Clin. exp. Immunol. (1990) 79, 123-129

Serological and histological characterization of thenew mutant strain of lpr mice, CBA/KlJms-lprcg/lprcg

M. KIMURA, H. MOHRI*, K. SHIMADA*, T. WAKABAYASHIt, Y. KANAI$ & A. MATSUZAWA§Departments of *Internal Medicine, tClinical Pathology, tMolecular Oncology; and §Laboratory Animal Research Centre,

Institute of Medical Science, University of Tokyo, Tokyo, Japan

(Acceptedfor publication 15 August 1989)

SUMMARY

CBA/KlJms-lprcg/lprcg mice with a novel mutation producing systemic lymphoproliferation wereinvestigated for their serological and histological characteristics. The mutant mice showed elevatedlevels ofserum immunoglobulin, Cl q-binding immune complexes and antibodies to nuclear antigenssuch as dsDNA and ssDNA and poly(ADP-ribose). In contrast, histopathological lesions, e.g.glomerulonephritis, vasculitis or interstitial pneumonitis, were not revealed by histological andimmunofluorescent examinations, except for lymphocytic infiltration in various organs. These resultssuggest that this mutant mouse strain may provide a new animal model for autoimmunity. However,further investigations are required to clarify whether this strain is unique as compared with otherwell-known lupus-prone strains ofmice with respect to serological and histological abnormalities andbecome to be a new model of systemic autoimmune disease.

Keywords lymphoproliferation autoimmunity

INTRODUCTION

Many studies of spontaneous murine autoimmune disease havebeen concentrated on NZB (Bielschowsky, Helyer & Howie,1959) and subsequently on (NZB x NZW)F1 mice (Helyer &Howie, 1963), which develop autoimmune haemolytic anaemiaand immune complex glomerulonephritis, respectively, in addi-tion to production of various autoantibodies. Genetical analy-ses have revealed that multiple genes are involved in the fullexpression of the disease in these models (Theofilopoulos &Dixon, 1985).

More recently, single-gene murine models for autoimmunedisease, such as MRL/MpJ-lpr/lpr (MRL-lpr) (Murphy &Roths, 1979), C3H/HeJ-gld/gld (C3H-gld) (Roths, Murphy &Eicher, 1984) and BXSB-Yaa (Murphy & Roths, 1979), havebeen introduced in the studies of the immunopathology andclinical treatment of human systemic lupus erythematosus(SLE). They may have advantages for the purpose of studyingthe primary aetiopathological event leading to the autoimmunedisease. Numerous functional abnormalities in these mice havebeen reviewed with a focus on those of T cells, B cells andmacrophages (Theofilopoulos & Dixon, 1985). To date, how-ever, no causative immunological abnormalities have beenrevealed. Recently, Matsuzawa (1988) established a new mutant

Correspondence: Mikio Kimura, Department of Internal Medicine,Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai,Minato-ku, Tokyo 108, Japan.

mutant mice lpr gld

strain of mice, CBA/KlJms-lprcg/lprcg (CBA-lprff), which de-velop systemic lymphoproliferation similar to that found in theabove-mentioned three strains of mice; Matsuzawa conductedextensive genetic studies and demonstrated that lymphoprolife-ration is controlled by a single autosomal recessive gene in themutant mice. More interestingly, the mutant gene has beenproved to be allelic with lpr and to complement gld in inductionof lymphoproliferation. Thus the gene was named lprcg (cg,complementing gld).

We have extensively investigated the natural courses ofserological and histological characteristics in the CBA-lprcg micein comparison with the non-mutant counterpart, CBA/KlJms-+ /+ (CBA-+ ). The results presented here have supported thatthe mutant mice are useful as a model of autoimmunity.

MATERIALS AND METHODS

MiceMutant CBA-lprcg and normal CBA-+ mice were bred andmaintained as described previously (Tanaka et al., 1987). Miceat indicated ages were killed by total blood collection from theheart under ether anaesthesia. Spleens were weighed wet andused as an indicator of the severity of systemic lymphoprolife-ration. Lungs, hearts, livers, kidneys, spleens, lymph nodes andother organs or tissues were fixed in 10% buffered formalinsolution for histology. Sera were isolated and stored at - 20°Cuntil use.

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M. Kimura et al.

Table 1. Spleen weights in mutant CBA-lprcg and normalCBA-+ mice

Weight (mg)*

Mice I month 3 months 6 months 12 months

CBA-lprcg 83+3 408+23 1027+ 163 1036+206CBA-+ 80+4 67+4 77+3 98+6

* Mean + s.e.m. from 11 to 26 mice.

Table 2. Serum immunoglobulin concentrations in 6-month-old mutant CBA-lprcg and normal CBA-+ mice

Serum concentration (mg/l)*

Mice IgM IgG

CBA-lprcg 1231 +98 26971 + 3063CBA-+ 627 + 33 5492+753

*Mean+ s.e.m. from five to six mice.

Serum immunoglobulinsSerum IgM and IgG concentrations were determined by singleradial immunodiffusions that were commercially available (TheBinding Site, Birmingham, UK).

Antibodies to nuclear antigensFluorescent anti-nuclear antibodies (FANA) of the sera were

assayed on mouse liver cryostat sections at a dilution of 1:10.Second antibody was FITC-conjugated anti-mouse IgG (H + L)(Miles-Yeda, Rehovot, Israel).

Antibodies to dsDNA, ssDNA and poly(ADP-ribose) were

determined by ELISA as described by Kanai et al. (1982).Briefly, purified nucleic acids were coated on 96-well microtitreplates that had been precoated with poly-L-lysine. These plateswere blocked with tris-buffered saline (TBS) (25 mM tris, 140 mMNaCl, pH 7 4) containing 5% fetal bovine serum (FBS) and0 05% tween 20. Sera were diluted 1: 50 with TBS containing 5%FBS. After each incubation, the plates were washed extensivelywith TBS containing 0-05% tween 20. Bound antibodies were

detected with alkaline phosphatase-conjugated anti-mouse IgM(j-chain specific) or anti-mouse IgG (y-chain specific) (Cappel,Malvern, PA). The substrate was p-nitrophenylphosphate(Sigma, St Louis, MO). Antibody activity was expressed as theabsorbance at 405 nm (ImmunoReader, Nippon InterMed,Tokyo, Japan).

Antibodies to extractable nuclear antigens (ENA) were

determined by double immunodiffusion using a commerciallyavailable test kit (MBL, Aichi, Japan).

Anti-erythrocyte antibodiesThe ordinary direct Coombs' test with rabbit anti-mouse IgG(H + L) (Bio-Yeda, Rehovot, Israel) was applied for the

Table 3. Positivity for fluorescent anti-nuclear antibodies(FANA)

Percent positivity*

Mice I month 3 months 6 months 12 months

CBA-lprcg 26-7 89-5 100 100CBA-+ 0 0 8-3 (0) 23-1 (154)

* Eleven to 26 mice tested in each group.All the mutant CBA-lprcg mice showed a homogeneous

occasionally mixed with a shaggy pattern.Some aged normal CBA-+ mice showed a pure speckled

pattern and figures in the parentheses show percent of micewith a homogeneous pattern.

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Fig. 1. Age-dependent production of antibodies to dsDNA in mutantCBA-lprig (-) and normal CBA- + (0) mice. (a) IgM anti-dsDNA; (b)IgG anti-dsDNA.

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CBA-lprcg (0) and normal CBA- + (0) mice. (a) IgM anti-ssDNA; (b)IgG anti-ssDNA.

measurement of anti-erythrocyte antibodies. Blood taken fromautoimmune graft-versus-host F1 mice served as a positivecontrol (Gleichmann, van Elven & van der Veen, 1982).

Immune complexesCirculating immune complexes were determined by the micro-titre plate Clq-binding method (Field et al., 1987) with slightmodifications using aggregated mouse IgG as a standard(Andrews et al., 1978). Human Clq was purchased from SankoJunyaku (Tokyo, Japan).

Proteinuria

Urinary protein was determined by Albustix (Miles-Sankyo,Tokyo, Japan). For the statistical analysis, the degree ofproteinuria was scored as follows 0, 0 mg/dl; 1, 30 mg/dl; 2, 100mg/dl; 3, 300 mg/dl; and 4, _ 1,000 mg/dl.

Histological examinationsFormalin-fixed paraffin-embedded sections of the major organs

were stained with haematoxylin and eosin (H + E), periodicacid-Schiff(PAS) and periodic acid silver methenamine (PAM).

Age (months)

Fig. 3. Age-dependent production of antibodies to poly(ADP-ribose) inmutant CBA-lprig (0) and normal CBA- + (0) mice. (a) IgM; (b) IgG.

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Fig. 4. Levels of circulating C q-binding immune complexes in mutantCBA-lprff (0) and normal CBA-+ (0) mice at 3, 6 and 12 months ofage.

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M. Kimura et al.

Table 4. Urinary protein excretion

Scored proteinuria*

Mice 1 month 3 months 6 months 12 months

CBA-lprcg 120+0-15 147+011 144+014 182+0 23CBA-+ 105+011 093+007 082+010 054+006P P>0 05 P<0O01 P<001 P<0 01

* Mean + s.e.m. from 11-26 mice. See Materials andMethods for score.

Renal immunofluorescenceCryostat sections (4 pm) of kidneys were stained with FITC-conjugated anti-mouse IgG (H + L) (Miles-Yeda). GlomerularIgG deposition was visually scored as -; ±; 1+; 2 + and 3+,and was regarded positive when it was ) 1.

RESULTS

Spleen weightDeterminations were carried out at the ages of 1, 3, 6 and 12months. Spleen enlargement was evident from 3 months of agein mutant CBA-lprcg, but it did not occur at all in normalCBA- + (Table 1). In mutant mice, spleen weights increased atup to 6 months of age and remained at a plateau thereafter. Thiswas partially explained by sporadic death of diseased mice after6 months of age (Matsuzawa et al., 1990). Lymph nodesenlarged in a similar course as did spleens (data not shown).Spleens were larger in female than in male mutant mice.

Serum immunoglobulinsDeterminations were conducted at the age of 6 months. SerumIgM and IgG levels in mutant mice were about two and fivetimes higher than those in normals, respectively (Table 2).

FANAAs shown in Table 3, 89-5% of the mutant sera at 3 months ofage and 100% of those at 6 and 12 months of age were positive.All positive samples provided a homogenous pattern, occasion-ally mixed with a shaggy pattern. In contrast, normal mousesera were almost negative for FANA until the age of 6 months.At 12 months of age, 23-1% of normal sera were judged aspositive because of appearance in a purely speckled, non-

homogeneous pattern in some individuals.

Anti-nucleic acid antibodiesBoth IgM and IgG antibodies to dsDNA were detectable invirtually all mutant CBA-lprcg mice examined, their levels beingmaximal at 6 months of age (Fig. la, b). The IgM anti-ssDNAantibody level reached a peak at 6 months of age (Fig. 2a),although the IgG anti-ssDNA tended to increase from 6 to 12months of age (Fig. 2b). Assay of IgM anti-poly(ADP-ribose)antibodies demonstrated some degree of the antigen binding innormal CBA-+ mice, as was previously seen in other normalmouse strains (Kimura et al., 1987). However, some mutantmice had clearly higher than normal titres of IgM anti-

poly(ADP-ribose) (Fig. 3a). IgG anti-poly(ADP-ribose) anti-bodies showed an age-related increment in titres from 3 to 12months of age (Fig. 3b).

Anti-ENA antibodiesTen, 8 and 10 sera, respectively, from 3-, 6- and 12-month-oldmutant CBA-lprcg were tested. All samples were negative foranti-ENA precipitins (data not shown). Human monospecificanti-RNP and anti-Sm sera (Centers for Disease Control,Atlanta, GA) included as positive controls produced clearprecipitin lines.

Anti-erythrocyte antibodiesAssay was conducted with the red blood cells from mutant miceat 1, 2, 3, 4, 6 and 12 months of age. All but one sample werenegative and the only positive sample was from one of 13 miceaged 12 months (data not shown). Normal mice at 1, 3, 6 and 12months of age were also negative. In contrast, samples fromgraft-versus-host F1 mice included as positive controls pro-duced positive reaction as reported previously (Gleichmann etal., 1982).

Immune complexesCirculating Cl q-binding immune complexes were determined at3, 6 and 12 months of age. As shown in Fig. 4, positive resultswere most frequently obtained at 6 months of age with sporadicoccurrence of high values at 3 and 12 months of age in mutantCBA-lprcg mice. All the sera from normal CBA-+ mice werenegative.

ProteinuriaStatistical analyses of proteinuria scores determined as de-scribed in Materials and Methods demonstrated significantlyhigher levels in 3-, 6- and 12-month-old mutant mice ascompared with normal mice (Table 4). However, this wasmainly due to the decreased proteinuria with age in normalCBA- + mice, rather than to an age-related increment inproteinuria in the mutant mice. Moreover, the proteinuria levelsof the mutant mice even at 12 months are not considered to beabnormally high (Kimura, van Rappard-van der Veen &Gleichmann, 1986).

HistologyLymphocytic infiltration began to appear at 3 to 4 months ofagein the lung, liver and kidney and as early as 1 month in thespleen. Details are described as follows: lymph nodes (Fig. Sa,b), in the early stage, atypical lymphoid cells proliferated ininter-follicular areas with the normal follicle structures retained,but in the late stage they replaced a whole lymph node. Thoseexpanding atypical lymphoid cells had either large vesicular ormedium-sized chromatin-rich nuclei of oval or irregular shape.The larger cell had a relatively prominent nucleolus. Spleen (Fig.Sc), in the early stage, atypical lymphoid cells proliferated at theperiphery of white pulps and gradually invaded both the centreof them and the red pulps with increased collagen fibres. Plasmacells proliferated occasionally about atypical lymphoid cells.Lung (Fig. Sd), atypical medium-sized lymphoid cells infiltratedperibronchial and/or perivascular areas. Liver (Fig. Se), atypicalmedium-sized lymphoid cells frequently infiltrated the portaltract and occasionally formed small foci in sinusoids. Kidney(Fig. Sf ), atypical medium-sized lymphoid cells massively

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The new mutant strain of lpr mice

Fig. 5. (a) Two-month-old female lymph node. Atypical large lymphoblastic cells proliferate massively but normal structures still remainpartly. x 36; H + E; (b) Two-month-old female lymph node ofhigher magnification. Atypical large lymphoblastic cells have a large vesicularnucleus with a relatively prominent nucleolus. x 73, H + E; (c) Two-month-old female spleen. Atypical large lymphoblastic cells proliferatein the peripheral area of the white pulp. x 36, H + E; (d) Six-month-old male lung. Atypical medium-sized lymphoid cells infiltrate theperivascular area. x 36, H + E; (e) Twelve-month-old male liver. Atypical medium-sized lymphoid cells markedly infiltrate the portal tractand lobule. x 36, H + E; and (f) Twelve-month-old male kidney. Atypical medium-sized lymphoid cells massively infiltrate the circumferenceof a relatively large artery. x 36, H + E.

infiltrated the circumference of a large artery in one kidney and C3H-gld (Roths et al., 1984) and vasculitis in MRL-lpr strainformed small foci in some places in the others. (Andrews et al., 1978) were absent in the mutant CBA-lprcg mice

Renal glomerular lesions were not revealed at all by H +E (data not shown).staining. PAS and PAM staining also did not show anyabnormalities in the glomerular basement membranes like Glomerular IgG depositionthickening or spike formation (data not shown). Other histolo- Kidneys from mice at 1, 3, 6 and 12 months of age weregical abnormalities such as interstitial pneumonitis reported in examined and percents of mice positive for glomerular IgG

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128 M. Kimura et al.

Table 5. Renal glomerular IgG deposition

Percent positivity*

CBA-lprcg 429 26 7 7-7 7 7CBA-+ 909 0 23 1 53-8

* Eight to 15 mice were tested in each group.One 1-month-old and two of 12-month-old

normal CBA- + mice showed 2 + degree offluorescence; the other positive cases in bothstrains were only of 1 + degree.

deposition are shown in Table 5. All positive CBA-lprcg micewere judged as such by 1 + degree of fluorescence. This was thecase for all positive CBA- + mice excluding some in which thefluorescence was graded as 2 +. The proportion of positive micedid not increase but rather tended to decrease with age in bothmutant and normal mice. Collectively, glomerular IgG deposi-tion was not significantly higher in the mutant mice as comparedwith the normal counterpart.

DISCUSSION

The newly established mutant strain of mice, CBA-lprcg, mayprovide a powerful tool for the study of human autoimmunediseases. This urged us to characterize the mutant mice serologi-cally and histologically and to clarify whether they showfeatures similar to or distinct from those of other pre-existingautoimmune mouse strains.

First, serum IgM and IgG concentrations were shown to beelevated at 6 months in the mutant CBA-lprcg, being about twoand five times higher than those in the non-mutant counterpart,CBA- +, respectively. The levels were comparable to thosereported in MRL-lpr (Theofilopoulos & Dixon, 1985; Andrewset al., 1978).

The percent of mice positive for FANA increased with age.All the sera from 6-month-old mutant mice showed titres equalto or above 1: 320 (data not shown) in a homogeneous patternwith occasional sprinkling of a shaggy pattern, suggesting thepresence of anti-DNA. In fact, antibodies to dsDNA andssDNA were detected by ELISA in the mutant mice, as in otherlupus-prone strains (Roths et al., 1984; Theofilopoulos &Dixon, 1985). Antibodies to poly(ADP-ribose) that are reportedto be useful for both diagnosis (Okolie & Shall, 1979) andmonitoring of the clinical activity (Morrow et al., 1982) ofhuman SLE were also detected in the mice, as in MRL-lpr(Kanai & Fujiwara, 1985). However, anti-Sm antibodies wereundetectable in this mouse strain, as contrasted with MRL(Eisenberg, Tan & Dixon, 1978) in which the antibodies weredetected by double immunodiffusion. However, it may be thatanti-Sm in the mutant mice were not detected due to the lowsensitivity of the test. Further studies will be needed using moresensitive methods.

The mutant CBA-lprcg mice were almost negative for anti-erythrocyte antibodies. In this respect, this mutant strain issimilar to MRL-lpr but different from NZB or female(NZB x W)F1 which produced significant anti-erythrocyte anti-bodies (Howie & Helyer, 1968; Andrews et al., 1978).

Only one 3-month-old and some 6- and 12-month-oldmutant mice tested showed elevated levels of circulating Clq-

binding immune complexes. Although not compared preciselyin this study, the levels and the positive frequency seemed lowerthan those of MRL-lpr as determined by Izui et al. (1984).

Despite the slightly but significantly larger amounts ofurinary protein in the mutant than in the normal mice,histological and immunofluorescent examinations did notreveal any significant abnormalities in the renal glomeruli. Thesame result has been reported in congenic lpr mice with geneticbackgrounds other than MRL (Izui et al., 1984; Kelley& Roths,1985), and this is the case for the gld gene originally found inC3H/HeJ strain (Roths et al., 1984). Thus, some other geneticalfactors represented by the background genes of the MRL strainmay be necessary to produce renal damage in addition toproduction of autoantibodies. Alternatively, the natures of theautoantibodies, e.g. IgG subclass, complement-fixing ability,avidity, fine specificity or electrical charge, may be crucial indetermining the pathogenicity.

Lymphoid cells infiltrating the lung, liver and kidney arevery likely derived from the expanding populations in the lymphnodes and spleen, because the cells have atypical features andthe infiltration became more extensive in parallel with enlarge-ment of the lymphoid organs. This issue may be correctlyaddressed by immunohistologic characterization of those lym-phoid cells, using appropriate antibodies to cell surface markers.

The mutant CBA-lprcg mice with lymphoproliferation showincreased serum immunoglobulin concentrations, Cl q-bindingimmune complexes, and autoantibodies to nuclear antigensincluding anti-DNA. However, they have almost no histopatho-logical lesions, e.g. glomerulonephritis, interstitial pneumonitisor vasculitis, except for lymphocytic infiltration in variousorgans. Considering the nature of this mutant gene (Matsuzawaet al., 1990), this newly found CBA-lprcg mice could be almostthe same as the lpr-congenic non-MRL mice (Izui et al., 1984).The findings reported here largely support this notion. Whetherunique features still exist in serological and histological aspectscompared with other autoimmune strains of mice remains to beelucidated.

ACKNOWLEDGMENTS

This work was supported in part by a Grant-in-Aid for Cancer Researchfrom the Ministry of Education, Science and Culture, Japan, and byspecial coordination funds from the Science and Technology Agency ofthe Japanese Government.

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