Seand. J. Immunol. 32, 21 28. 1990 Expression of Heterozygous lpr Gene in MRL Mice II. Acceleration of Glomerulonephritis, Sialadenitis, and Autoantibody Production H. CARLSTEN. A. TARKOWSKI, R. JONSSON & L.-A. NILSSON Departments of Medical Microbiology, Rheumatology, Clinical Immunology, and Oral Pathology, University ofGoteborg, Sweden Carlslen. H.. Tarkowski. A., Jonsson. R. & Nilsson. L--A. Expression of Heterozygous lpr Gene in MRL Mit-e. U. Acceleration of Glomerulonephritis. Siaiadenilis. and Auioiintibody Production. Scand. J. Immunol. 32, 21-2«. 1990 Recently we showed that not only homozygous MRL Ipr/lpr mice but also hclcro/ygous MRL -l-.'lpr mice display defective antigen- and mitogen-driven T-cett responses as well as polyclonal B-ccll activation compared with congeneic MRL +/+ mice. In Ihis study we examined the impact of Ihe heterozygous lpr gene on organ pathology in kidneys, joints, and salivary gktnds. as well as serum levels of immunoglobulins and autoantibodies in young and old MRL mice. Only 1 out of 17 heterozygous Ipr-bearing MRL mice developed clinically oven renal disease with significant proteinuria and haematuria during the iirst year of life. However, examination of Ig and C3 deposits in glomeruli of kidneys from these mice revealed that the expression ofthe heterozygous Ipr gene in M RL mice accelerates glomerulonephritis- In addition, histological examination ofthe submandibular salivary glands showed an increased focus score in heterozygous MRL mice at 4 5 months of age compared wiih that of matched congeneie -1-/ + mice. In contrast, no signs of arthropathy were registered in the heterozygous Ipr-bearing MRL mice. Hetero7ygous MRL mice displayed an expanding tymphoid system as evaluated by significantly increased spleen and lymph nodeweightscompared with thoseof matched MRL -I-/ + mice. Further evidence for immunomodulatory properties of the heterozygous lpr gene was obtained when analysing serum levels of IgG. IgM. and autoantibodies. Thus, heterozygous MRL -H/lpr mice produced significantly higher levels of both Ig and autoantibodies than matched MRL +/+ mice. We conclude that the expression ofihe heterozygous lpr gene in MRL mice results in acceleraiion ofthe autoimmune proces.s. giving rise lo precocious clinical disease. Dr Hau.s Carlsten. Department uf Medical Microttiotogy. University ofGotehorg. Gutdhed.sgatan 10. S-413 46 Gotehorg. Sweden MRL Ipr/Ipr (MRL/l) mice constitute a model for huniati systemic lupus erylheniatosus (SLE) and rheumatoid arthritis (RA) [3, 10, 17]. The charac- teristics of the autoimmune disease of MRL/l mice include massive lymph node enlargement, immune complex-mediated glomerulonephritis. arthritis, sialadenitis, vasculitis, and high serum levels ofa variety of autoantibodies [3, 25]. The major accelerating factor for the disease develop- ment in MRL/1 mice is the presence of ihe homozygous Ipr (lytnphoproliferative) gene. Thus eongeneic MRL + / + mice lacking this gene develop only a late-life mild autoimmunity [3]. The presence of Ihe homozygous lpr gene in MRL/1 miee has previously been regarded as mandatory for lymphoproiiferation [17], T-cell dysfunction [2, 6, 21], polyclonal B-cell acti- vation, and the early organ involvement found in this mouse strain. Recently, we showed that not only MRL/1 but also heterozygous Ipr-bearing MRL mice display defective antigen- and mito- gen-driven T-cell responses as measured by impaired delayed-type hypersensitivity (DTH) and proliferative response lo concanavalin A (Con A) as well as polyclonal B-cell aetivation as measured by an increased frequency of immuno- globulin-producing splenic cells [5]. In this report we present further evidenee for accelerated 21
Transcript
Seand. J. Immunol. 32, 21 28. 1990
Expression of Heterozygous lpr Gene inMRL MiceII. Acceleration of Glomerulonephritis, Sialadenitis, andAutoantibody Production
H. CARLSTEN. A. TARKOWSKI, R. JONSSON & L.-A. NILSSON
Departments of Medical Microbiology, Rheumatology, Clinical Immunology, and Oral Pathology,University ofGoteborg, Sweden
Carlslen. H.. Tarkowski. A., Jonsson. R. & Nilsson. L--A. Expression of Heterozygous lpr Genein MRL Mit-e. U. Acceleration of Glomerulonephritis. Siaiadenilis. and AuioiintibodyProduction. Scand. J. Immunol. 32, 21-2«. 1990
Recently we showed that not only homozygous MRL Ipr/lpr mice but also hclcro/ygousMRL -l-.'lpr mice display defective antigen- and mitogen-driven T-cett responses as well aspolyclonal B-ccll activation compared with congeneic MRL + / + mice. In Ihis study weexamined the impact of Ihe heterozygous lpr gene on organ pathology in kidneys, joints, andsalivary gktnds. as well as serum levels of immunoglobulins and autoantibodies in young and oldMRL mice. Only 1 out of 17 heterozygous Ipr-bearing MRL mice developed clinically oven renaldisease with significant proteinuria and haematuria during the iirst year of life. However,examination of Ig and C3 deposits in glomeruli of kidneys from these mice revealed that theexpression ofthe heterozygous Ipr gene in M RL mice accelerates glomerulonephritis- In addition,histological examination ofthe submandibular salivary glands showed an increased focus score inheterozygous MRL mice at 4 5 months of age compared wiih that of matched congeneie -1-/ +mice. In contrast, no signs of arthropathy were registered in the heterozygous Ipr-bearing MRLmice. Hetero7ygous MRL mice displayed an expanding tymphoid system as evaluated bysignificantly increased spleen and lymph nodeweightscompared with thoseof matched MRL -I-/ +mice. Further evidence for immunomodulatory properties of the heterozygous lpr gene wasobtained when analysing serum levels of IgG. IgM. and autoantibodies. Thus, heterozygousMRL -H/lpr mice produced significantly higher levels of both Ig and autoantibodies thanmatched MRL + / + mice. We conclude that the expression ofihe heterozygous lpr gene in MRLmice results in acceleraiion ofthe autoimmune proces.s. giving rise lo precocious clinical disease.
Dr Hau.s Carlsten. Department uf Medical Microttiotogy. University ofGotehorg. Gutdhed.sgatan10. S-413 46 Gotehorg. Sweden
MRL Ipr/Ipr (MRL/l) mice constitute a model forhuniati systemic lupus erylheniatosus (SLE) andrheumatoid arthritis (RA) [3, 10, 17]. The charac-teristics of the autoimmune disease of MRL/lmice include massive lymph node enlargement,immune complex-mediated glomerulonephritis.arthritis, sialadenitis, vasculitis, and high serumlevels ofa variety of autoantibodies [3, 25]. Themajor accelerating factor for the disease develop-ment in MRL/1 mice is the presence of ihehomozygous Ipr (lytnphoproliferative) gene.Thus eongeneic MRL + / + mice lacking thisgene develop only a late-life mild autoimmunity[3]. The presence of Ihe homozygous lpr gene in
MRL/1 miee has previously been regarded asmandatory for lymphoproiiferation [17], T-celldysfunction [2, 6, 21], polyclonal B-cell acti-vation, and the early organ involvement found inthis mouse strain. Recently, we showed that notonly MRL/1 but also heterozygous Ipr-bearingMRL mice display defective antigen- and mito-gen-driven T-cell responses as measured byimpaired delayed-type hypersensitivity (DTH)and proliferative response lo concanavalin A(Con A) as well as polyclonal B-cell aetivation asmeasured by an increased frequency of immuno-globulin-producing splenic cells [5]. In this reportwe present further evidenee for accelerated
21
22 //. Carlsten et al.
autoimmunity in heterozygous lpr-beaHng MRLmice as demonstrated by organ pathology andautoaniibody production.
MATERIALS AND METHODS
Miee. MRL/Mp-lpr/lpr (MRLI ) and MRL/Mp--1-/4- (MRL -i-l-\-) mice, originally obtained froniHarlanOlac farm. UK., were bred und maintained in ihcanimal facility of ihc Departments of Medical Micro-biology and Clinical Immunology. University of Golc-borg. Sweden. MRL 1 and MRL -l-/-t- mice weremaled. and the Fl offspring ohtained were named lpr;+ and -(-.ipr. The Iirst allele quoted refers to the oneinherited from Ihe mother. Mice were housed 5-10 ineach cage under standard conditions of temperatureand light They were fed standard laboratory chow andwater ad libitum.
Experlmentat design. Mice were electively killed at2-3.4-5. or 8 12 months of age. The weights ol" the totalbody and spleen as well as brachial and cervical lymphnodes were studied in the two older groups ofmice. Serawere collected and stored at - 20 C until use. To avoidthe possible variauon of experimental conditions, allseroiogical assays and histotogical examinations wereperformed simultaneously.
Examination of urine and scrntogical axsays. Everythird week freshly voided urine samples were analysedfor the presence of albumin and haemoglobin using astandard dipstick (Redia test. BoehnngcrA Co.). Micewere eonsidered to have developed significant prctein-uria or haemuturia when the readings for albumin oreryihrocytes were >-l-2.
Scrum levels of IgG and IgM were measured by theradial immunodilTusion technique [16]. Antisera speei-lic tor IgG and IgM and the IgG and IgM standardswere purchased from Meloy Laboratories (Springfield.Va, USA).
Scrum levelsoflgG and IgM rheumatoid factor (RF)were measured by dilfusion-in-gel (DIG-ELISA)enzyme-linked immunosorbent assay as previously de-scribed 122J.
Levels of antibody to denatured (ss) DNA weremeasured by ELISA using methylated bovine serumalbumin (BSA) (lO/ig/ml) lo precoat wells followed byeoating with 50/ig,'ml of heat-denatured (boiled for 20min. then cooled rapidly on ice) ealf thymus DNA(Sigma, St Louis, Mo.. USA). All sera were incubated induplieate and at several dilutions. To measure levels andclass specificity of anti-ssDN A bound to the solid phase.affinily-purified and biotinytated F(ab'): fragmenis ofgoat anti-mouse IgG (Jackson Laboratories. Baltimore.Pa. USA) diluted 1 :3000 in PBS Tween 20 were usedfollowed by stepwise addition of avidin horseradishperoxidase 0.5 /Jg/ml (Sigma) and 2.5 tng;m! of theenzyme substrate 2.2-azlnobis-(3-ethyl-ben7othiamlinesulphonic acid) (Sigma) in citrate buffer. pH 4,2.containing 0.0075"'!. H:On. The absorbanee was mea-sured inaTiterlek Muttiscan photometer (Flow labora-tories. McLean. Va. USA) at 414 nm. All optical density(OD) values were converted to antigen-specific arbi-trary units (AU) with calibration curves based on the
OD obtained from serial dilutions ofa reference pool ofsera. The calibration curves were constructed with acomputer program based on weighted logit-log models[15.20],
Examination of joinis. .salivary gland.s. and kidneys.After the mice were bled, the bilateral submandibularsalivary glands, ihe righl kidney, and the right hindiimbwere removed. The salivary glands and hindiimb speci-mens were fi.ied and stored in 4"'., paraformaldehydeuntil sectioning. After sectioning and staining withhaematoxylin and eosin. the sections of salivary glandswere blindly evaluated by a focus scoring as previouslydescribed [Ll]. This scoring is based on enumeration ofthe number of Inflammatory cell infiltrates (foci) con-sisting of at least 50 mononuclear cells per mm'. Thehiddlimb specimens of S- to 12-month-old MRL 4-,'lprand MRL -I-/-I- mice were demineralized. and aftersectioning and staining with haematoxylin and eosin theseclions were evaluated as previously described [24].Synovitis was defined as proliferation involving three ormore synovial lining cell layers. The kidney specimenswere rapidly frozen and stored al -7O'C unlii section-ing. Glomerular deposits of IgG and C3 were visualizedby direct immunojluorescence on cryoslat sectionsusing F(ab'): fragments of fluorescein isothiocyanate-(FITC) conjugaled goal anti-mouse Ig (Dakopatts,Denmark) and anti-mouse C3 antibodies (CappelLaboralories. Cochraneville. Pa. USA) respectively.The intensity of staining in mesangium and peripheralloop areas was scored accordingly from 0 (backgroundstaining) to 3 (maximal staining) [23).
Anuly.sis of vett phenotypes. Freshly isolated spleencells were stained wilh FITC-labelled antibodies toThy-l (Sera-Lab. Crawley Down. UK'). CDS (Lyt-2).and CD5 (Lyt-1) (Becton Dickinson, Mountain View.Calif.. USA), and wilh phycoerythrin-conjugated anti-bodies to CD4 (L3T4) (Becton Dickinson). The fre-quency of cells expressing a given phenolype was thenanalysed on a FACSTAR (Becton Dickinson) andexpressed as the percentage of gated mononuclear cells.
Statistical analysis. Diflerences between means weredetermined by the Student two-tailed f-test.
RESULTS
The numbers and Ihe gender of the mice withdifferent lpr gene status included in this study arelisted in Table L Al 8 months of age a majority ofthe MRL/1 miee were already dead. None of theMRL -I-/-H mice died spontaneously, whereasone male MRL +/!pr mouse died at II months ofage. The outcome for -|-/lprandlpr/+ mice in thesame age group did not differ significantly.Female MRL mice, independently of Ipr genestatus, displayed lower total body weights andslightly but not statistically significantly higherserum levels of immunoglobulins and autoanti-bodies. as well as increased salivary focus scoring,compared with those of age- and strain-matched
Heterozygous lpr Gene in MRL Mice 23
TABLE 1. Numbers of mice of different Ipr gene status and genderincluded in the present study
MRL Ipr/lprMRL +/lprMRL lpr/-fMRL -I-/-F
2-3 months
Male
36
3
Female
38
3
4-5
Male
7449
months
Female
7548
8 12
Male
665
months
Female
54
males. Since the numbers of mule and female mieewere similar in all strains, we present the results asmean values of individually analysed mice ofpools of males and females and -I-/lpr and lpr/-t-(altogeiher named -H/ipr) in each age group.
L\ niphoprotiferation
In rniceetectively killed at 4-5 and 8-12 monthsof age the weights of body, spleen, and cervicaland brachial lymph nodes were registered. Whencomparing the total body weights for MRL micewith difFerent Ipr gene status, no significantdifferences were obtained (Table II). In contrast,both the spleen and lymph node weights ofheterozygous Ipr-bearing mice were significantlyincreased at 4-5 and 8-12 months compared withthose of matched -I-/-I- mice. As expected. 4-to 5-month-oid MRL Ipr/lpr mice displayed a four- lonine-fold increase in spleen and lymph nodeweight compared with those of heterozygous and+ /-I- mice (Table II).
Renal di.^ease
At4 5monllisofage, 12 out of 14 MRL Ipr/lprmice displayed signilicant albuminuria and hae-maluria. None ofthe MRL -t-/-l- mice showedpathological urine easts even when analysed at 8-12 months, [n contrast, one female MRL -I-/Iprmouse displayed signiftcant albuminuria and hae-maturia starting at 8 months of age. Glomerulardeposits of Ig and C.I were anlysed in 4- to 5- and8- to 12-month-old mice using the direct itrtmuno-fluorcscence technique. In 4- to 5-month-oldheterozygous MRL -t-/lpr mice, intermediatemean glomerular intensity scores (Ig 1.00 + 0.71and C3 0.56 + 0.71) were registered in between thehigh ones for Ipr/lpr mice (Ig 2.11 +0.33[P< 0.005] and C3 2.00 ±0.50 [/>< 0.001]) and Ihelow ones for + / + mice (Ig 0.22 + 0.44 [/'<0.05]and C3 0,22±0.44 [NS]) (Fig. la and b). Further-more, in 8- to 12-month-old mice the meanglomerular intensity score for C3 in heterozygousMRL mice (0.94 + 0.97) was significantly higher(P<0.05) compared with thai of MRL 4-/ +
TABLE IL Weighls ol' body, spleen, and cervical and brachiul lymph nodes in MRL mice withdifTerent lpr gene status at 4 5 and 8 12 monlhs of age
Total body weight (g) Spleen weight (g) Lymph node weight (g)
*P<0.05. "P<O.Ol. ***P<0.005,****P<Q,OOl.NS. not significant.
24 H. Carlsten et at.
HRLlpr/lpr• MRL-/lpr
HRL-/-
13 MRLIpr/lprMRL'/lpr
• MRL-/'
£ 04-5 menII
ant1-C3FIG, I. The immunotluoresccnce intensity of :inti-Ig- and anti-C3-stained kidney specimens from 4- to 5- and 8- to 12-month-old MRL.!mice wilh difTerent lpr gene status. Intensity ofstaming tn mesangiumand penpheral loop areas was scored from 0 (hackground staining) to 3(maximal staining). The staples and error bars represent mean intensityscore+ SD. The dots represent the intensity scores for individuallyanalysed kidney specimens.
mice (0.22 + 0.44). However, when comparingglomerular deposits of Ig iti heterozygous(1.59+0.87) and + / + (I.I I+0.93) tnice, nostatistically significant difference was obtained(Fig. la and b).
Sialadenitis and synovitis
The degree of focal sialadenitis was measuredat 4-5 and 8-12 months of age by focal scoring.Four- to 5-monih-old homozygous lpr and hetero-zygous + /lpr mice displayed a similar focus score(0.40 + 0.20). significantly higher (P<0.05) thanthat of matched congeneic + / + mice (Fig. 2). Inthe older group no statistical difference wasoblained when comparing MRL +/lpr(0.29+0.16) and MRL + / + mice (0.24 + 0.12).
Articular and synovial tissue from 8- to 12-month-old heterozygous +/lpr and congeneic+ ; + mice was slained with haematoxylin andeosin and analysed for the occurrence of synovitisand pannus formation. Mild synovial prolifer-ation of the lining layer was found in 2 out of 9(22%) MRL + / + mice and in 4 out of 17 (24%)MRL -t- lpr mice. Pannus formation or destruc-tion of cartilage was not demonstrated in any ofthe sections.
Serologieat analyses and T-cetl phenotype.^
Serum levels of IgG and IgM as well asantibodies to ss-DNA and rheumatoid factorwere analysed at 2-3. 4-5. and 8-12 months ofage. In Table III it is shown that, at all ages
ca
Heterozygous lpr Gene in MRL Miee 25
D• •
• MRLlpr/lpr
• MRL'/lpr
P MRL*/*
DO
I-̂ -"-1 months )- 12 months
FIG. 2. Focal sialadenitis, expressed as the number of inflamma-tory cell infiltrates (foci) consisting of at least 50 mononuclear cells,in 4- Io5-and8- to i 2-month-old M RL mice witli dilTereni lpr genestatus. The staples and error bars represent mean locus score + SD.The dots represent the focus scores for individually analysedsubmandibular gland specimens.
studied, the levels of IgG and IgM and anti-ssDNA were significantly increased in sera fromheterozygous MRL +/lpr mice compared withthose of eongeneic 4-/+ mice. When comparingserum levels of rheumatoid faetor in heterozy-
gous + /lpr and in -|-/+ mice, the former groupdisplayed a slightly increased production, whichhowever was statistically significant only for IgGclass at 2-3 months of age. Table III shows alsothat 2- to 3- and 4- to 5-month-old homozygous
TABLE III. Serological analyses of the content of IgG and IgM (single radial imtnunodilTusioii. g/l),rheumatoid factor of IgG and IgM classes (DIG-ELISA. mm) and anti-ss-DNA of IgG class (FLISA.arbitrary units. AU) in MRL mice with different lpr gene status at 2 ?. 4 5, and 8-12 months of age
Serum Ig (gjt)MRLlpr/lpr
MRL + 'Ipr
MRL +/ +
RE(mm)MRLlpr/lpr
MRL -(-/lpr
MRL +/ +
Anti~ss-DNA (AU)MRLlpr/lpr
MRL +/lpr
MRL +/ +
2 3 months
43,4±7.2
I4.6±5.7
6.1+4.4
9.2 + 5,3NS
6.7 ±1.3
4.5 + 0.6
160 + 40
21+8*•«
5±l
IgG
4-5 months
71.3 ±30.6
I7.l±6.7
M.7 + 6,1
13.5 + 3.7
6.9 + 0.7NS
6.1 ± 1.4
604 ±440
24±5
8±3
8-12 months
—
21.5±7.l
15,5 ±5.0
—
6.0+1.1NS
5.1 ±0.6
—
3I±9
11±3
2-3 months
0,32 + 0.06•*
0.24 + 0,06*
0.13 ±0.10
7.8±1.3
6.3+1.2NS
6.3 ±1.7
ND
ND
ND
IgM
4-5 months
0.65 + 0.45•
0.27 ±0.09**
O.I5±O.IO
8.5+1.8NS
6.9-1-2.5NS
5.5+1,3
ND
ND
ND
8 12 months
—
0-33 ±0.11***
0.14 + 0,08
—
6.6+1.3NS
6.5±1.2
—
ND
ND
\, ***P<0.0Q5.''"*P<0.00\.NS, not significant; ND. not done.
26 //, Carlsten el al.
TABLE IV. Numbers of splenic MNCx 10* (±SD) expressing Thy-l. CD5. CLM. and CD8phenotypes in MRL mice with dilTerent tpr gene status. The percentage ofMNC expressing thegiven phenotype is shown in p;irentheses
MRL Ipr/lpr mice diplayed strotigly elevatedserum levels of IgG and IgM as well as autoanti-bodies compared with those of heterozygous and+ /-t- mice.
The frequencies of splenic T-cell phenotypesThy-I (pan-T). CD5 ' . CD8 ' (dass I-restricied).and CD4 * (elass H-restrieted) were analysed in 4-lo 5- and 8- to I2-nionth-old mice. The frequen-cies of Thy-l- and CD5-cxpressing mononuclearcells (MNC) were higher in MRL/1 mice com-pared wilh those of MRL +/lpr and MRL + / +(Table IV). In contrast, no such differences wereobtained upon comparison of the frequencies ofCD4- and CD8-expressing cells in ihe abovemouse strains. However, since the total numberof MNC per spleen was significantly increased inthe Ipr-bearing mice (cotnpared with congeneic+ /-f mice), consequently the total numbers ofMNC expressing a given phenotypic celt-surfacetnarker in MRL/1 and MRL + /lpr mice werehigher (Table IV).
DISCUSSION
It has previously been claimed that the lympho-proliferative (lpr) gene is a strictly recessiveautosomal gene. This assumption was based onresults showing lhat heterozygous Ipr-bearingMRL mice resemble congeneic MRL -I-/-I- micein terms of disease pattern, longevity, and pheno-typic and functional characteristics of their lym-phocytes [17, 25]. However, in contrast to theseearlier observations, we recently demonstratedthat heterozygous Ipr-bearing MRL mice displaydcfectiveantigen-and mitogen-driven T-cell reac-tivity as well as polyclonal B-cell activation [5]. Inthis report we demonstrate further evidence for
accelerated autoimmunity in heterozygous MRL-h/lpr mice compared with that of congeneicMRL + / + mice.
Increased production of autoantibodies haspreviously been demonstrated in heterozygousIpr-bcaring B6 [12] and MRL mice [5] comparedwith that in congeneic -!-/•+ mice. The presentreport is however the first to show that heterozy-gous MRL +;lpr tnice display acceleratedautoimmuniEy as manifested by organ involve-ment. Significantly increased glomerular depositsof Ig and C3 as well as aggravated sialadenitisspeak in favour of true promoting effects, by theexpression of the heterozygous lpr gene, on theautoitntnune disease and not only for triggeringof autoimmune phenomena.
It is known that background genes are of greatimportance for the expression ofthe homozygouslpr gene. Thus, there is a great variation in thedegree of lymphadenopathy, longevity, andseverity of autoimmune disease when the Ipr geneis back-crossed into other mouse strains [7,14,19,26], For instance. Kelley & Roths [14] showedthat homozygous Ipr-bearing MRL and AKRstrains developed glomerulonephritis. whereasC3H and B6 did not. In the same report it wasalso demonstrated that young (4-8 months)MRL -\-1 + mice displayed more severe renalpathology compared with that of older (8-12months) homozygous Ipr-bearing C3H and B6mice. Not only glomerulonephritis but also siala-denitis appears in MRL -+/+ mice [11]. Incontrast, spontaneously occurring arthritis hasnever been described in MRL + / + mice [10],Thus, our present results showing acceleratedglomerulonephritis and sialadenitis but nochange in articular pathology in MRL +/Iprmice indicate that the expression ofthe heterozy-
Heterozygous tpr Gene in MRL Mice 21
gous lpr gene promotes only the already existingautoimmune manifestations found in MRL +/-|-mice.
We have previously reported that heterozygousM RL + /lpr mice display an expanding lymphoidsystem, the observation being based on increasednumbers of MNC/spleen compared with those ofmatched MRL + / + mice [5]. This fmding isfurther extended by the present study showingnot only increased spleen but also lymph nodeweights in heterozygous MRL mice. It should benoted, however, that the lytnph node enlargementis several-fold increased in homozygous MRL/1mice compared with that of heterozygous mice. Insome earlier studies analysing lymph node en-largement in mouse strains with dilTerent lpr genestatus, only palpation or abdominal inspection ofthe nodes was performed [9, I?]. Thus, it is notsurprising if a slightly increased weight of lymphnodes in heterozygous mice was not observed.
When breeding F! hybrids of Ipr/lpr and +/-I-mice it is of course of interest to delect anypossible maternal effects. Janchez ei at. found,when analysing autoantibody production inheterozygous Ipr-bearing B6 mice, that anti-nuclear and anti ss-DNA antibodies appearedearlier in 4-/lpr than in lpr/+ mice [12]. Theauthors also discussed a possible mechanismmediated by fetal and posl-natal autoantibodyexposure through the placenta and milk respect-ively, resulting in suppressive etfects in the lpr/-l-offspring. In contrast to their results, we did notfind any significant differences in any of theanalysed parameters when comparing MRL -\-lIpr and lpr/-(- tnice (Ref. 5 and the presentreport). Thus, it is not yet clearly elucidatedwhether or not maternal expression of the homo-zygous Ipr gene results in retarding cfTect onautoimmunity in the FI hybrids.
MRL + / + mice display normal T-cel! re-sponses to mitogens and antigens, whereas MRL/Imice are defective in their reactivity [1, 2,4, 6, 18,,21. 28]. We have previously demonstrated thatheterozygous MRL +-/Ipr mice display an inter-mediate proliferative response in spleen cell cul-tures containing Con A and cutaneous DTHreactivity to oxazolone [5]. Therefore weaddressed the question of whether this variationin T-cell functions in MRL mice with different Iprgene status correlated to any differences in thephenotypic characteristics of splenic T cells. Anincrease in the frequencies of pan-T-cell markers(Thy-l and CD5) was noted in homozygous Ipr-
bearing mice compared with -I-/Ipr and -t-/-l-mice. but we failed to demonstrate any significantdifference in the frequencies of CD4- and CDK-expressing spleen cells between MRL mice withdifferent Ipr gene status. Our results are inaccordance with earlier studies showing that theexpanding lymphocyte population in MRL/1mice expresses Thy-1. 2 and dull Ly-1 (CD5) [25].However. Wofsy et al. [27] demonstrated thatlymphocytes from lymph nodes and spleen inMRL/1 mice largely lack the CD4 expression ontheir surface. This finding has, however, not beenfully confirmed in other tnore recently presentedreports [24]. It is possible that analyses of thephenotypic characteristics of T cells in MRL/Imice show a significant variation depending onthe environment, the source of the tymphoid cells,the enumeration technique, and the monoclonalantibodies used.
In summary, the results of the present studydemonstrate that the expression of heterozygousIpr gene in MRL mice not only promotes autoim-mune phenomena but also accelerates the organpathology. It could thus beeoncluded that the Iprgene, at least on an MRL background, is notstrictly recessively inherited as regards the acce-lerating effects on murine lupus.
ACKNOWLEDGMENTS
We would like to thank Mrs Lena Svensson forher expert technical assistance a.s well as BjornKjellson for helping us with FACS analyses. Thisstudy was supported by the Goteborg MedicalSociety, the Swedish Soeicty of Medicine, theSwedish Association against Rheumatism, theKing Gustav Vs 80 years foundation, theSwedish Life Insurance Companies, and theSwedish Medical Research Council.
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Received 18 December 1989Accepted in revised form 5 March 1990
