia Patologica I,

Centro di Microscopia Elettronica, Turin, Ita1.v

The localization of human neutrophil cationic protein (NCP) antigens in 14 patients with systemic lupus erythematosus (SLE) glomerulonephritis was investigated by immu- nofluorescence, using a rabbit anti-NCP serum. The specificity of the antiserum was demonstrated by its ability to bind exclusively to cytoplasmic structures of human poly- morphonuclear neutrophils (PMN), but not of lymphocytes, monocytes, or platelets. Furthermore, the immunofluorescence reaction was abolished by absorption of the anti- serum with purified NCP. In 7 patients with active SLE, severe proteinuria, and nephrotic syndrome, NCP were detected in glomerular capillary walls (GCW). The glomerular deposits of NCP were associated with a loss of glomerular polyanions (GPA). as revealed by colloidal iron and Alcian blue staining. The PMN, present in the peripheral blood of these patients, were markedly depleted of NCP. The depletion of NCP in circulating PMN, and the presence of NCP deposits in GCW with concomitant loss of GPA are consistent with the interpretation that NCP are released in raivo and that they directly contribute to the development of glomerular injury and proteinuria in pa- tlents with active SLE glomerulonephritis.

INTRODUCTION

Fixed anionic sites in glomerular capillary wall (GCW)’ are known to influence glomerular permeability to plasma proteins (1- 8). Several studies have shown the distribution and the nature of the glomerular polyanions (GPA) (5, 7-12). The glomerular perfusion with polycations induces spreading of foot processes, oblit- eration of slit pores, and reduction of GPA (13, 14). Since polyanions or neutral molecules show none of these effects, Seiler et al. (13, 14) hypothesized that anionic sites, not only condition glomerular permeability to anionic molecules such as albumin, but also maintain, by electrostatic repulsive forces, the structural organization of the foot processes. In rats injected with aminonucleoside of puromycin, the epithelial changes do not result from proteinuria, but are due to

’ This work was supported by CNR, Rome Ctr. 80.00602.04. ? Abbreviations used: GCW, glomerular capillary wall; GPA, glomerular polyanions; GBM,

glomerular basement membrane; IC, immune complexes: SLE, systemic lupus erythematosus: CP. cationic proteins: PMN, polymorphonuclear neutrophils: NCP, neutrophil cationic proteins: TT-BSA. T&buffered Tyrode’s solution with bovine serum albumin.

0090- 1229/82/090299- 16$01.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

300 CAMUSSI ET AL.

the primary loss of GPA induced by the drug (15- 17). Moreover, the chronic administration of aminonucleoside is followed by loss of GPA, collapse of GCW, plasma protein accumulation in the subendothelial part of the glomerular base- ment membrane (GBM) and in the mesangium and, finally, by glomerular sclerosis (17). In nephrotoxic glomerulonephritis, the loss of GPA precedes the onset of proteinuria (18), a finding which further supports the contention that neutraliza- tion of GPA leads to altered charge-selective permeability. Furthermore, the de- position of immune complexes (IC) in the GCW is associated with loss and/or redistribution of GPA (19, 20). In mice with systemic lupus erythematosus (SLE) nephritis and proteinuria, the passage of plasma proteins across the GCW is caused by nonuniform increases in glomerular permeability (20), related to a focal loss of GPA (19).

A number of cationic proteins (CP) has been identified as being released from a variety of inflammatory cells such as platelets (21, 22), macrophages (23), and polymorphonuclear neutrophils (PMN) (24-26). Among these mediators, three CP, isolated and purified from rabbit PMN (MW 12,000), have been shown to promote an increase in vascular permeability. A fourth cationic component (MW 5000) induces degranulation of, and histamine release from, mastocytes (24).

Evidence for an active release of CP from stimulated rabbit PMN has been provided (25). More recently, Olsson and Venge purified and characterized human neutrophil cationic proteins (NCP) from leukemic leukocytes (26) with a molecu- lar weight ranging from 21,000 to 29,000 and we have shown their active release from IC-stimulated normal human PMN (27, 28).

NCP display several important biological activities as they increase vascular permeability when injected intradermally (28); they activate the complement cas- cade with generation of anaphylatoxin activity in serum (29), have a bactericidal capacity (30), cause basophil degranulation and release of mediators (28), have chemotactic activity (29), aggregate PMN in vitro, cause neutropenia in Iho, and induce the release of platelet-activating factor (PAF) from PMN (27, 31, 32).

We have previously shown an in vi\‘0 release and urinary excretion of NCP in acute phases of SLE glomerulonephritis (33). The present study describes the localization of NCP in glomeruli of patients with active SLE glomerulonephritis, the concomitant loss of GPA in GCW and of NCP in PMN of the peripheral blood. These findings support the hypothesis that NCP are released in viva and exert a direct pathogenetic effect on the glomeruli of these patients.

MATERIALS AND METHODS

Preparation of Cells

Leukocytes, obtained from peripheral blood of normal donors, were collected in siliconized plastic tubes containing 5 x 10M3 M (final concentration) of ethyl- enediaminetetraacetate (EDTA), pH 7.2. After separation by the Hysopaque- Ficoll step gradient (34), 95% pure PMN were prepared, as previously described (27), from the granulocyte-erythrocyte-containing sediment by subsequent gela- tin sedimentation, osmotic shock and finally washed three times with TT-BSA (no

NEUTROPHIL CATIONIC PROTEINS AND SLE NEPHRITIS 301

Caz+, no Mg2+)3 to avoid appreciable platelet contamination (less than 1%). Finally, PMN were resuspended in TT-BSA.

Plastic-adherent, mononuclear cells (98% pure) were obtained from the high- density mononuclear-rich fraction of the Hysopaque - Ficoll gradient by allowing cell adherence in 35mm-diameter plastic petri dishes, as previously described (32). Adherent cells were characterized as monocytes by esterase staining (35), coupled with inhibition by sodium fluoride (36). Washed human platelets were obtained as previously described (37). Briefly, human blood was collected on citric acid-citrate-dextrose (for 9 ml of blood, 1 ml of a solution of 8 g. of citric acid monohydrate, 22 g of citrate trisodium dehydrate, and 22.3 g per liter of distilled water was used). Platelets were washed in TT-BSA, no Ca’+, no Mg2+, centrifuged twice at 1OOOg for 15 min on a cushion of erythrocytes, separated by centrifugation at 90g for 15 min, and finally resuspended in TT-BSA.

Purification of Human Cationic Proteins

A. Neutrophil cationic proteins. NCP were obtained from PMN as previously described (27). Briefly, the sonicates (1 x 108/ml of PMN) were extracted with 5 vol of 0.2 M acetate buffer, pH 4, at 4” C for 3 hr. After centrifugation at 40,OOOg for 30 min, the supernatant was collected and recentrifuged under similar condi- tions. The supernatant was dialyzed for 48 hr against 0.01 M phosphate buffer, pH 8.5, then chromatographed on a 1.5 x 30-cm DEAE-Sephadex A-50 (Pharmacia Fine Chemicals, lot 3537, Uppsala, Sweden) column equilibrated with the same buffer. After elution at pH 8.5, NCP were concentrated to approximately 5 ml with an Amicon ultrafiltration unit, using a UM 2 membrane, then dialyzed against 0.1 M acetate buffer, pH 3.7. The whole procedure of purification was carried out at 4°C to avoid digestion by proteolytic enzymes. Molecular sieve filtration was performed on a 2.6 x loo-cm Sephadex G-100 (Pharmacia Fine Chemicals, lot 9069) column, equilibrated with 0.1 M acetate buffer pH 3.7. The flow rate of the column was 12 mVhr and 3-ml fractions were collected. The molecular weight of NCP was estimated according to Andrews’ method (38), using as reference sub- stances BSA (66,000) (Boehringer GmbH, lot 19602991, Mannheim, West Ger- many) labeled with ‘251, hemoglobin (64,000) (Sigma Chemical Co., lot H-2707, St. Louis, MO.) peroxidase (40,000) (Grade II, Boehringer, lot 127361), cytochrome c (12,000) (Type XY, from rabbit heart, Sigma, lot C-9137), and glucagon (3600) (crystalline, Sigma, lot 4250) labeled with 1251. The electrophoretic identification of NCP was performed on cellulose acetate strips in 0.05 M acetate buffer, pH 4.0, according to Olsson and Venge (26). NCP were also examined by polyacrylamide gel electrophoresis (gel concentration 8%)) carried out in Tris -glycine - sodium

Ii Composition of buffers was as follows. Tyrode’s: KCl, 2.6 x lo-” M: MgCl,6H,O, 1.0 x lo--” M: NaCI, 1.37 x 10-i M: NaHCO,, 1.0 X lo-* M; CaClZ6H,0, 3.0 X 10m3 M: glucose 0.1%. Tris-buffered Tyrode’s (TT): Tyrode’s with 1 x 10e3 M Tris, used instead of bicarbonate. When indicated, 0.25% bovine serum albumin (BSAl was added to TT (‘IT-BSA). When calcium and magnesium ions were

omitted in TT-BSA, the latter is designated as TT-BSA, no Ca*+ no Mg”+. Phosphate-buffered saline

(PBS): 0.01 M phosphate buffer, NaCl, 0.15 M. All solutions were buffered at pH 7.4 at room temper- ature.

302 CAMUSSI ET AL.

dodecyl sulfate (SDS) buffer, 0.025 44, pH 8.8, at room temperature for 90 min at 200 V and 30 mA (39). Proteins were stained with Coomassie brilliant blue R250, 0.2% (w/v), in methanol:acetic acid:water (9:2:9, v/v), the dye-free solvent being used as washing solution. IgG (IgG Standard, Boehringer, lot TRA-07) heavy (52,000) and light (22,000) chains, lysozyme (crystalline, from egg white, Boehringer, lot 107255), and carboxypeptidase B (Boehringer, lot 15265) were used as marker polypeptides for determination of molecular weights. The five NCP components, exhibiting on analytical electrophoresis a cathodal mobility higher than that of lysozyme, had a molecular weight ranging from 25,000 to 32,000 by the Andrews’ method (38) and ranging from 17,000 to 25,000 in four distinct bands by SDS-polyacrylamide gel electrophoresis (Fig. 1) (27). The five components displayed biological activity, based on the blueing reaction induced in the skin of rabbits injected with Evans’ blue (28) (minimal effective dose 2.5 pg protein) (27) and on in vitro basophil degranulation (28) (minimal effective dose 0.25 pg protein) (27). The antigenic inoculum used to immunize rabbits was ob- tained by pooling several homogeneous preparations of NCP.

B. Platelet cationic proteins. CP were obtained from sonicated platelets ac- cording to the technique described by Nachman et al. (22), consisting in the separation of CP from the acid extracts by anion exchange chromatography and gel filtration using sequentially DEAE and Sephadex G-75 (Pharmacia) columns

FIG. 1. Polyacrylamide gel electrophoresis of NCP (2) (gel concentration 8%), performed in Tris-glycine-sodium dodecyl sulfate buffer, 0.025 M, pH 8.8, at room temperature for 90 min at 200 V and 30 mA. IgG heavy (52,000) and light (22,000) chains and lysozyme (14,500) were used as reference substances (1). NCP show separate bands with molecular weights ranging from 17,000 to 25,000.

NEUTROPHIL CATIONIC PROTEINS AND SLE NEPHRITIS 303

(22). Platelet CP were used to exclude immunologic cross-reactivity with serum from rabbits immunized to NCP.

Preparation of Rabbit Anti-Human NCP Serum

Rabbit antiserum to purified NCP was prepared by injection of 14 mg protein of purified NCP in complete Freund’s adjuvant (Behring Institut, lot 43-C, Marburg, West Germany) at various sites (foot pads, neck, limbs) in New Zealand white rabbits weighing 2-2.5 kg. At 3-week intervals, the animals twice received a booster injection (14 mg protein) and the animals were bled one week after the last injection. The pooled anti-NCP serum was fractionated by ammonium sulfate precipitation (40) and sequentially absorbed with the following substrates: 0, Rh-negative erythrocytes, platelets, and lymphocytes pooled from the same donors of PMN (1 x 10’ cells/ml of serum) for 30 min at room temperature and overnight at 4°C. The antiserum was either labeled with fluorescein isothiocyanate (41) (Miles Laboratories, lot 64-284, Elkart, Ind.), according to the method of Johnson and Dorling (41) or used as such in experiments of indirect immunofluo- rescence (42). The anti-NCP serum was able to react with the intracytoplasmic granules of peripheral blood PMN from unrelated normal donors (Fig. 2A) as seen by indirect (titer > 1024) or direct immunofluorescence techniques. The binding of the antiserum to the intracytoplasmic granules of PMN was blocked by preab-

FIG. 2. Fluorescence photomicrographs of peripheral blood leukocytes, stained with fluorescein- conjugated serum from rabbits immunized with NCP. Identification of the cell type was allowed by nuclear staining with ethidium bromide and by phase contrast microscopy. PMN from unrelated nor- mal donors (A) gave an intense, green-colored, granular cytoplasmic staining. Erythrocytes, present in the preparation, were not stained and not visible. The cytoplasm of both lymphocytes (B) and mononuclear cells (C) were negative, only the nuclei being stained bright red. The figures are repre- sentative of 10 experiments. (A, B, C, x 1000).

304 CAMUSSI ET AL.

sorption with purified NCP. No reactivity with human erythrocytes (Fig. 2A), washed or unwashed platelets, lymphocytes (Fig. 2B) or mononuclear cells in peripheral blood smears (Fig. 2C) or with purified plastic-adherent macrophages was observed. By immunofluorescence, the antiserum did not react with normal human skin or kidney tissue. Studies of immunoprecipitation by double im- munodiffusion (43) against normal human plasma, purified y globulin (Cohn Frac- tion II, Sigma, lot HG-II), and CP purified from platelets showed absence of cross-reactivity. In contrast, the anti-NCP serum reacted with purified NCP. These studies demonstrated that serum from rabbits immunized to NCP specifi- cally reacted with NCP.

Patients

Fourteen patients (13 females and 1 male), aged 19 to 65 years (mean 32), satisfying the criteria of the American Rheumatism Association for the diagnosis of SLE (44), were selected for this study. Patients with drug-induced, lupus-like or overlapping syndromes were not included in this study. The clinical activity was independently assessed at the time of the renal biopsy by two investigators on the basis of the following signs and symptoms (44): fever, active rash, serositis, ar- thritis, central nervous system involvement, and active renal disease. As de- scribed in a previous publication (37), patients were divided into three groups on the basis of various degrees of disease activity: Grade I (active) comprised pa- tients with at least three or more of the above-cited symptoms. In addition, all of them had depressed C3 and C4 serum levels, high titers of antinuclear antibodies (45) with a homogeneous pattern, high titers of anti-native DNA antibodies (46) and of circulating IC (47,48). Furthermore, these patients had a leukopenia of less than 4000 white blood cells per cubic millimeter while they were not exposed to drugs known to induce leukopenia. Grade II (moderately active) comprised pa- tients with one or two of the above-cited symptoms. Patients lacking any of these symptoms or signs of serological SLE activity were assigned to Grade III.

Eight normal human kidney specimens, obtained by open, surgical biopsy and six kidney specimens, obtained by needle biopsy from patients with Berger’s glomerulonephritis, were used as controls in the immunofluorescent and histo- logical studies.

Staining of Intracytoplasmic Cationic Proteins in Peripheral Blood Smears

In order to assess the intracytoplasmic CP content of PMN from SLE patients in respect to controls, direct immunofluorescence using anti-NCP serum was performed on peripheral blood smears. The intensity of the immunofluorescence staining of the intracytoplasmic PMN granules was graded from 0 to 3 by two independent observers unaware of the pathologic findings. A minimum of 50 PMN per slide was evaluated.

Histological and Pathological Study of Renal Tissue

All 14 patients were submitted to percutaneous renal biopsy. The renal tissue was divided into two parts, one used for light and the other for immunofluores-

NEUTROPHIL CATIONIC PROTEINS AND SLE NEPHRITIS 305

cence microscopy. Tissue for light microscopy was fixed in Serra’s solution (60% ethyl alcohol, 12% phormol, and 10% glacial acetic acid) for 2 hr, postfixed in 95% alcohol for 2 hr, and embedded in paraffin. The sections were stained with hema- toxylin and eosin and periodic acid-Schiff (PAS) reaction and the histological findings were classified according to Appel er al. (49) in five classes as indicated herein: Class I, normal kidneys; Class II, mesangial changes; Class III, focal and segmental glomerulonephritis; Class IV, diffuse proliferative glomerulonephritis: Class V, membranous glomerulonephritis. Immunofluorescence was performed on renal tissue snap-frozen in liquid nitrogen. Sections, 2-4 pm thick, were obtained in a cryostat. After washing in PBS, the sections were incubated with fluoresceinated rabbit antisera specific for human IgG, IgM, IgA, C3, Clq, C4, and fibrinogen (Behringwerke). In order to amplify the fluorescence staining reaction for NCP, an indirect immunofluorescence test was performed using goat anti- rabbit IgG (Behringwerke), labeled with fluorescein isothiocyanate. In prelimi- nary experiments, the goat anti-rabbit IgG serum, preabsorbed with human y globulins, was unable to stain the renal sections from normal as well as from SLE individuals. The renal sections were examined with a Zeiss microscope equipped with a HBO-200 high-pressure mercury vapor lamp, using a GB 12 exciter filter and a KV 490 barrier filter. A minimun of four glomeruli per section (range 4-23) was examined. The intensity of the fluorescent staining was evaluated indepen- dently by two observers and the intensity and extent were graded from 0 to 3. When staining for NCP was present, serial sections were incubated with antise- rum previously absorbed with purified NCP to assess the specificity of the reac- tion. PMN present in the glomeruli were quantitated independently by two ob- servers. The PMN index was evaluated by counting the total number of intra- glomerular PMN divided by the number of glomeruli, and expressed the number of PMN per glomerulus.

Staining of Glomerular Polyanions

The presence of GPA was evaluated by the colloidal iron (50) and the Alcian blue (14) staining techniques. Staining was performed on 4-pm-frozen or paraffin embedded sections of renal tissue. For the colloidal iron staining, after rinsing (30 set) in 12% glacial acetic acid aqueous solution, the sections were stained for 1 hr and 30 min in the colloidal iron solution, followed by four (3 min each) rinses in 12% glacial acetic acid. The pH of both the staining and the rinsing solutions was between 1.1 and 1.3 to insure selective staining of acidic polysaccharides without coloration of nucleic acids. The Prussian blue color was developed for 20 min in a freshly prepared mixture of equal parts of 2% hydrochloric acid and 2% potassium ferrocyanide. Carballumen (15 min) was used as counterstain. The amount of staining was evaluated independently by two examiners who were unaware of the pathologic findings in the renal sections. The intensity and extent of GPA staining were semiquantitatively evaluated and expressed as the number of glomeruli which exhibited segmental or total loss of GPA (Table 1). Specimens from eight normal subjects and from six patients with Berger’s glomerulonephritis served as controls. Furthermore, duplicate sections from each specimen were not exposed

306 CAMUSSI ET AL.

to colloidal iron but were processed with the HCl-potassium ferrocyanide part of the colloidal iron method in order to rule out nonspecific staining due to naturally occurring hemosiderin. In nine patients, GPA were also evaluated by the Alcian blue staining technique, a more selective but weaker stain (50) that invariably provided results comparable to those obtained by the colloidal iron technique.

RESULTS

As shown in Table 1, the study by direct and indirect immunofluorescence showed deposits of NCP in glomeruli of seven out of fourteen patients with SLE. The specificity of the immunofluorescence staining was confirmed by the loss of the reaction when the antiserum was absorbed with purified NCP. The deposits of NCP were localized in a linear, segmental manner along the peripheral part of the GCW (Figs. 3B, D, F) and markedly differed from the granular or patchy deposits of immunoglobulins (Figs. 3A, C, E) or complement. In contrast, aside from the segmental pattern, the deposits of NCP resembled those of polyanions detected by colloidal iron or Alcian blue stainings in normal human glomeruli (Figs. 4A, B).

Both the intensity and extent of NCP deposits were greater in glomeruli of patients with grade I of SLE activity. The glomeruli of these patients also had the greater loss in GPA (Figs. 4C-F), a loss that was especially marked in patients with severe proteinuria and nephrotic syndrome (Table 1). The loss in GPA, however, appeared more widespread than the deposits of NCP.

The study by light microscopy of glomeruli with NCP deposits revealed no correlation with local accumulation of PMN. Only in two patients (cases 3 and 13, Table l), with absence of NCP in GCW, were CP detected in the cytoplasm of PMN present in the glomeruli. In patients with grade I of SLE activity and with deposits of NCP in GCW (cases 6- 12, of Table l), the PMN present in glomeruli (PMN index: 2.45 t 1.48, mean + 1 SEM) failed to stain for NCP. Likewise, the PMN present in the peripheral blood of these patients appeared markedly depleted of NCP (Fig. 5B), as compared with PMN from normal individuals (Fig. SA).

Deposits of NCP were never detected in glomeruli from normal subjects or from patients with Berger’s glomerulonephritis. These glomeruli always showed normal amounts of polyanions, as detected by the colloidal iron or Alcian biue techniques (Table 1).

DISCUSSION

The results of the present study show that deposits of NCP and loss of GPA occur concomitantly in the GCW of patients with active SLE glomerulonephritis, proteinuria, and nephrotic syndrome. These findings are associated with depletion of NCP in circulating PMN.

SLE glomerulonephritis is a prototype of IC disease (51). In this condition, PMN are attracted and stimulated by IC to release lysosomal enzymes (52) and PAF (31, 32). These mediators may contribute to a local inflammatory reaction and to increase glomerular permeability. It has been shown that in SLE the in- teraction between IC and PMN occurs during the active phases of the disease (49) and results in neutropenia, a sign of disease activity (44). Our previous work

TABL

E 1

GI~O

MER

~.TI

AR

Lo

ch1

IZAT

ION

OF

NCP

DEPO

SITS

AN

D Lo

ss

OF

NEG

ATIV

E CH

ARGE

S IN

PA

TIEN

TS

WIT

H LU

PIC

NEPH

RITI

S CA

TEGO

RIZE

D AC

CORD

ING

TO

CLIN

ICAL

AC

TIVI

TY

Imm

unof

luor

esce

nce

Case

s

Anti-

hum

an’

Hist

olog

ic Ne

phro

tic

Prot

einur

ia Gr

ade

SLE

Anti-

NCP

Anti-

NCP”

cla

ss”

synd

rom

e (g

/24

hr)

activ

ity

on

PMN”

kG

kM

lg

.4

C3

Clq

C4

F Di

rect

In

dire

ct

SLE

(11

C.A.

(2

) P.

G.

(31

T.G

. (4

) V.

C.

(5)

G.F.

(6

) C.

R.

(7)

L.A.

(8

) B.

M.

(9)

P.C.

(1

0)

M.M.

(1

1)

P.A.

(1

2)

V.E.

(1

3)

G.A.

(1

4)

C.L.

Cont

rols

Berg

er’s

GN

(6)

NHK

(8)

I 11

II IV

III

III

IV

IV

IV

IV

IV

IV

IV

III - -

No

0.5

No

3.4

No

1.5

Yes

3.8

Yes

3.5

Yes

5.0

Yes

5.0

Yes

6.5

Yes

9.2

No

3.5

Yes

4.8

Yes

4.5

No

2.5

No

2.8

No

No

<OS

co.5

III

111

III II II II III II I I I I II I

N.D.

0

0 N.

D.

+1

0 +3

+1

+1

t2

+3

+1

+3

+2

0

t2

+4

13

N.D.

f3

fl

N.D.

+2

+2

+1

+3

t2

fl

+3

+3

+1

t3

t2

+2

+3

t2

+2

0 0

N.D.

+2

t1

+2/+

3g

t 11

20

0 -

+3

0 0

0 +1

0 +I

0 +3

+1

+1

t2

t2

+3

+3

+I

3-I

t 213

0 0

0 00

0 +2

+2

+1

i-2

0

+2

0 0

+1

+3

t1

0 t2

t3

13

+I

+3

t4

t1

+2

fl

+3

+1

0 +1

t2

0

0 t4

f4

+2

0

t3

t4

t1

+2

+2

t2

t2

t2

t3

i-2

t2

t2

+1

t3

+I

0 t1

+1

+1

1-

2

t2/3

g 0

0 +1

0

000

0 0

0 0

0 0

0 0

0 0

+1

+2

t2

t2

t2

t3

t2

+2

t1

t3

+I

t2

t2

t3

0 0

0 0

0 0

0 0

PMN

index

0’

O/5

015

0 O/

S 01

5 0.

14

O/5

O/5

0.15

01

5 O/

5 0

O/l8

51

18

1.84

I/7

31

7 2.

14

7118

11

/18

2.12

11

4 2/

4 1.

5 O

il1

1111

1 1.

75

8119

51

19

4.77

31

23

17/2

3 1.

66

l/S

415

2.14

o/

9 21

9 0

116

3/6

o-o.

379

1129

41

29

O-0.

208

0151

o/

5 I

Loss

of

G

PA’

Tota

l Se

gmen

tal

r( Cl

assif

icatio

n ac

cord

ing

to

Appe

I ef

nl.

(49)

. ’

Intra

cyto

plas

mic

NCP

were

de

tect

ed

by

dire

ct

imm

unof

luor

esce

nce

(see

M

ater

ials

and

Met

hods

). ’

Glom

erula

r de

posit

s of

Ig

G,

IgM,

IgA,

C3

, Cl

q,

C4,

and

fibrin

ogen

(F

l we

re

evalu

ated

by

im

mun

oflu

ores

cenc

e us

iug

spec

ific

rabb

it an

tiser

a.

d NC

P de

posit

s we

re

dete

cted

by

in

dire

ct

and

dire

ct

imm

unof

luor

esce

nce

usin

g sp

ecifi

c ra

bbit

anti-

NCP

seru

m.

Patie

nts

3 an

d I3

pr

esen

ted

stai

ning

in

the

cy

topl

asm

of

fe

w int

raglo

mer

ular

cells

. ’

Thes

e nu

mbe

rs

were

de

rived

fro

m

the

form

ula:

No.

of

intra

glom

erula

r PM

NINo

. of

gl

omer

uli.

’ GP

A we

re

stud

ied

by t

he

collo

idal

iro

n (5

01 a

nd

Alcia

n bl

ue

(14)

st

aini

ng

tech

niqu

es.

Resu

lts

were

ex

pres

sed

as th

e nu

mbe

r of

glo

mer

uli

with

eit

her

segm

enta

l or

to

tal

loss

of

GPA

on

the

tota

l nu

mbe

r of

gl

omer

uli.

For

cont

rols,

th

e nu

mbe

r of

glo

mer

uli

is g

iven

as t

he

tota

l su

m

of

glom

erul

i co

unte

d on

ea

ch

spec

imen

fro

m

diffe

rent

su

bjec

ts.

GN.

glom

erul

onep

hritis

: NH

K.

norm

al hu

man

kid

ney.

” Ra

nge

of

scor

es.

- -

.- ,..

-

.- -

- --

-

- -

1-1

-

-.

_ .I

I

_ -,

I.

-

.*

- ._

. .I

.-

-

308 CAMUSSI ET AL.

FIG

. 3.

Im

mun

oflu

ores

cenc

e m

icrog

raph

s of

glo

mer

uli

from

thr

ee

patie

nts

with

gr

ade

I SL

E ac

tivity

. A,

C,

E re

pres

ent

glom

erul

i fro

m

patie

nts

9, 1

0,

i 12

. res

pect

ivel

y (T

able

1). T

he g

lom

erul

i we

re

stai

ned

with

flu

ores

cein

-con

juga

ted

rabb

it an

ti-hu

man

Ig

G.

Patc

hy o

r gr

anul

ar

depo

sits

of h

uman

Ig

G a

re

pres

ent

alon

g G

CW

. B,

D,

and

F ag

ain

repr

esen

t gl

omer

uli

from

pat

ient

s 9,

10,

12,

resp

ectiv

ely

(Tab

le 1)

. The

se

glom

erul

i we

re

stai

ned

with

flu

ores

cein

- 3

conj

ugat

ed

rabb

it an

ti-N

CP.

N

ote

the

segm

enta

l, lin

ear

depo

sits

of N

CP

in p

erip

hera

l G

CW

. A,

B,

D,

E, X

400;

C

, X2

50;

F, X

100

0.

2 r, CiJ

310 CAMUSSI ET AL.

FIG

. 4.

St

aini

ng

of G

PA

by t

he c

ollo

idal

iro

n te

chni

que

in 4

-pm

-thic

k,

para

ffin-

embe

dded

re

nal

tissu

e.

A an

d B

show

de

nse,

ra

il-lik

e st

aini

ng

of t

he

F G

CW

(ar

rows

). C

, D

, E,

F s

how

glom

erul

i fro

m t

hree

diff

eren

t ne

phro

tic,

grad

e I

SLE

patie

nts

(cas

es 9

, 10,

12

of T

able

1). I

n C

, th

e re

dist

ribut

ion

of G

PA,

m

mai

nly

loca

lized

at

the

per

iphe

ry

of g

lom

erul

ar

loop

s (a

rrows

) is

vis

ible

. In

D

, se

gmen

tal

stai

ning

in

a m

arke

dly

scle

rose

d gl

omer

ulus

an

d in

E a

nd

F.

mz

com

plet

e di

sapp

eara

nce

of C

PA

are

seen

. A,

X3

00:

B,

x 10

00:

C.

D,

E, F

, x4

00.

i =i

;;

312 CAMUSSI ET AL.

FIG. 5. Photomicrographs of peripheral PMN obtained from unrelated normal donors (A) and (B) from a Grade I SLE patient (case 9 of Table I). The PMN were stained with fluorescein-conjugated rabbit anti-NCP. In A (example of score +3), there is staining of the cytoplasm (see also Fig. 2A). In B (example of score +l), a marked reduction of the cytoplasmic staining is observed. This figure is representative of 12 experiments performed in several patients with Grade I SLE A, B. x 1000.

indicated that NCP are released in the circulation during active stages of SLE (33). This finding is in agreement with the observation, reported in this paper, that in patients with acute episodes of SLE circulating PMN are depleted of their CP content, as compared to PMN of normal individuals.

The concomitant deposition of NCP and loss of fixed negative charges in GCW is consistent with the hypothesis that NCP may bind and neutralize GPA. Recent studies indicate that GPA are localized on the plasma membrane of endothelial and epithelial cells of GCW and, especially in the laminae rarae interna and ex- terna (8- 10). In view of the fact that lysozyme, a cationic protein, when injected into a normal rat binds and neutralizes GPA (14), it can be assumed that NCP, released from PMN during the acute stages of SLE, may also bind and neutralize GPA. Consistent with this interpretation is the linear and segmental staining pat- tern induced by fluorescein-conjugated NCP antibodies in the peripheral GCW of SLE patients, a pattern different from the granular and patchy staining obtained in the same kidneys with fluoresceinated antibodies to immunoglobulins and com- plement.

NCP deposits were detected in the glomeruli of six patients with diffuse prolif- erative glomerulonephritis (Class IV) and of one patient with focal proliferative glomerulonephritis (Class III) and were associated in 4/5 patients with grade I activity, but only in 2/5 patients with grade II and in l/4 with grade III. All patients showing glomerular NCP deposits had proteinuria (more than 3.5 g/24 hr) and, except one, all were nephrotic. In contrast, in 97 patients negative for NCP

NEUTROPHIL CATIONIC PROTEINS AND SLE NEPHRITIS 313

deposits, proteinuria was lower than 3.5 g/24 hr and no nephrotic syndrome oc- curred. The association between the deposition of NCP and active SLE, pro- teinuria as well as nephrotic syndrome suggests a pathogenetic relation between these events. However, while NCP binding was invariably associated with the loss of GPA (7/7 patients), this appeared more widespread than the NCP deposits. It is possible that the amount of NCP, sufficient to neutralize GPA, may be too low to be detected by immunofluorescence. It is also conceivable that cationic mediators other than NCP may have been released in the circulation and fixed by GCW. The most likely candidates are CP released from platelets. Platelet aggre- gation can be induced by IC (53) or by PAF (54, 55). Platelet could initiate the glomerular inflammatory (56) injury or amplify an inflammatory reaction initiated by NCP. Further studies are needed to clarify this issue.

The depletion of CP in circulating PMN, the presence of NCP deposits in GCW with the concomitant loss of GPA seems to suggest that NCP are released in V~VO from stimulated PMN and contribute to the pathogenesis of glomerular injury and proteinuria in patients with active SLE glomerulonephritis.

ACKNOWLEDGMENT

The authors wish to thank Professor G. A. Andres for his help.

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Received December 8. 1981: accepted with revisions March 13. 1982.