Establishment of Monoclonal Anti-Retroviral gp70 Autoantibodies from MRL/lpr Lupus Mice and Induction of Glomerular gp70 Deposition and Pathology by Transfer into Non-Autoimmune Mice

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Establishment of Monoclonal Anti-Retroviral gp70 Autoantibodies

from MRL/

lpr

Lupus Mice and Induction of Glomerular gp70

Deposition and Pathology by Transfer into

Non-Autoimmune Mice

NOBUTADA TABATA,

1

MASAAKI MIYAZAWA,

1,2

* RYUICHI FUJISAWA,

2

†

YUMIKO A. TAKEI,

2

HIROYUKI ABE,

1_AND

KEIJI HASHIMOTO

1,3

Department of Immunology

1

and Third Department of Internal Medicine,

3

Kinki University

School of Medicine, Osaka-Sayama, Osaka 589-8511, and Department of Pathology,

Tohoku University School of Medicine, Sendai 980-8575,

2

Japan

Received 6 October 1999/Accepted 1 February 2000

Several strains of mice, including MRL/MpJ mice homozygous for the Fas mutant

lpr

gene (MRL/

lpr

mice),

F

1

hybrids of New Zealand Black and New Zealand White mice, and BXSB/MpJ mice carrying a Y-linked

autoimmune acceleration gene, spontaneously develop immune complex-mediated glomerulonephritis. The

involvement of the envelope glycoprotein gp70 of an endogenous xenotropic virus in the formation of

circu-lating immune complexes and their deposition in the glomerular lesions have been demonstrated, as has the

pathogenicity of various antinuclear, antiphospholipid, and rheumatoid factor autoantibodies. In recent

genetic linkage studies as well as in a study of cytokine-induced protection against nephritis development, the

strongest association of serum levels of gp70–anti-gp70 immune complexes, rather than the levels of

antinu-clear autoantibodies, with the development and severity of glomerulonephritis has been demonstrated,

sug-gesting a major pathogenic role of anti-gp70 autoantibodies in the lupus-prone mice. However, the

pathoge-nicity of anti-gp70 autoantibodies has not yet been directly tested. To examine if anti-gp70 autoantibodies

induce glomerular pathology, we established from unmanipulated MRL/

lpr

mice hybridoma clones that secrete

monoclonal antibodies reactive with endogenous xenotropic viral

env

gene products. Upon transplantation, a

high proportion of these anti-gp70 antibody-producing hybridoma clones induced in syngeneic

non-autoim-mune and severe combined immunodeficiency mice proliferative or wire loop-like glomerular lesions.

Further-more, deposition of gp70 in glomeruli and pathological changes were observed after intravenous injection of

representative clones of purified anti-gp70 immunoglobulin G, demonstrating pathogenicity of at least some

anti-gp70 autoantibodies.

Several strains of mice such as MRL/MpJ mice homozygous

for the Fas mutant

lpr

gene (MRL/

lpr

mice), F

1

hybrids of New

Zealand Black (NZB) and New Zealand White (NZW) mice

[(NZB

⫻

NZW)F

1

], and BXSB/MpJ mice carrying a yet

un-defined Y-chromosome-associated autoimmune acceleration

gene (

Yaa

) spontaneously develop an autoimmune syndrome

closely resembling human systemic lupus erythematosus (SLE)

(1, 4, 35, 41). Both human and murine SLE are serologically

characterized by elevated levels of multiple autoantibodies (1,

4, 20, 35). These include antibodies (Abs) reactive with DNA

and other nuclear components, Abs to extracellular matrices

and cytoplasmic proteins, and in mice Abs reacting to the

major envelope glycoprotein (gp70) of an endogenous

xeno-tropic retrovirus that is expressed as a normal constituent of

mouse serum (8). These autoantibodies and resultant

circulat-ing immune complexes (IC) have been implicated in the

de-velopment of fatal glomerulonephritis. However, not all

auto-antibodies are primary pathogens (20, 39); in some cases they

may instead be a secondary consequence of tissue damage.

Several different approaches have been used to delineate the

relationship between types of autoantibodies and the

develop-ment of renal pathology. Several different clones of anti-DNA

antibodies have been shown to induce glomerular lesions

as-sociated with immunoglobulin (Ig) deposition and/or

protein-uria when transferred into non-autoimmune mice (13, 20, 36,

38). On the other hand, recent genetic analyses using simple

sequence length polymorphisms as positional markers have

identified several chromosomal loci in linkage with the

devel-opment and severity of the renal disease. Interestingly, one of

the genetic linkage analyses performed by using (NZB

⫻

NZW)

F

1

⫻

NZW backcross mice (40) demonstrated that the loci

linked with anti-gp70 Ab production, rather than those

asso-ciated with levels of antinuclear Ab, had the strongest

influ-ence on the development of glomerulonephritis. In a similar

study performed with C57BL/6

⫻

(NZW

⫻

C57BL/6.

Yaa

)F

1

backcross mice (26), association of serum levels of gp70 IC

with severe glomerulonephritis was much stronger than that

between levels of IgG anti-DNA autoantibodies and the renal

disease. In addition, transgenic expression of interleukin-4

(IL-4) in the (NZW

⫻

C57BL/6.

Yaa

)F

1

mouse model of SLE

resulted in almost complete protection against the

develop-ment of lupus-like nephritis in association with the lack of

IgG3 production and marked decrease in the amount of serum

gp70–gp70 IC, while the serum concentrations of

anti-DNA IgG were not markedly reduced (25). These data suggest

that autoantibodies reactive to endogenous retroviral gp70

comprise the major pathogenic Abs in the mouse models of

* Corresponding author. Mailing address: Department of

Immunol-ogy, Kinki University School of Medicine, 377-2 Ohno-Higashi,

Osaka-Sayama, Osaka 589-8511, Japan. Phone and fax: 81 723-67-7660.

E-mail: masaaki@med.kindai.ac.jp.

† Present address: Department of Microbiology, Kinki University

School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan.

4116

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lupus nephritis. However, suggested pathogenicity of anti-gp70

autoantibodies has not yet been directly proven. Therefore, we

decided to develop a new screening system and establish from

MRL/

lpr

mice hybridoma clones that secrete monoclonal Abs

(MAbs) reactive with endogenous xenotropic virus

env

gene

products. MRL mice were chosen so that passive transfer into

syngeneic mice of hybridoma cells and MAbs were more easily

performed than in the cases of the F

1

hybrid models with a

complex genetic background.

Tryptic peptide mapping analyses of gp70 molecules eluted

from IC revealed that the serum gp70 involved in the

produc-tion of circulating IC both in (NZB

⫻

NZW)F

1

and MRL/

lpr

mice is structurally related to the envelope glycoprotein of an

infectious NZB xenotropic virus (5, 12). Subsequent studies

have shown that almost all strains of mice, healthy and SLE

prone, produce endogenous xenotropic viral gp70 in the liver

as an invariable serum constituent, and its expression is

con-trolled as an acute-phase reactant (8). A cDNA clone encoding

the serum gp70 was isolated from the liver of a

lipopolysac-charide (LPS)-injected NZB mouse, and Northern blot

analy-ses confirmed the expression of this message as an acute-phase

reactant (29). Therefore, we used this cDNA clone, along with

the

env

gene from an infectious molecular clone of NZB

xe-notropic virus (21), for in vitro expression of the endogenous

retroviral

env

gene products to screen anti-gp70 Ab-producing

hybridoma cells. Resultant hybridoma clones established from

unmanipulated MRL/

lpr

mice induced severe glomerular

le-sions upon transplantation into syngeneic (BALB/c

⫻

MRL)F

1

and severe combined immunodeficiency (SCID) mice.

More-over, purified IgG molecules of representative anti-gp70

auto-antibodies induced glomerular deposition of gp70 and renal

pathology when injected intravenously (i.v.) into

non-autoim-mune mice.

MATERIALS AND METHODS

Mice.The original breeding pairs of MRL/MpJ-⫹/⫹(MRL/⫹) and MRL/lpr

mice were purchased from The Jackson Laboratory, Bar Harbor, Maine. These strains of mice were maintained by sister-brother mating in our animal facilities under specific-pathogen-free conditions. BALB/cCrSlc, NZW/NSlc, and C57BL/ 6CrSlc (B6) mice were purchased from Japan SLC, Inc., Hamamatsu, Japan, and (BALB/c⫻MRL/⫹)F1hybrid mice were bred in our animal facilities. C.B-17/

Icr-scid/scid(SCID) mice were produced from the breeding pairs originally donated by S. Ikehara, Kansai Medical University, Moriguchi, Japan, and were kindly provided by M. Nose, Tohoku University School of Medicine. All animal experiments described in this report were approved by the institutions and performed under the guidelines of our animal facilities.

NZB xenotropic virus-producing cells.NZB-AR cells that are chronically infected with a biological clone of NZB xenotropic virus were kindly provided by L. Evans, Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, Hamilton, Mont. Control uninfected Mv1Lu mink lung cells were purchased from the American Type Culture Collection, Manassas, Va. Expression of xenotropic murine leukemia viralenvgenes and their chimeras in recombinant vaccinia viruses.Vaccinia virus transfer vectors used for the expression of mouse retrovirusenvgenes and their chimeras were constructed as described previously (10, 17, 18). The structures of the expressedenvgenes and their chimeras are diagrammatically presented in Fig. 1. Plasmid clones pGP6-8, containing the gp70 cDNA isolated from a LPS-injected NZB mouse liver (29), and pNZB9-1, containing the whole permuted infectious molecular clone of an

[image:2.612.62.542.73.336.2]

NZB xenotropic virus, IU-6 (21), were used as sources of endogenous xenotropic virusenvgene sequences. Amino acid sequence analyses have revealed only three substitutions near the C terminus of gp70 between these twoenvgene products, although the C terminus of the transmembrane portion (p15E) contains five additional substitutions (21, 29), four of which are located within the R peptide that is cleaved from the mature transmembrane protein (31). ASalI oligonucle-otide linker (New England Biolabs, Beverly, Mass.) was ligated onto both ends of the 2.2-kbAccI-HaeII fragment harboring the entireenvsequence and a part of the long terminal repeat (LTR) isolated from pGP6-8 (Fig. 1), and the modifiedenv-containing fragment was recloned into the uniqueSalI site of the

FIG. 1. Diagrammatic representation of the liver-derived gp70 cDNA and viralenvgenes and their chimeras expressed in recombinant vaccinia viruses. The nucleotide sequence of the gp70 cDNA isolated from an LPS-injected NZB mouse liver (29) is 99% homologous to that of the infectious NZB xenotropic virusenv

gene (21), except for several base changes clustered near the gp70/p15E cleavage site and in the 3⬘flanking region and LTR (□), which are reflected by the indicated differences in restriction sites. The SFFVenvgene (

,

) is a product of natural recombination between endogenous polytropic and exogenous Friend ecotropic viruses with a large in-frame deletion (ƒ_{) encompassing the 3⬘portion of gp70- and the 5⬘portion of p15E-encoding regions. Dashed lines represent vector-derived sequences.} A,AccI; B,BamHI; E,EcoRI; Ha,HaeII; Hc,HincII; Hd,HindIII; K,KpnI; S,SmaI; V,EcoRV; X,BstXI; AAAAA, poly(A) tail.

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vaccinia virus expression vector pSC11-SS (10). This construct was used to generate a vaccinia virus-NZB liver cDNA recombinant. TheHincII-SmaI frag-ment harboring the entireenvgene and portions of thepoland LTR from pNZB9-1was reconstructed in pBluescript-KS(⫹) vector from purifiedHin cII-EcoRI andEcoRI-SmaI fragments (Fig. 1), and the uniqueAccI site was re-placed with aBamHI linker (New England Biolabs). ABamHI-digested frag-ment containing the entireenvgene and a part of the LTR was cloned into the uniqueBglII site of the previously described modified vaccinia virus expression vector pSC11-SB (17), resulting in the generation of a vaccinia virus-infectious NZB xenotropic virusenvgene recombinant. Theenvclones derived from the infectious NZB xenotropic virus and those derived from the NZB liver gp70 cDNA were easily distinguishable by the presence of a few different restriction sites (Fig. 1).

For the construction ofenvgene chimeras, a plasmid clone (BT4-1a3 [42]) that contains a permuted infectious molecular clone of the Friend spleen focus-forming virus (SFFV) was used as a source of nonxenotropicenvsequences (Fig. 1). A vaccinia virus recombinant expressing the whole SFFVenvgene has been described elsewhere (18). TheEcoRI-SmaI fragment from pNZB9-1was

sub-cloned into pBluescript-KS(⫹) and was ligated with the 1.3-kbHindIII-EcoRI fragment harboring the 3⬘portion of thepoland the 5⬘portion of the SFFVenv

genes from BT4-1a3. TheBamHI-digested fragment containing the entire chi-mericenvgene was then inserted to pSC11-SB at the uniqueBglII site. The resulting construct was used to generate a recombinant vaccinia virus that ex-pressed the chimeric SFFV-NZB xenotropic virusenvgene. For the construction of a reciprocal chimera, the uniqueKpnI site in the LTR of BT4-1a3 was replaced with theBamHI linker, and the 1.8-kbBamHI-digested fragment har-boring the entire SFFVenvgene was subcloned into pUC19. TheHincII-EcoRI fragment containing the 5⬘portion of the infectious NZB xenotropic virusenv

gene was ligated to theEcoRI-KpnI (BamHI) fragment of the subcloned SFFV

envgene, taking advantage of the uniqueHincII site in the vector, and theAccI site upstream of the initiation site of NZB xenotropic virusenvgene was replaced with a BamHI linker to insert theBamHI-digested fragment containing the chimericenvgene into pSB11-SB. The resultant plasmid was used to generate a recombinant vaccinia virus that expressed the chimeric NZB xenotropic virus-SFFVenvgene. Recombinant vaccinia viruses were produced by homologous recombination as described elsewhere (10, 17, 18). A recombinant vaccinia virus expressing the influenza virus hemagglutinin (HA) gene (30) was used as a negative control throughout the experiment.

Production and screening of hybridoma cells.Spleen and lymph node cells were prepared aseptically from unmanipulated MRL/lprmice. P3/NSI/1-Ag4-1 (NS-1) and P3X63Ag8.653 (8.653) myeloma cells were purchased from the American Type Culture Collection and used as fusion partner cells. Hybridoma cell fusion, hypoxanthine-aminopterin-thymidine selection, and cloning by col-ony formation in fibrin gels were performed as described previously (16, 24). For immunofluorescence detection of the reactivities of hybridoma Abs to expressed

envgene products, monkey CV-1 cells were grown in wells of 96-well tissue culture plates, infected with a recombinant vaccinia virus at 100 to 200 PFU/well for 20 to 36 h, and incubated at 4°C overnight with a hybridoma culture super-natant added at 100␮l/well. Culture supernatants were then aspirated, and the wells were washed twice with phosphate-buffered balanced salt solution (PBBS) (3) containing 2% fetal bovine serum (FCS), and once with PBBS not containing FCS. Cells in each well were fixed with methanol, blocked with 10% skim milk, and stained with a 1/150 dilution of fluorescein isothiocyanate (FITC)-conju-gated goat anti-mouse Ig Ab (Cappel, Organon Teknika Corporation, West Chester, Pa.) as described elsewhere (17). For observation, the plates were placed upside-down under a Zeiss Axioplan fluorescence microscope (Zeiss, Overkochen, Germany). Antinuclear Ab activity was detected by treating unin-fected CV-1 cells with methanol before incubating them with hybridoma-derived Ab. Methanol-fixed cells in wells of 96-well plates were washed with phosphate-buffered saline (PBS), blocked with 10% skim milk, and incubated with Ab as described above. To prepare representative immunofluorescence photographs, CV-1 cells were grown on glass coverslips and processed similarly.

Hybridoma cells producing reference MAbs that react with various mouse retrovirusenvgene products (2, 22, 23) were kindly provided by B. Chesebro, Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infec-tious Diseases. Hybridoma cell line N-S.7, producing mouse IgG3 reacting with sheep red blood cells (SRBC), was purchased from the American Type Culture Collection. Another IgG3-producing hybridoma clone, 11, reactive with mumps virus nucleoprotein (37), was kindly provided by Y. Ito, Department of Micro-biology, Mie University School of Medicine, Tsu, Japan. Ig isotypes of MAbs were determined by an Ouchterlony immunodiffusion method using an isotype-specific Ab kit (The Binding Site, Birmingham, United Kingdom) as described previously (16, 24). The above-described reference MAbs and others of previ-ously defined isotypes were used as controls in the Ig isotype determination.

Western blotting.Western blotting analyses of polypeptide specificity of the Abs was performed as described previously (17, 19, 22, 23), using extracts from NZB-AR and control Mv1Lu cells. In brief, cells were washed four times with ice-cold PBS and incubated with 0.5% NP-40 in 50 mM Tris-buffered saline (pH 7.4) containing 10 mM EDTA, 5 mMn-ethylmaleimide, 1 mM phenylmethyl-sulfonyl fluoride, and 0.002% leupeptin at 4°C for 15 min. The supernatant was collected after centrifugation at 15,000⫻gfor 10 min. The extract was mixed with an equal volume of 4% sodium dodecyl sulfate (SDS) sample buffer (17, 19)

without a reducing agent and was subjected to SDS-polyacrylamide gel electro-phoresis. Proteins separated through 7.5% polyacrylamide gels were transferred onto polyvinylidene difluoride membranes (Immobilon; Millipore Corporation, Bedford, Mass.) as described previously (17, 19), and the blotted membrane was blocked with 10% skim milk. Incubation with MAb and detection of bound Ab by using biotinylated horse anti-mouse Ig secondary Ab and avidin-biotinylated peroxidase complex (Vector Laboratories, Burlingame, CA) has been described elsewhere (17, 19).

For the detection of serum gp70, sera from NZW, (BALB/c⫻MRL/⫹)F1, and

B6 mice were mixed at 1:20 with the SDS sample buffer containing no reducing agent, and serum proteins were separated through 7.5% polyacrylamide gels and blotted as described above. Serum gp70 molecules were detected with biotin-conjugated anti-gp70 MAb 24-6 by chemiluminescence reaction using horse-radish peroxidase-conjugated streptavidin (Vector Laboratories) and ECL⫹

reagent (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturers’ instructions.

Transfer of hybridoma cells or purified Abs into mice and pathological anal-yses.Hybridoma cells were grown in Dulbecco’s modified Eagle medium sup-plemented with glucose (4.5 g/liter [final concentration]), gentamicin sulfate (50 mg/liter), and 10% FCS, washed twice with PBBS, and resuspended in PBBS at 107_{cells/ml. (BALB/c⫻MRL/⫹)F}

1and SCID mice were transplanted

intra-peritoneally (i.p.) with 1⫻107_{to 2⫻10}7_{hybridoma cells after a pretreatment}

with a 0.5-ml/mouse i.p. dose of 2,6,10,14-tetramethylpentadecane (pristane; Aldrich Chemical Co., Inc., Tokyo, Japan) given 1 to 3 weeks prior to hybridoma transplantation. Serum concentrations of IgG in transplanted SCID mice were measured by single radial immunodiffusion assays using isotype-specific antisera (anti-mouse IgG2a and anti-mouse IgG3; Zymed Laboratories, Inc., South San Francisco, Calif.) as described previously (34). For purification of a clonal anti-gp70 IgG, hybridoma cells were grown in a serum-free medium (Hybridoma SFM; Gibco BRL, Rockville, Md.) in 4-liter spinner flasks, and culture super-natants were concentrated by using a tangential flow ultrafiltration system (Mini-tan II; Millipore Corporation). IgG was purified by protein A-Sepharose (Am-ersham Pharmacia Biotech) affinity chromatography as described previously (19, 24). Special care was taken to perform the purification aseptically at room temperature. Purified MAbs dissolved in PBBS at 0.5 to 1.0 mg/ml were injected into the tail vein after removing possibly contaminating Ig aggregates by centrif-ugation at 10,000⫻gfor 15 min.

The methods of preparation and staining of formalin-fixed, paraffin-embedded tissue sections and specimens for electron microscopy have been described else-where (19, 34). A part of the kidneys from each mouse was snap frozen in a mixture of dry ice and acetone after being embedded in O.C.T. compound (Miles Scientific, Naperville, Ind.), and frozen sections were prepared as described previously (16, 19, 24). For immunofluorescence detection of mouse IgG and C3 in frozen sections, FITC-conjugated goat anti-mouse IgG and anti-mouse C3 Ab (Cappel, Organon Teknika Corporation) were used. To detect the deposition of retroviral gp70, MAbs specific for xenotropic viralenvgene products, 24-6 and 24-9 (23), were purified as described above and labeled with biotin (19, 24). Localization of the biotinylated anti-xenotropic viral envelope MAb was visual-ized by using the avidin-biotinylated peroxidase complex (Vector Laboratories) as described previously (16, 24).

Histopathologic severity of each glomerular lesion in periodic acid-Schiff (PAS)-stained sections was semiquantitatively determined according to previ-ously described criteria (34), and an average index of glomerular pathology (IGP) was calculated by examining⬎20 glomeruli per mouse. In brief, grade 1 was given when there was apparent increase in the number of mesangial cells (⬎3 nuclei in a single separate section of a mesangial area) but no inflammatory cell infiltration into capillaries, grade 2 was given when cellular components were increased in at least one capillary lumen, and grade 3 was given when obliteration of at least one capillary lumen with fibrin- or collagen-containing materials was observed. Grade 0 means that none of the above histologic changes were ob-served in a glomerulus in question. Mice in which⬎80% of examined glomeruli showed significant histologic changes (IGPⱖ1) or in which 30 to 80% of glomeruli showed severe histologic changes (IGPⱖ2) are designated nephritic in this study. Incidences and average IGP were statistically compared with those of control mice by Fisher’s exact probability test and by Student’sttest, respec-tively.

RESULTS

Expression of endogenous xenotropic viral

env

cDNA and

establishment of MAbs reactive with the

env

gene product

from MRL/

lpr

mice.

A DNA fragment containing the entire

env

gene sequence from the cDNA clone isolated from a

LPS-injected NZB mouse was inserted into a vaccinia virus

expres-sion vector, and a recombinant vaccinia virus that expressed

the liver-derived xenotropic viral envelope glycoprotein was

constructed (Fig. 1). The whole

env

gene from a molecular

clone of an infectious xenotropic virus isolated from an NZB

mouse, IU-6, was also expressed in another vaccinia virus

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combinant as a positive control. Reactivities of a panel of

antiretroviral MAbs previously established from

non-autoim-mune mice (2, 22, 23) to these two xenotropic viral

env

gene

products showed no difference (Fig. 2 and Table 1), reflecting

their almost identical amino acid sequences.

Fifteen separate hybridoma clones were selected from a

total of three fusions of spleen and lymph node cells, using

eight unmanipulated, female MRL/

lpr

mice, both for reactivity

of secreted Ab with CV-1 cells expressing the liver-derived

gp70 cDNA and for lack of reactivity to cells infected with the

control vaccinia virus-influenza virus HA recombinant, as

ex-emplified in Fig. 2. Of these, three were established from the

first fusion performed by using spleen and lymph node cells

from two 2.5-month-old female mice and 8.653 myeloma cells,

seven others were established from the second fusion in which

four 2.5 month-old female mice and NS-1 myeloma cells were

used, and the remaining five clones were derived from the third

fusion performed by using two 4.5-month-old female mice and

NS-1. An additional clone, 17D7.1, was similarly selected from

a fusion made with spleen and lymph node cells of two

4.5-month-old male MRL/

lpr

mice and 8.653 myeloma cells. A few

nonproducer clones were also established from these fusions,

as represented by clone 4E9.1 in Table 2, and were used as

negative controls in the following experiment along with the

fusion partner cells. During the initial screening procedure for

Ab-producing hybridoma cells, wells containing antinuclear Ab

were also observed at roughly the same frequency as those

containing anti-gp70 Ab; however, none of the MAbs selected

for reactivity to the xenotropic viral

env

gene product

cross-reacted with nuclear antigens in the immunofluorescence

as-say. Western blotting analysis confirmed the reactivity of these

MAbs with the whole

env

gene product gp85 (gp70 plus p15E),

which was detected from the lysate of NZB-AR cells

chroni-cally infected with an NZB xenotropic virus but not from the

lysate of uninfected Mv1Lu cells (Fig. 3). Although Abs

reac-tive with the surface components of CV-1 cells other than

expressed gp70 were eliminated through the screening

proce-dure, by selecting MAbs reactive with the plaques of

gp70-expressing cells but not with the surrounding uninfected CV-1

cells (Fig. 2), a few bands other than that of gp85 were readily

detectable with some of these MAbs in blots of both NZB-AR

and uninfected Mv1Lu cell lysates, suggesting possible

cross-reactivity with normal cellular components.

[image:4.612.117.491.72.244.2]

The 16 MAbs reactive with the xenotropic viral

env

gene

products were further analyzed for reactivities to the products

of the SFFV

env

gene and the chimeras between NZB

xeno-tropic virus and SFFV

env

genes (Fig. 1 and Table 2) for rough

epitope mapping. The SFFV

env

gene is a naturally produced

recombinant with a large deletion encompassing the

C-termi-nal one-fourth of the gp70 and the N-termiC-termi-nal half of the

transmembrane p15E (42). Its gp70 sequence is unrelated to

that of Friend murine leukemia virus, but the N-terminal

one-FIG. 2. Antigenic characterization of expressedenvgene products using a panel of MAbs. Representative immunofluorescence micrographs of infected CV-1 cells are presented. 24-6, 24-8, 514, and 603 are anti-retroviral envelope MAbs of previously defined virus type and polypeptide specificities (2, 22, 23), whose reported reactivities to different types of mouse C-type retroviruses are given in parentheses; 12H5.1 is a representative MAb newly established from MRL/lprmice in this study. Note that only the foci of cells infected with a relevant recombinant vaccinia virus, not the uninfected cells surrounding the foci, are stained.

TABLE 1. Reactivities of reference MAbs of known specificity to various murine leukemia virus

env

gene products

Hybridoma clone

Virus type, polypeptide specificitya

Reactivity with CV-1 cells expressingb_:

NZB liver

gp70 cDNA NZB IU-6env SFFVenv NZBenv⫹SFFV SFFVenv⫹NZB Influenzavirus HA

24-6

Xeno

⫹

Poly, gp70

⫹

⫹

⫺

⫹

⫺

⫺

24-8

Eco

⫹

Poly, gp85

⫺

⫺

⫺

⫺

⫺

⫺

514

Poly, gp70

⫺

⫺

⫹

⫺

⫹

⫺

603

Xeno, gp70

⫹

⫹

⫹

⫹

⫹

⫺

11

Mumps, NP

⫺

⫺

⫺

⫺

⫺

⫺

a_{Virus type specificities of reference MAbs are summarized from the previous reports (2, 22, 23). Xeno, xenotropic viruses; Poly, polytropic viruses; Eco, ecotropic}

viruses; NP, nucleoprotein.

b_{Tested by indirect immunofluorescence assays using CV-1 cells infected with an indicated recombinant vaccinia virus as target cells as shown in Fig. 2. Assays were}

repeated at least three times for each MAb, with consistent results.

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[image:4.612.52.552.605.694.2]

third is most similar to endogenous polytropic viruses (31, 42),

a type distinct from xenotropic viruses. Eight of the MRL/

lpr

-derived MAbs (17D7.1 through 42D3.2 in Table 2) reacted

with the products of the both xenotropic viral

env

genes but

lost reactivity when the 5

⬘

one-third of the NZB xenotropic

viral

env

gene was replaced at the

Eco

RI site with the

corre-sponding portion of SFFV

env

. Based on their reactivity to the

reciprocal chimera (NZB xenotropic viral

env

-SFFV

env

), they

are most likely to react with epitopes located in the N-terminal

[image:5.612.55.551.91.307.2]

one-third of xenotropic viral gp70. On the other hand, two

clones, 37C4.1 and 42D3.1, reacted with the products of the

whole NZB xenotropic viral

env

and the SFFV

env

-NZB

xe-notropic viral

env

chimera but not with the products of the

SFFV

env

and the NZB xenotropic viral

env

-SFFV

env

chi-mera. Therefore, they seem to recognize epitopes located in

the C-terminal portion of the xenotropic viral

env

gene

prod-ucts. Six other clones were reactive to the products of

xeno-tropic viral and SFFV

env

genes and both of the chimeras.

TABLE 2. Characteristics of anti-xenotropic viral MAbs established from MRL/

lpr

mice and incidence and severity of

glomerular lesions induced by transplantation of the hybridoma cells

a

Hybridoma clone Heavy-chain_isotype

Reactivity with CV-1 cells expressing: Glomerular pathology NZB liver

gp70 cDNA NZB IU-6env SFFVenv NZBenv⫹SFFV SFFVenv⫹NZB Influenzavirus HA Incidence Avg IGPSEM ⫾

8.653

(⫺)

⫺

⫺

⫺

⫺

⫺

⫺

1/7

0.42

⫾

0.10

NS-1

(⫺)

⫺

⫺

⫺

⫺

⫺

⫺

0/7

0.12

⫾

0.03

4E9.1

b

(nonproducer)

(⫺)

⫺

⫺

⫺

⫺

⫺

⫺

0/6

0.22

⫾

0.12

17D7.1

␮

⫹

⫹

⫺

⫹

⫺

⫺

0/17

0.52

⫾

0.05

34B4.1

␮

⫹

⫹

⫺

⫹

⫺

⫺

0/8

0.34

⫾

0.08

58C5.1

␮

⫹

⫹

⫺

⫹

⫺

⫺

10/12

c

1.07

⫾

0.08

e

12H5.1

␥3

⫹

⫹

⫺

⫹

⫺

⫺

12/13

c

2.28

⫾

0.18

e

36D1.1

␥2a

⫹

⫹

⫺

⫹

⫺

⫺

0/11

0.23

⫾

0.05

37C6.1

␥2a

⫹

⫹

⫺

⫹

⫺

⫺

10/14

c

0.96

⫾

0.06

e

42B4.1

␥2a

⫹

⫹

⫺

⫹

⫺

⫺

0/8

0.49

⫾

0.05

e

42D3.2

␥2a

⫹

⫹

⫺

⫹

⫺

⫺

1/7

0.54

⫾

0.11

f

6F12.3

␮

⫹

⫹

⫹

⫹

⫹

⫺

0/12

0.30

⫾

0.06

7C6.3

␮

⫹

⫹

⫹

⫹

⫹

⫺

1/7

0.49

⫾

0.09

51C4.1

␥3

⫹

⫹

⫹

⫹

⫹

⫺

1/4

0.75

⫾

0.15

f

51D1.1

␥3

⫹

⫹

⫹

⫹

⫹

⫺

14/19

c

1.20

⫾

0.12

e

59C4.1

␥3

⫹

⫹

⫹

⫹

⫹

⫺

4/8

0.88

⫾

0.16

f

60A5.1

␥3

⫹

⫹

⫹

⫹

⫹

⫺

6/9

d

1.01

⫾

0.09

e

37C4.1

␥3

⫹

⫹

⫺

⫺

⫹

⫺

12/12

c

2.10

⫾

0.19

e

42D3.1

␥2a

⫹

⫹

⫺

⫺

⫹

⫺

0/10

0.49

⫾

0.06

a_{Reactivities of MAbs were tested by indirect immunofluorescence assays as described for Table 1. Criteria for histopathologic diagnosis of glomerulonephritis and}

the standards for determination of the IGP are described in Materials and Methods. The incidence of nephritis and IGP for each MAb were statistically compared with those for relevant fusion partner cells.

b_{Three other nonproducer or non-anti-gp70 hybridoma clones were similarly tested for potential pathogenicity, and none induced significant glomerular pathology}

in transplanted (BALB/c⫻MRL/⫹)F1mice.

c_{P⬍0.003 by Fisher’s exact probability test.} d_{0.01⬍P⬍0.02 by Fisher’s exact probability test.} e_{P⬍0.001 by Student’sttest.}

f_{0.001⬍P⬍0.01 by thettest.}

FIG. 3. Representative results of Western blotting assays showing virus polypeptide specificities of the MAbs. Mr, molecular mass markers, with positions indicated in kilodaltons at the left; U, uninfected mink Mv1Lu cells; AR, NZB-AR cells chronically infected with a biological clone of NZB xenotropic virus. N-S.7 is a negative control IgG3 specific for SRBC; 603 is a positive control IgM specific for xenotropic viral gp70. The arrowhead indicates bands of the viralenvgene product, gp85 (gp70 plus p15E).

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[image:5.612.135.470.520.692.2]

These MAbs, therefore, may recognize epitopes common to

different types of mouse retrovirus

env

gene products. It is

notable that these latter MAbs showed more prominent

cross-reactivity to normal cellular components in Western blotting as

exemplified by clones 51D1.1 and 603 in Fig. 3.

Pathogenicity of gp70-reactive autoantibodies produced

from transplanted hybridoma cells in non-autoimmune mice.

To test possible pathogenicity of these MAbs reactive with

retroviral gp70, each hybridoma clone was injected i.p. into

syngeneic (BALB/c

⫻

MRL/

⫹

)F

1

mice that had been injected

with a single i.p. dose of pristane to facilitate hybridoma

trans-plantation. Injected mice were killed before dying of a tumor

burden, and the organs were examined histopathologically.

The average interval between pristane injection and organ

removal was 23.8 days, and that between hybridoma

transplan-tation and organ removal was 14.3 days. Six of the 16

hybrid-oma clones induced in syngeneic (BALB/c

⫻

MRL/

⫹

)F

1

mice

significant glomerular lesions at a considerable frequency,

while the transplantation of fusion partner cells or a control

nonproducer clone did not induce significant pathology at this

early stage after a single pristane treatment (Fig. 4 and Table

2). Another clone, 59C4.1, induced histologically evident

glo-merular lesions (Fig. 4l) at a low frequency (in four of eight

mice). Among the six clones that consistently induced

glomer-ular lesions, four IgG3-producing hybridoma clones, 12H5.1,

37C4.1, 51D1.1, and 60A5.1, caused diffuse and histologically

more severe glomerular pathology compared with other

anti-gp70 hybridomas when transplanted into (BALB/c

⫻

MRL/

⫹

)F

1

mice. The glomerular lesions induced by transplantation

of hybridoma clone 12H5.1 were characterized by

intracapil-lary proliferation and/or infiltration of cells with granular

sub-endothelial and intracellular deposition of IgG and C3 (Fig. 4b

to d). Massive deposition of fibrin was also demonstrated in

affected glomeruli by phosphotungstenic acid-hematoxylin

stain-ing (not shown). On the other hand, hybridoma clone 37C4.1

induced diffuse lupus-like glomerular lesions characterized by

light microscopic wire loops and massive subendothelial IgG

deposition (Fig. 4g and h). Two other IgG3 clones, 51D1.1 and

60A5.1, induced proliferative glomerular lesions at a high

in-cidence with dilatation of capillary lumina and occasional

ac-cumulation of red cell fragments (Fig. 4k). One clone (58C5.1)

out of five IgM- and another (37C6.1) from five

IgG2a-pro-ducing hybridoma cells also induced proliferative glomerular

lesions at a high frequency (Table 2).

Induction of glomerular lesions in transplanted SCID mice

and deposition of gp70 in glomeruli.

To exclude the possibility

that the induction of glomerular lesions by transplantation of

the hybridoma cells was due to host immune responses against

hybridoma-derived Abs or cellular components, or a result of

autoantibody production in response to pristane injection, we

next injected three representative clones of the hybridoma cells

into SCID mice. Both hybridomas 12H5.1 and 37C4.1 induced

in the transplanted SCID mice glomerular lesions that were

histologically similar to the lesions induced in the F

1

mice (Fig.

4e, f, i, and j). The presence of xenotropic viral gp70, along

with IgG and complement, was demonstrated in glomerular

lesions of SCID mice transplanted with either one of the two

pathogenic hybridoma clones (Fig. 4f and j), suggesting the

deposition of gp70 IC.

On the other hand, an IgG2a-producing anti-gp70

hybrid-oma clone, 36D1.1, did not induce significant nephritic lesions

in SCID mice. Since differential measurement of the

concen-trations of IgG produced from transplanted hybridoma cells

was impractical in immunocompetent (BALB/c

⫻

MRL/

⫹

)F

1

mice, serum concentrations of hybridoma-derived IgG were

determined by single radial immunodiffusion in SCID mice. At

the time the transplanted SCID mice were killed for

histopath-ologic examination, average serum concentration of IgG2a in

hybridoma 36D1.1-bearing mice was 6.7 mg/ml, while that of

IgG3 in hybridoma 12H5.1-bearing mice was 5.3 mg/ml. In

some SCID mice transplanted with hybridoma 36D1.1 cells,

higher serum concentrations of IgG2a such as 16.6 mg/ml were

observed.

Induction of gp70 deposition and glomerular pathology by

injecting purified MAb.

To further exclude the possibility that

the glomerular lesions were induced by products of the

hybrid-oma cells other than Ig, IgG3 molecules purified from culture

supernatants of hybridoma cells 12H5.1 and 51D1.1 were

in-jected i.v. into syngeneic (BALB/c

⫻

MRL/

⫹

)F

1

mice. A single

injection of purified 12H5.1 induced minimal glomerular

pa-thology; however, when purified 12H5.1 IgG3 (0.25 mg/mouse)

was injected for 3 consecutive days and the kidneys were

ex-amined 2 days after the final injection, diffuse granular

depo-sition of retroviral gp70 in the glomeruli was observed by

immunohistochemical staining (Fig. 5c and d) along with IgG.

No gp70 deposition was observed when control anti-SRBC

IgG3 was injected in the same manner (Fig. 5e). Histologic

changes characterized by PAS-positive depositions in the

mes-angial area were also observed in all the mice injected with

purified 12H5.1 IgG (Fig. 5g) but not in those injected with

purified anti-SRBC IgG (Fig. 5f). Repeated injection of

puri-fied 51D1.1 on the same schedule resulted in slight expansion

of mesangial areas and minimal deposition of gp70. However,

when purified 12H5.1 and 51D1.1 were mixed and injected for

3 consecutive days as described above, apparently more severe

glomerular pathology characterized by edema and

PAS-posi-tive deposits in the mesangial areas and some capillary walls

was observed (Fig. 5h).

Thus, these results directly indicate that at least some

mono-clonal anti-gp70 autoantibodies induce, in the absence of other

cellular products, glomerular deposition of gp70 and renal

pathology.

Differences in glomerular pathology in mice expressing high

and low levels of serum gp70.

To further examine the

possi-bility that injected anti-gp70 autoantibodies were involved in

the formation of immune complexes with serum gp70, purified

MAb 12H5.1 was injected i.v. into three different strains of

mice that are known to express high or low levels of serum

gp70. NZW mice have been shown to express the highest level

of serum gp70 among several different strains tested (14), while

B6 mice express a very low level of gp70 in their sera (15).

These differences in the amount of expressed serum gp70 were

also confirmed by Western blotting, along with the expression

of a relatively large amount of serum gp70 in (BALB/c

⫻

MRL/

⫹

)F

1

mice (Fig. 6a). Although NZW and B6 mice are

not syngeneic to BALB/c and MRL backgrounds in which the

hybridoma cells were produced, mouse IgG3 constant regions

contain extremely limited polymorphisms (32), and no

sero-logically definable IgG3 allotypes have been reported. Thus,

induction of anti-allotypic immune responses by injecting

pu-rified IgG3 molecules is very unlikely.

When IgG3 molecules purified from culture supernatants of

12H5.1 or control N.S-7 hybridoma cells were injected for 3

consecutive days as described above, all of the 10 NZW and 8

(BALB/c

⫻

MRL/

⫹

)F

1

mice injected with 12H5.1 IgG3

de-veloped focal but significant glomerular pathologies

character-ized by thickening of the capillary walls, cell proliferation, and

inflammatory cell infiltration (Fig. 6b and c). On the other

hand, significant pathologic changes were not observed in the

B6 mice injected with purified 12H5.1 IgG3 (Fig. 6d). Control

N.S-7 IgG3 did not induce significant glomerular lesions in any

of the three strains of mice. Furthermore, no deposition of

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gp70 was demonstrated in the kidneys of B6 mice after

injec-tion of purified 12H5.1 IgG3.

DISCUSSION

In this study, we established from unmanipulated MRL/

lpr

lupus mice hybridoma clones that secrete MAbs reactive with

[image:7.612.81.525.71.493.2]

the endogenous xenotropic viral

env

gene products. The MAbs

were selected both for reactivity with CV-1 cells expressing the

xenotropic viral

env

cDNA and for lack of reactivity with the

same cells expressing the influenza virus HA gene. Specificities

of the established MAbs were further confirmed by Western

blotting and immunofluorescence assays using CV-1 cells

ex-pressing chimeric

env

genes between NZB xenotropic virus and

FIG. 4. Representative kidney pathology of hybridoma-transplanted mice. (a) (BALB/c⫻MRL/⫹)F1mouse transplanted with 8.653 fusion partner cells. PAS

staining,⫻70. (b) (BALB/c⫻MRL/⫹)F1mouse transplanted with hybridoma cells 12H5.1. PAS staining,⫻70. Note the extreme expansion of the glomeruli compared

to those in panel a, which shows normal glomeruli at the same magnification. Sizes of tubules and of the nuclei of tubular epithelial cells are not different in panels a and b, but glomeruli are markedly enlarged in panel b. Granular deposition of fibrin in the affected glomeruli was also shown when phosphotungstenic acid-hematoxylin staining was applied (not shown). (c) Immunofluorescence staining with FITC-conjugated anti-mouse IgG of a fresh-frozen section taken from a representative (BALB/c⫻MRL/⫹)F1mouse transplanted with hybridoma cells 12H5.1. Use of FITC-conjugated anti-mouse C3 resulted in a similar pattern of staining.

(d) Electron micrograph showing an affected glomerulus of a representative (BALB/c⫻MRL/⫹)F1mouse transplanted with hybridoma 12H5.1. Cells occupying the

capillary lumina (❉) are filled with numerous electron-dense granules. Arrows indicate subendothelial deposits along the basement membrane. Bar⫽2␮m. (e) SCID mouse transplanted with hybridoma cells 12H5.1. PAS staining,⫻140. (f) Immunoperoxidase staining of a fresh-frozen section prepared from a SCID mouse at 3 days after transplantation of hybridoma 12H5.1. Purified MAb 24-9 (23) was biotinylated to detect the presence of xenotropic viralenvgene products. (g) (BALB/c⫻

MRL/⫹)F1mouse transplanted with hybridoma cells 37C4.1 showing typical wire loop lesions. PAS staining,⫻175. (h) Electron micrograph of the kidney from a

(BALB/c⫻MRL/⫹)F1mouse transplanted with hybridoma cells 37C4.1. Bar⫽2␮m. Note the dense subendothelial deposits consistent with light microscopic wire

loops along the basement membrane. (i) SCID mouse transplanted with hybridoma cells 37C4.1. PAS staining,⫻140. (j) Dense linear deposition of mouse C3 in a representative glomerulus from a SCID mouse transplanted with hybridoma 37C4.1. Similar deposition of mouse IgG and xenotropic viral gp70 in the affected glomeruli was also demonstrated in fresh-frozen sections of the transplanted SCID mice. (k and l) (BALB/c⫻MRL/⫹)F1mice transplanted with one clone of hybridomas 51D1.1

and 59C4.1, respectively. PAS staining,⫻140. Note PAS-positive deposition and expansion of the mesangial areas in panel k and cell proliferation (arrowheads) and occlusive changes (arrow) of capillaries in panel l. Lesions similar to those in panel l were observed in the mice transplanted with hybridoma 60A5.1 (not shown).

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Friend SFFV. About one-half of the gp70-reactive MAbs

es-tablished from MRL/

lpr

mice lost reactivity when a part of the

xenotropic viral

env

gene was replaced with the corresponding

portion of SFFV

env

, thus confirming the presence of antigenic

epitopes within the xenotropic viral gp70. On the other hand,

[image:8.612.141.464.68.584.2]

some other MAbs similarly established from MRL/

lpr

mice

were reactive to both the xenotropic viral and SFFV

env

gene

products. These latter MAbs, exemplified by clone 51D1.1,

tended to show reactivities to several protein bands other than

gp85 that were common to uninfected Mv1Lu mink cells and

FIG. 5. Photomicrographs showing gp70 deposition in glomeruli and pathology induced in mice injected with purified anti-gp70 MAb. (a [⫻85] and b [⫻175]) Representative frozen sections taken from a female MRL/lprmouse showing granular deposition of gp70 in glomeruli; immunoperoxidase staining with MAb 24-6. (c [⫻85] and d [⫻175]) Representative frozen sections taken from a (BALB/c⫻MRL/⫹)F1mouse injected with purified 12H5.1 IgG3; immunoperoxidase staining with

MAb 24-6. Note that all four glomeruli seen in panel c (arrows) exhibit gp70 deposition. Deposits of gp70 seem to localize along mesangial cells (d). (e) Representative frozen section taken from a control (BALB/c⫻MRL/⫹)F1mouse injected with purified N-S.7 IgG3. No gp70 deposition was observed. (f) Representative glomeruli

of a control (BALB/c⫻MRL/⫹)F1mouse injected with purified N-S.7 IgG3; PAS staining,⫻175. (g) Representative glomerular pathology induced by injection of

purified 12H5.1 IgG3 in (BALB/c ⫻MRL/⫹)F1 mice; PAS staining,⫻175. Note expansion of mesangial areas with PAS-positive deposits (arrowhead). (h)

Representative glomerular pathology induced in (BALB/c⫻MRL/⫹)F1mice by injecting a mixture of purified 12H5.1 and 51D1.1 IgG3; PAS staining,⫻350. Note

expansion of mesangial spaces between the capillaries and subendothelial hyaline deposits (arrowhead).

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NZB-AR cells chronically infected with an NZB xenotropic

virus in Western blotting. Possible cross-reactivity of these

gp70-reactive antibodies with normal cellular components and

its potential roles in the development of autoimmune lesions,

described as molecular mimicry (6), might be worth pursuing.

Glomerular lesions were induced in non-autoimmune mice

by transplanting single clones of hybridoma cells producing

gp70-reactive Abs. At least 7 of the 16 separate hybridoma

clones established from unmanipulated MRL/

lpr

mice induced

histopathologically significant glomerular lesions, and

deposi-tion of xenotropic viral gp70 along with IgG and C3 was

dem-onstrated in the lesions induced by transplantation of the two

representative hybridoma clones into SCID mice. Direct

in-volvement of the anti-gp70 MAb, rather than possible

second-ary host immune responses to the transplanted hybridoma cells

including anti-idiotypic Ab production, in the induction of

glo-merular pathology was demonstrated by successful induction in

SCID mice of glomerular lesions that were similar to those

induced in (BALB/c

⫻

MRL/

⫹

)F

1

mice.

It has been shown that a single i.p. injection of pristane

induces in non-autoimmune BALB/c mice production of

anti-nuclear ribonucleoprotein and anti-Su autoantibody

produc-tion and proliferative glomerulonephritis (27, 28). However,

the development of autoantibody production and nephritis

took months after pristane injection (27). On the other hand,

our mice were killed and examined within 5 weeks after a

single pristane injection, and thus it is unlikely that the

injec-tion of pristane alone was responsible for the development of

glomerular lesions in the hybridoma-transplanted mice. In fact,

control mice transplanted with the fusion partner cells or a

nonproducer clone of hybridoma cells established from MRL/

[image:9.612.85.520.72.410.2]

lpr

mice after an injection of the same pristane dose showed

only minimal pathologic changes in the kidneys (Fig. 4 and

Table 2). Reproduction of severe glomerular pathology in

SCID mice that should not produce any Ab in response to

pristane injection (Fig. 4) also supports the notion that

pristane-induced autoantibody production is not the major

pathogenetic factor in this transplantation model. It should be

noted that possible production of anti-gp70 autoantibodies in

the above-described pristane-induced model of nephritis has

not been examined. Thus, the presence of the pristane-induced

model neither contradicts nor supports possible pathogenicity

FIG. 6. Differences in serum gp70 expression and glomerular pathology induced after injection of purified 12H5.1 IgG3 in NZW, (BALB/c⫻MRL/⫹)F1, and B6

mice. (a) Results of Western blotting assays showing the expression of serum gp70 in three different strains of mice. Sera were diluted 1:20 into SDS sample buffer without a reducing reagent and boiled for 5 min; 10␮l of each boiled mixture was loaded into a well of 7.5% polyacrylamide gel. Plasma from a 4 month-old female MRL/lprmouse (Lpr), which should contain a large amount of gp70–anti-gp70 immune complexes, was used as a positive control. As reported previously (14, 15), NZW mice expressed a high level of serum gp85 (gp70 plus p15E), gp70, and a degradation product gp45 (arrowheads), while their expression in B6 mice was low. (BALB/c⫻

MRL/⫹)F1mice (F1) expressed an intermediate level of serum gp70. Mr, biotinylated markers, with positions indicated in kilodaltons at the left. (b to d) Representative

photomicrographs taken from kidney sections of NZW (b), (BALB/c⫻MRL/⫹)F1(c), and B6 (d) mice injected with purified anti-gp70 IgG3, 12H5.1; hematoxylin

and eosin staining,⫻300. Note apparent thickening of the capillary walls (arrowheads) and inflammatory cell infiltration (arrow) in panel b and marked increase in glomerular cellularity and evident neutrophilic infiltration in panel c.

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of anti-gp70 autoantibodies. It is also possible, however, that

production of some cytokines, especially IL-6, either from the

hybridoma cells or from host tissues in response to pristane

injection and/or hybridoma transplantation, might have

con-tributed to the development of glomerular pathology. The

slight increase in the index of glomerular pathology in mice

transplanted with 8.653 myeloma cells (Table 2) might be

ex-plained by this mechanism. However, representative clones of

the anti-gp70 MAb did induce glomerular deposition of gp70

and significant pathology in non-autoimmune mice when

in-jected as purified IgG, clearly eliminating possible

pathoge-netic effects of cellular products other than Ig.

Possible involvement of gp70–anti-gp70 immune complexes

in the induction of the currently described Ab transfer models

was indicated by the demonstration of gp70 deposition in

af-fected glomeruli along with IgG and C3 (Fig. 4 and 5). This

finding was supported by the demonstration of differences in

glomerular pathology induced by injection of purified

anti-gp70 MAb 12H5.1 in NZW and B6 strains of mice that are

known to express high and low serum levels of gp70,

respec-tively (Fig. 6). Thus, mice expressing high levels of serum gp70

developed apparently more severe glomerular pathology after

injection of 12H5.1 IgG3, while B6 mice expressing minimal

serum gp70 did not develop glomerular lesions even when the

same amount of purified anti-gp70 IgG3 was injected. These

results support the possibility that injected anti-gp70 MAb

produced immune complexes with serum gp70 before being

deposited into kidney glomeruli. Further studies including

measurements of serum immune complexes are required to

correlate possible production of gp70–anti-gp70 immune

com-plexes and the development of glomerulonephritis.

It is noteworthy that anti-gp70 IgG3-producing hybridoma

clones induced histologically evident glomerular lesions at an

apparently higher frequency than IgM- and IgG2a-producing

clones did (Table 2). Since it is difficult to differentially

mea-sure the amount of IgG produced from transplanted

hybrid-oma cells in immunocompetent (BALB/c

⫻

MRL/

⫹

)F

1

mice,

concentrations of hybridoma-derived IgG were determined for

a limited number of hybridoma clones in transplanted SCID

mice. Average concentrations of serum IgG were in the same

range among the mice transplanted with a representative

IgG2a-producing hybridoma cells and those bearing

represen-tative IgG3-producing cells. Although hybridoma-derived Ab

concentrations were not measured in every transplanted

ani-mal, it is unlikely that IgM- and IgG2a-producing hybridoma

cells, but not IgG3-producing ones, selectively lose their

Ab-producing ability soon after transplantation. Therefore, IgG3

anti-gp70 MAbs may have higher pathogenic potentials than

IgM and IgG2a anti-gp70 MAbs. In fact, the importance of the

IgG3 isotype in the induction of glomerular lesions has been

demonstrated in spontaneous and induced models of MRL/

lpr

mice (7, 34). The importance of IgG3 isotype in the

pathogen-esis of mouse SLE was also demonstrated in a recent study (25)

in which transgenic expression of IL-4 protected (NZW

⫻

C57BL/6.

Yaa

)F

1

lupus mice from fatal glomerulonephritis in

association with a lack of IgG3 and strong reduction in the

serum levels of gp70 IC. It has been suggested that IgG3 MAbs

of specific yet undefined physicochemical properties might

in-duce glomerular lesions in non-autoimmune mice when

pro-duced from transplanted hybridoma cells, regardless of their

antigenic specificity (9, 33, 34). One can argue that an

ex-tremely high serum concentration of IgG3 would be achieved

when hybridoma cells were transplanted, and cryoprecipitating

activity of IgG3 molecules induced the observed glomerular

lesions. However, it should be noted that hybridoma clones

secreting MAbs with strong cryoprecipitating activity were

clearly distinguished from noncryogenerating or less

cryogen-erating anti-gp70 clones through the screening procedure and,

thus eliminated from the present study, because the former

actually caused fine granular precipitates throughout the

bot-toms of culture wells after overnight incubation with target

cells at 4°C. In addition, none of the mice transplanted with our

anti-gp70 Ab-producing hybridomas, either of IgG3 or another

isotype, developed purpuric skin lesions, a typical

manifesta-tion of cryoglobulinemia (7, 11). These findings, along with the

demonstration of gp70 deposition in the induced glomerular

lesions (Fig. 4 and 5) and differences in the pathogenicity of

injected anti-gp70 MAbs in strains of mice expressing high and

low levels of serum gp70 (Fig. 6), strongly suggest that cognate

binding of anti-gp70 MAbs to serum gp70 is mainly responsible

for their ability to induce glomerular pathology.

Because gp70 was expressed from a cloned cDNA in our

experiments, and different isolates of endogenous mouse

retro-viruses are readily available, these anti-gp70 MAbs might

be-come very useful in identifying Ab-binding epitope structures

and in analyzing the ontogenic origins of

autoantibody-produc-ing cells. Our preliminary analyses usautoantibody-produc-ing chimeras between

NZB xenotropic viral and SFFV

env

genes has shown that

one-half of the anti-gp70 autoantibody clones including the highly

pathogenic 12H5.1 recognize epitopes located within the

N-terminal 150 amino acids of the xenotropic viral gp70. Amino

acid sequences are rather homologous in this region between

NZB xenotropic virus and SFFV (21, 29, 42). The major

dif-ference consists of an insertion of four consecutive amino acids

in NZB xenotropic viral gp70, in addition to several scattered

amino acid substitutions. Construction of recombinant vaccinia

viruses that express gp70 minigenes and use of synthetic

oli-gopeptides may lead to the identification of epitope structures

recognized by pathogenic anti-gp70 MAbs in the near future.

ACKNOWLEDGMENTS

We thank M. Nose for providing SCID mice and scientific advice,

M. P. Gorman for reviewing the manuscript, and J. Nishio, H.

Shi-waku, E. Kondoh, and Y. Akahoshi for technical assistance.

Some of the recombinant vaccinia viruses described in this report

were constructed during M. Miyazawa’s stay at the Rocky Mountain

Laboratories, Hamilton, Mont., under the financial support of B.

Chesebro. This work was supported in part by grants from the

Minis-tries of Education, Science and Culture and of Health and Welfare of

Japan and from the Cell Science Foundation.

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