Thymic Microenvironment and NZB Mice: The Abnormal Thymic Microenvironment of New Zealand Mice Correlates with Immunopathology

Clinical Immunology Vol. 90, No. 3, March, pp. 388 –398, 1999 Article ID clim.1998.4655, available online at http://www.idealibrary.com on

Thymic Microenvironment and NZB Mice: The Abnormal Thymic Microenvironment of New Zealand Mice Correlates with Immunopathology Yuichi Takeoka,*,† Nobuhisa Taguchi,*,† Brian L. Kotzin,‡,§ Sean Bennett,‡ Timothy J. Vyse,‡ Richard L. Boyd,¶ Mitsuru Naiki,† Jin-emon Konishi,† Aftab A. Ansari,\ Leonard D. Shultz,** and M. Eric Gershwin*,1 *Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis, School of Medicine, Davis, California 95616; †Institute of Bio-Active Science, Nippon Zoki Pharmaceutical Co. Ltd., Yashiro, Hyogo 673-1461, Japan; ‡Division of Basic Sciences, National Jewish Medical and Research Center, Denver, Colorado 80206 ; §Departments of Medicine and Immunology, University of Colorado Health Science Center, Denver, Colorado 80262; ¶Department of Pathology and Immunology, Monash University, Prahran, Victoria, 3181 Australia; \Department of Pathology, Emory University, School of Medicine, Atlanta, Georgia 30322; and **Jackson Laboratory, Bar Harbor, Maine 04609

There are distinct microenvironmental abnormalities of thymic architecture in several murine models of SLE defined using immunohistochemistry and a panel of mAb dissected at thymic epithelial markers. To address the issue of the relationship between the thymic microenvironment and autoimmunity, we studied backcross (NZB 3 NZW) F1 3 NZW mice in which 50% of offspring develop nephritis associated with proteinuria and anti-DNA antibodies. We reasoned that if thymic abnormalities are associated with development of disease, the correlation of abnormalities with lupus-like disease in individual backcross mice will form the foundation for identification of the mechanisms involved. In parallel, we directed a genetic linkage analysis, using markers previously shown to be linked to nephritis and IgG autoantibody production, to determine if such loci were similarly associated with microenvironmental changes. Our data demonstrate that all (NZB 3 NZW) F1 3 NZW backcross mice with disease have microenvironmental defects. Although the microenvironmental defects are not sufficient for development of autoimmune disease, the severity of thymic abnormalities correlates with titers of IgG autoantibodies to DNA and with proteinuria. Consistent with past studies of (NZB 3 NZW) F1 3 NZW mice, genetic markers on proximal chromosome 17 (near MHC) and distal chromosome 4 showed trends for linkage with nephritis. Although the markers chosen only covered about 10 –15% of the genome, the results demonstrated trends for linkage with thymic medul1 To whom correspondence and reprint requests should be addressed at Division of Rheumatology/Allergy and Clinical Immunology, University of California at Davis, One Shields Avenue, TB 192, School of Medicine, Davis, CA 95616-8660. Fax: 530-752-4669. Email: [email protected].

1521-6616/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

lary abnormalities for loci on distal chromosome 4 and distal chromosome 1. We believe it will be important to define the biochemical nature of the molecules recognized by these mAbs to understand the relationships between thymic architecture and immunopathology. © 1999 Academic Press Key Words: thymic microenvironment; thymus; lupus; autoimmunity. INTRODUCTION

The unique capacity of the thymus for generating functionally mature, self-MHC-restricted, yet self-tolerant T cells is accomplished by the migration and interaction of precursor T cells with the cell lineages that comprise the thymic microenvironment. The thymic microenvironment encompasses epithelial cells and stromal cells which express unique arrays of cell surface molecules that provide the signals which support T cell development and maturation. A study of thymic microenvironment organization and content reveals that there is a great deal of cell lineage diversity which contributes to the complex structural nature of this organ (1). Important components of the thymic microenvironment include the epithelial cells which line the sinusoids within the subcapsule, cortex, and medulla and are thought to initiate thymopoiesis (1–3). These epithelial/stromal cell lineages, by virtue of their differential cell surface expression of MHC class I and II antigens, along with a variety of yet to be defined molecules in distinct thymic locations, play critical roles in negative/positive selection of T lymphocytes (4, 5). The finding that a number of murine models of human systemic lupus erythematosus (SLE), including New Zealand Black (NZB), (NZB 3 NZW) F1, MRL/ MP-Faslpr , C3H/HeJ-Fasgld, and BXSB/MpJ-Yaa mice,

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express thymic tissue abnormalities has led to the thesis that such abnormalities may be the basis for dysregulated positive/negative selection leading to the autoimmune disease in these mice (6 –9). In efforts to formally address this issue, use was made of backcross (NZB 3 NZW) F1 3 NZW mice in which about 50% of the offspring have been previously documented to develop renal disease associated with proteinuria and high titers of anti-single strand (ss) and -double strand (ds) DNA antibodies (11, 12). It was reasoned that if thymic abnormalities underlie development of autoimmune disease, studies of such abnormalities, and the characterization of clinical disease in individual backcross mice, will form the foundation upon which future studies on the identification of the gene(s) and the cellular and molecular mechanisms could be based. Such a study was therefore carried out and is the basis of this report. MATERIALS AND METHODS

Mice. Parental female or male (NZB/BlnJ 3 NZW/ LacJ) F1 and NZW/LacJ mice (after herein referred to as B/WF1 and NZW mice) were obtained from the Jackson Laboratory (Bar Harbor, ME) and were maintained in the Animal Resource Service of the University of California at Davis (Davis, CA). The backcross mice resulting from breeding of B/WF1 mice and NZW mice were derived in our laboratory and followed for 12 months or until disease expression. The backcross mice with proteinuria levels greater than or equal to 100 mg/dl and/or significant anti-ds DNA autoantibody levels (see below) on two consecutive test dates were regarded as positive. The backcross mice that survived to the end of the 1-year period with no evidence of detectable proteinuria or anti-DNA antibody were designated as negative. Only female offspring of the matings between B/WF1 3 NZW or NZW 3 B/WF1 backcross mice were used for the studies reported herein. Thymectomy. At 1 month after birth, the backcross mice were thymectomized under anesthesia induced by inhalation of methoxyflurane (Metofane; PitmanMoore, Mundelein, IL) and intraperitoneal injection with pentobarbital sodium (Nembutal; Abbott Laboratories, North Chicago, IL). Thymectomy at this age does not influence disease; nonetheless sham thymectomy controls were included. A group of backcross mice that were neither anesthetized nor surgically manipulated served as intact controls. The study encompassed a total of 102 thymectomized, 30 sham-operated, and 20 intact backcross mice. Monoclonal antibodies (mAbs). A panel of three mAbs with previously defined specificity for mouse thymic stromal (MTS) elements were used in the present study. The MTS mAbs were prepared from the fusion of P3-NS-1-Ag4-1 (NS-1) cells with spleen cells or popli-

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teal lymph node cells from LOU/M rats immunized with enriched mouse thymic stromal cell suspensions (1, 6). These mAbs detect molecules expressed by thymic epithelial cells including pan epithelium (MTS1), subcapsular and medullary epithelium (MTS10), and cortical epithelium (MTS44). The mAbs, MTS1, MTS10, or MTS44, have been classified as I, II, and IIB, respectively, according to the criteria previously established and termed as clusters of thymic epithelial staining (13, 14). Immunohistochemistry. Thymic tissue was snapfrozen in dry ice-cold 2-methylbutane and embedded in Tissue-tec (Miles Laboratories, Inc., Elkhart, IN). Freshly cut sections (5 mm) were mounted on clean glass slides coated with poly-L-lysine (Poly-prep slides; Sigma Chemical Co., St. Louis, MO) and rapidly airdried. The slides were stored at 280°C until used for immunohistochemical staining. The sections were fixed with acetone at 220°C for 5 min before staining and were incubated for 30 min at room temperature (RT) with normal goat serum diluted 1:5 in 0.05 M Tris-buffered saline (TBS) pH 7.6 to block nonspecific staining. Thymic sections were then stained using an avidin– biotin technique based on the system of Wood and Warnke (15). Individual thymic sections were incubated with the appropriate and optimal dilution of MTS mAbs for 1 h at RT in a moist chamber, washed three times in TBS for 5 min with gentle shaking, incubated with biotinylated goat anti-rat Ig (Tago Inc., Burlingame, CA) diluted 1:200 in TBS for 30 min at RT, and washed with TBS. The alkaline phosphataseconjugated streptavidin– biotin complex (AB complex/ AP) (Dako, Carpinteria, CA) was freshly prepared and applied to sections for 30 min at RT. After washing in TBS, the substrate (Vector Red; Vector Inc., Burlingame, CA) in 0.1 M TBS, pH 8.2, was applied to the sections with levamisole to block endogenous alkaline phosphatase activity. After being washed in TBS, the tissues were mounted with glycerol in phosphate-buffered saline (mounting medium; Sigma Diagnostics, St. Louis, MO) and coverslipped. The slides were viewed using an Olympus BH-2 microscope and the cortex and medulla were subjectively evaluated and graded as follows: 0, no abnormalities detected; 11, mild abnormalities or under 33% of the cells involved; 21, moderate abnormalities or over 33% but under 66% of the cells involved; 31, severe abnormalities or over 66% of the cells involved), using previously defined criteria (7, 8). A total score was calculated by addition of the respective scores of the cortex and medulla. These methods have been described in more detail elsewhere, including data that the natural thymocytotoxic autoantibodies produced in NZB mice do not mask the epitopes detected by the MTS monoclonal antibodies (6 – 8).

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ELISA for anti-DNA antibodies. Blood was collected from the retroorbital venous plexus of individual backcross mice monthly until they were 5 months old and biweekly thereafter. Nonheparinized tubes (Scientific Products, McGraw Park, IL) were utilized to collect blood which was allowed to clot at room temperature for 1.5–2 h, and centrifuged for 2 min at 1000g and the sera were collected and frozen at 220°C until use. IgG antibodies to single strand (ss) or double strand (ds) DNA were assayed by ELISA as previously described (16). Briefly, to prepare dsDNA for anti-DNA ELISA, calf thymus DNA (Sigma Chemical) was reconstituted to 0.5 mg/ml in PBS and frozen in aliquots at 220°C. To prepare ssDNA, dsDNA was boiled 15 min and then immediately placed in an ice-water bath with occasional mixing. DNA-coated plates were prepared as described (15). Individual wells of Immulon-1 microtiter plates (Dynatech Laboratories; Chantilly, VA) were coated with 50 ml of 10 mg/ml methylated bovine serum albumin (Sigma Chemical) in PBS and left overnight at 4°C and washed three times with PBS. Fifty microliters of 2.5 mg/ml calf thymus ssDNA or dsDNA was then added to appropriate individual wells and left overnight at 4°C. Twofold dilutions of the serum to be tested each in triplicate were added at a starting dilution of 1/200 in a volume of 50 ml/well in PBS/BSA. The sera were allowed to incubate for 2 h at room temperature and the microtiter wells were then washed three times with PBS/Tween. Horseradish peroxidase-conjugated goat anti-mouse IgG (Caltag Laboratories, South San Francisco, CA) was diluted with PBS/BSA at 1:1000, and 100 ml was added to each well and then incubated for 2 h at RT. After being washed three times with PBS/Tween, the reaction was developed with 50 ml of 0.55 mg/ml 2,29-azino-bis-3-ethylbenthiazoline-6sulfonic acid (Sigma Chemical) in citrate buffer (pH 4.2) including 0.03% (v/v) H2O2. After 30 min, the reaction was stopped by the addition of 50 ml of 5% (w/v) sodium dodecyl sulfate (Boehringer Mannheim; Indianapolis, IN). Controls consisted of known positive and negative sera. Results were read at 405 nm using a Thermomax reader (Molecular Devices, Inc., Menlo Park, CA). Proteinuria. Each of the backcross mice was evaluated monthly until 5 months old and biweekly thereafter for proteinuria using tetrabromphenol blue paper (Albustix, Miles, Inc., Elkhart, IN). Urine samples were graded colorimetrically from 11 to 41 corresponding to approximate protein concentrations of 30, 100, 300, and 2000 mg/dl, respectively. Urine samples with negative (,30 mg/dl) or trace determination of protein urea were considered normal. Evaluation of autoimmune disease. Mice with severe (21 or greater) proteinuria or reactivity of sera

diluted 1/200 with an O.D. 405 three standard errors greater than controls of IgG type anti-dsDNA antibody on two or more consecutive time points before 12 months of age were designated as positive for autoimmune disease. This value is the mean plus 3 SD of control sera. Mice with negative or trace determination of proteinuria, or negative for anti-dsDNA with no clinical evidence of disease at 12 months of age, were designated as not expressing lupus-like disease (negative phenotype). Mice were sacrificed either after two determinations of positive anti-DNA and proteinuria or at 12 months. At that time, liver DNA was isolated and studied as described below. Genetic mapping using simple sequence length polymorphisms (SSLP). Liver DNA was used for genetic mapping using SSLP. Oligonucleotides flanking simple sequence repeats were either purchased (Research Genetics, Huntsville, AL) or synthesized at the National Jewish Molecular Resource Center using an Applied Biosystems model 392 DNA synthesizer. In this study, we focused on a subset of NZB markers (loci) that have been shown to be linked to nephritis and/or IgG autoantibody production in previous studies of New Zealand hybrid mice (11, 12, 17–19). Markers mapped in this study are indicated as follows: D1Mit106 (chromosome 1, 85 centiMorgans from the centromere), Crp (1,94), D1Mit155 (1,109), D4Mit17 (4, 25), D4Mit9 (4, 44), D4Mit70 (4, 65), D4Mit343 (4,75), D7Nds5, (7,23), D7Mit16 (7,40) D7Mit125 (7,50), D13Mit95 (13,66), D17Mit35 (17, 144), D17Mit50 (17, 23), D18Mit16 (18,58), and D19Mit13 (19,33). The sequences of the primers utilized can be found at the internet address http://wwwgenome.wi.mit.edu/. Amplification of the simple sequence repeats was achieved by the polymerase chain reaction (PCR) in a PTC-100 thermal cycler (MJ Research, Watertown, MA). PCRs (20 ml) generally utilized 35 cycles of 30 s at 94°C, 1 min at 55°C, 30 s at 72°C. After amplification, 10 –15 ml of product was loaded onto a 15% polyacrylamide gel (Bio-Rad MiniProtean II) and electrophoresed at 12V/cm for 2 to 4 h. The PCR products were visualized by ethidium bromide staining and ultraviolet transillumination (254 nm). The animals were then scored as either B/W (heterozygous) or WW (homozygous) for each marker. The positions of the SSLP markers (and genetic loci) with respect to the centromere are given in accordance with the Mouse Chromosome Committee Reports obtained through the Encyclopedia of the Mouse Genome, Mouse Genome Database, The Jackson Laboratory (Bar Harbor, ME) (URL:http://www.informatics.jax. org). Statistical analysis. The linkage of a particular locus (B/W or WW) with thymic abnormalities, renal disease (proteinuria), or IgG anti-DNA antibodies was quantified by x2 analysis, using a standard [2 3 2]

NZB, THYMIC MICROENVIRONMENT

contingency matrix, after mice were categorized as positive or negative for the phenotype being studied (20). Mice were analyzed for a particular phenotype and grouped into discrete sets without knowledge of the genotype. For anti-DNA antibodies, mice were grouped on the basis of tertiles as previously reported (12, 19). The extreme phenotype sets, designated positive or negative, were then compared to the genotype data by x2 analysis. Mice analyzed in the present study are distinct from our previous analyses of (NZB 3 NZW)F1 3 NZW backcross mice (11, 12, 17), and linkage analysis was directed to a limited set of markers that cover approximately 10 –15% of the genome. Therefore, statistical thresholds recommended for genome-wide scans (21) were not considered appropriate for the current study. Loci were considered to be linked to a trait if a previously mapped locus was confirmed in the present study at P , 0.01 (x2 . 6.63) (21). A trend for linkage was considered to be present if one of the selected loci showed linkage at P , 0.05 (x2 . 3.8). Correlation coefficients and calculated significance were determined using Pearson’s linear regression analysis for relation between thymic abnormality and level of anti-dsDNA antibody, or using Spearman rank correlation analysis for relation between thymic abnormality and severity of proteinuria. The regressions were determined by using the look-up function in the StatView 4.0 program (Abacus Concepts, Berkeley, CA). The incidence of disease defined as severity of proteinuria or high level of anti-dsDNA antibody was statistically analyzed by using the Mann–Whitney U test or Student’s t test in the StatView 4.0 program or the Microsoft Excel program (Microsoft Corp., Redmond, WA). RESULTS

Studies of thymic abnormalities. Individual thymic lobes of each backcross mouse were histologically classified as described. Of the 102 backcross mice examined, 17 (16.7%) showed a staining pattern similar to that seen with normal thymic tissue from nonautoimmune mice. However, 85/102 (83.3%) had distinct thymic abnormalities as defined by the MTS antibodies (Table 1). Sera and urine from each of these 102 mice were screened for the presence of proteinuria and for anti-dsDNA antibodies. As shown in Table 1, consistent with previously reported data, 41/102 mice had significant anti-dsDNA antibodies and 49/102 had significant proteinuria. Of interest was the finding that none of the mice with normal thymic architecture had detectable anti-dsDNA antibodies and only 3 of these 17 had (low levels of) proteinuria. Thus, all 41 mice who had elevated anti-dsDNA antibody levels also demonstrated thymic abnormalities. Similarly, the majority (47/49) of the mice with proteinuria also showed thymic abnormalities.

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The frequencies of backcross control mice (shamthymectomized and intact) whose sera and urine showed anti-dsDNA antibodies and proteinuria, as expected, were approximately 50%. It is assumed that the frequencies that showed thymic abnormalities in these control sham-thymectomized and intact mice were similar to that in the thymectomized mice. These data confirm previous studies (11, 19) and are consistent with the possibility that thymic abnormalities and autoimmune disease are under similar genetic control. However, it is important to note that thymic abnormalities alone do not by themselves lead to clinical disease but must nonetheless contribute to clinical disease. Correlation of anti-ss or -dsDNA IgG isotype antibodies and thymic abnormalities. In efforts to distinguish whether severity of disease (in the form of anti-ss or -dsDNA IgG antibodies) correlated with specific patterns of thymic tissue abnormality (i.e., pathology and abnormal staining with the MTS antibodies within the cortex, medulla, or thymic tissue), thymic tissue from individual mice was scored blindly. Analysis of the data obtained showed that increasing titers of either anti-dsDNA (Fig. 1A) or anti-ssDNA (Fig. 2A) correlated with increased degrees of thymic abnormalities. Thus, individual mice with severe to moderate thymic abnormalities had the highest titers of anti-ss or -dsDNA antibodies. As seen in Figs. 1B and 2B, such a correlation was also documented when titers of anti-ss or -dsDNA antibodies were analyzed against total scores of thymic abnormalities (see Materials and Methods). Of interest was the observation that sublocalization of thymic abnormalities (i.e., abnormality within the cortex, capsular region, or medulla) in individual backcross mice did not correlate with titers or anti-ss or -dsDNA antibodies suggesting that specific subregion pathology is not the basis for the increased titers observed. Correlation of proteinuria with thymic abnormalities. Individual backcross mice demonstrated levels of proteinuria ranging from 0 (0 mg/dl) to 41 (2000 mg/dl). Of interest was the finding that while mice with low to moderate thymic abnormalities showed a correlation with increased levels of proteinuria (Fig. 3A), those with the most severe thymic abnormality had lower relative levels of proteinuria. Again, this pattern of higher levels of proteinuria in mice with moderate thymic abnormality and lower proteinuria in those with the most severe thymic abnormality was also seen when total scores of thymic microenvironmental abnormality was compared to levels of proteinuria (Fig. 3B). The reason for this decreased level of proteinuria in mice with the most severe thymic abnormality is not clear but it may be due to the fact that some backcross mice with the most severe thymic abnormality were sacrificed when they expressed high titers of anti-DNA

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TABLE 1 Thymic Abnormalities and Disease Expression Group

Total No.

Thymectomized

102

Control Sham-operated Unmanipulated

50 30 20

MTS staining

Number of dsDNA IgG-positive/totala

Number of proteinuria-positive/totala

Normal 17/102 (16.7%) Abnormal 85/102 (83.3%) Severe 60/85 (70.6%) Mild 23/85 (27.1%) NTc NT NT

41/102 (40.2%)b 0/17 (0%) 41/85 (48.2%) 31/60 (51.7%) 10/23 (43.5%) 24/50 (48.0%) 14/30 (46.7%) 10/20 (50.0%)

49/102 (48.0%) 3/17 (17.6%) 46/85 (54.1%) 32/60 (53.3%) 15/23 (65.2%) 27/50 (54.0%) 16/30 (53.3%) 11/20 (55.0%)

a

Number of positive/number of each group. Percentage of positive in each group. c NT, not tested. b

antibody but before the development of significant proteinuria. There also did not appear to be any correlation between select thymic subregion (cortex vs medulla) abnormality and values of proteinuria. Regression analysis of data on thymic abnormality and the incidence of disease. The relationship between abnormalities of thymic microenvironment and the titer of IgG anti-dsDNA antibodies or severity of proteinuria in individual mice was determined by calculating the regression coefficient (Figs. 4 and 5). Lin-

ear regression analysis showed that thymic microenvironment abnormalities were significantly correlated with both elevated levels of IgG type anti-dsDNA antibodies (r 5 0.402, P , 0.001, n 5 102, Fig. 4) and severity of proteinuria (r 5 0.197, P 5 0.013, n 5 102, Fig. 5). Two mating schemes to produce the backcross mice (B/WF1 3 NZW and NZW 3 B/WF1) were utilized in the present study. However, there was no difference noted in the frequency of mice with thymic abnormal-

FIG. 1. ELISA titers of serum IgG anti-dsDNA antibodies from backcross mice with thymic cortical (A, open columns) or medullary abnormalities (A, shaded columns). B shows the titers of the backcross mice which were evaluated on the basis of total thymic abnormalities (solid columns). IgG anti-dsDNA antibodies were significantly higher in mice who had severe or moderate abnormalities in the medulla (A) and in mice who had a severe abnormality in the cortex (A). Columns and bars represent the mean titers and SE. *P , 0.05, **P , 0.005, ***P , 0.001 compared to titers of backcross mice with a normal thymus (Student’s t test).

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FIG. 2. ELISA titers of serum IgG anti-ssDNA antibodies from backcross mice with thymic cortical (A, open columns) or medullary abnormalities (A, shaded columns). B shown the titers of the backcross mice evaluated on the basis of total thymic abnormalities (solid columns). IgG anti-ssDNA antibodies were significantly higher in mice who had severe or moderate abnormalities in the medulla or cortex (A) and in mice with higher total assessments of thymic abnormalities (B). Columns and bars represent the mean titers and SE measured using ELISA. *P , 0.05, **P , 0.005, ***P , 0.001 compared to titers of backcross mice with normal thymus in each group (Student’s t test).

ities or incidence of the disease between either of these mating schemes (data not shown). Moreover, the backcross mice expressed four different phenotypes with hair and eye colors (dark brown hair and black eyes, light brown hair and black eyes, yellow hair and red eyes, and white hair and red eyes), but these color phenotypes did not correlate with thymic abnormality, level of anti-dsDNA antibodies, or proteinuria (data not shown). Analysis of loci linked with thymic abnormalities, nephritis, and anti-DNA antibodies. We directed a linkage analysis to a subset of NZB loci that showed linkage to nephritis and IgG autoantibody production in previous backcross studies of New Zealand hybrid mice (11, 12, 19). We were especially interested in loci that were linked to nephritis and/or anti-DNA production. Although the markers selected for the present analysis only covered about 10 to 15% of the genome and the number of backcross mice studied was relatively small, trends for linkage with these disease traits were apparent in the present study (Table 2). For example, in our previous study of (NZB 3 NZW)F1 3 NZW backcross mice, loci on distal chromosome 4 and proximal chromosome 17 (close to MHC) provided the strongest linkage with nephritis. These loci also showed trends for linkage with nephritis in the present study with x2 values of 4.9 and 5.3, respectively. In addition, a locus on chromosome 7 showed a trend for

linkage with IgG anti-dsDNA production as noted in our previous study of such backcross mice (19). Interestingly, the trend in the present study was for homozygosity for the NZW allele, which was the opposite in the previous study (19). The trend for linkage with a chromosome 7 locus was for NZW homozygosity rather than for inheritance of the NZB allele. Since thymic abnormalities are determined primarily by NZB genes, we would not expect this locus to contribute to this trait. If anything, linkage of an NZW locus with antiDNA antibodies or nephritis might tend to obscure the correlation of these disease traits with thymic abnormalities. Hence, we would not expect the contribution determined by NZW homozygosity to contribute to thymic abnormalities. The analysis for loci linked with thymic abnormalities (Table 2) was limited by the relatively small number of animals with completely normal thymus histology. For example, only 19 backcross mice were scored as lacking histological abnormalities. Still, we noted a trend for linkage of thymic medullary abnormalities with markers on distal chromosome 4 (maximal at D4Mit343) in a chromosomal interval similar to that linked with nephritis. In an attempt to compare groups that were more evenly distributed, we combined mice with no or 11 abnormalities in the negative phenotype group. This failed to strengthen the linkage with chromosome 4 loci and instead, resulted in a trend for

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FIG. 3. Severity of proteinuria in backcross mice. A shows the severity of proteinuria in backcross mice with abnormalities in cortex (open columns) or medulla (shaded columns). B shows the severity of proteinuria in backcross mice as a total assessment of thymic abnormalities (solid columns). The severity of proteinuria was significantly high in mice who had mild or moderate abnormalities in the medulla or cortex (A) and in mice with a severe total degree of abnormalities (B). Columns and bars represent the mean proteinuria and SE *P , 0.05, ***P , 0.001 compared to mice with normal thymus (Mann–Whitney U test).

linkage with Crp on chromosome 1 at Crp. An NZB locus on distal chromosome 1 at this position (named Nba2) has been mapped previously in several different

crosses (reviewed in (17)). No trend for linkage with thymic cortical atrophy was observed (32 mice had normal thymic cortical histology). When total thymic

FIG. 4. Correlation between the total thymic microenvironment abnormality and titer of IgG type anti-dsDNA antibody. The thymic abnormalities strongly correlated with elevation of titer (linear regression analysis: r 5 0.402, P , 0.001, n 5 102).

FIG. 5. Correlation between the total thymic microenvironment and severity of proteinuria. The thymic abnormalities were correlated with proteinuria (linear regression analysis: r 5 0.197, P 5 0.013, n 5 102).

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TABLE 2 Loci Linked with Thymic Abnormalities, Proteinuria, and Anti-DNA Antibody Levels in (NZB 3 NZW)F1 3 NZW Backcross Mice Positive Locus

Position

B/W

WW

B/W

WW

x2

P value

D4Mit343 Crp

4, 75 1, 94

36 24

26 14

6 13

13 23

4.1 5.4

0.04 0.02

—

—

—

—

—

—

—

1, 94 1, 94

29 22

20 14

8 8

17 17

4.9 5.0

0.03 0.03

D4Mit70 D17Mit35

4, 65 17, 14

40 37

22 23

12 9

18 17

4.9 5.3

0.03 0.02

D7Mit16

7, 40

14

23

22

13

4.5

0.03

D7Mit16

7, 40

13

20

21

11

4.5

0.03

Phenotype Thymic medullary abnormalities 0 versus 11, 21, or 31 0 or 1 versus 21 or 31 Thymic cortex atrophy 0 versus 11, 21, 31 Total thymic abnormalities 0 or 1 versus $21 0 or 1 versus $31 Nephritis 0 versus $21 Anti-ssDNA ,0.9 versus .1.5 units Anti-dsDNA ,0.4 versus .1.0 units

Negative

None Crp Crp

Note. Loci listed showed linkage with the respective trait at P , 0.05 (x2 . 3.8).

abnormalities were considered, the group scored as histologically normal was too small for analysis. If the numbers of mice with 0 and 11 scores were combined and compared to those with higher scores, a trend for linkage with a locus on distal chromosome 1 (at Crp) was found. This locus was similar in position to that found for thymic medullary abnormalities. Although none of these linkages reached statistical thresholds recommended for confirming loci in genome-wide scans (21), the trends for linkage are impressive, considering the number of mice analyzed and the small number of markers mapped. DISCUSSION

Our laboratories undertook a study of individual backcross mice resulting from the matings of B/WF1 3 NZW mice. The rationale for the study was based on two previously published findings. First, it has been previously shown that approximately 50% of such backcross mice develop clinical autoimmune disease (11), and disease susceptibility has been mapped to several different loci as a complex genetic trait (11, 12, 17). Second, we have previously shown that the autoimmune lupus-prone NZB and B/W mice show marked thymic microenvironmental defects as defined by ultrastructural studies and by staining patterns with the use of a novel set of mAbs (MTS) (6 – 8). These MTS antibodies show patterns of staining in normal nonautoimmune thymic tissue which are distinct from that seen in the NZB and B/W F1 mice. The thymus tissue is known to initiate thymopoiesis and several lines of data have shown that it is within this tissue that

negative/positive selection occurs (9, 10). It has thus been proposed that abnormal selection during T cell development leading either to the escape of autoreactive T cells and/or decrease in T cells that regulate such autoreactive T cells may be the basis for the lupus disease in such mice (6 – 8). These lines of data and concepts led us to ask whether the thymic tissue abnormality at 1 month of age correlates with autoimmune disease seen in individual backcross mice. The results from the studies reported herein show that (1) all mice with lupus-like disease have thymic tissue abnormalities; (2) the thymic tissue abnormality by itself is not sufficient for the development of clinical disease; (3) the degree of severity of thymic abnormality correlates with increasing titers of both ss- and dsDNA IgG antibodies; (4) the degree of severity of thymic abnormality in general correlates with increasing levels of proteinuria; (5) there did not appear to be a correlation between clinical disease and a specific thymic subregion (cortex vs medulla) abnormality. Since .50% of the backcross mice show thymic abnormalities, clearly this specific dysfunction cannot be due to a single gene or set of linked genes. Genetic mapping studies also support this latter conclusion. Interestingly, NZW mice have been previously shown to have a normal thymic microenvironment using these same reagents (8). We have not studied the relationship of the thymic abnormalities to the natural thymocytotoxic autoantibodies produced in NZB mice; this would be an interesting issue for further study. SLE is a chronic autoimmune disorder characterized by antibodies to double stranded DNA, glomerulone-

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phritis, and multiorgan involvement. Autoreactive T and/or B cells appear in peripheral sites in patients or animal models with SLE. It has been shown that IgG type anti-DNA antibodies are important in the pathogenesis of lupus nephritis (22–24). Moreover, the production of anti-dsDNA antibodies and the development of disease is a T-cell-dependent process (22). Multiple in vitro and in vivo experiments have established that T cells promote anti-DNA antibody production by activated B cells in SLE patients and animals (25, 26). Although it remains unclear which T cell specificities are most important in augmenting anti-DNA antibody production, it is clear that T cells play an important role in the pathogenesis of SLE. Modifications in the thymic architecture could disrupt either or both positive and negative selection. Such selection is mandated, presumably through their contact with underlying cortical epithelial cells including thymic nurse cells, which express not only both MHC class I and II molecules but also a variety of other cell surface molecules (27). Electron microscopic studies suggest that isolated thymic nurse cells are the sites of thymic apoptosis as evidence for the tissue locale whereby negative selection occurs (10). A variety of mechanisms have been proposed to account for positive/negative selection of T cells, including T cell receptor density, relative affinity of the TCR for peptide bound MHC class I/II molecules, and expression or lack thereof of costimulatory/cell adhesion molecules and their cognate receptors (28 –30). In addition, an epithelial cell line has been shown to form thymic nurse cells in vitro and has the functional capacity to induce thymocyte apoptosis in vitro (9). Although the function of thymic medullary epithelial cells is unclear, clonal deletion is manifest in this region and the epithelial cells can induce tolerance in the form of anergy (31–34). Medullary epithelial cells also form stromal cell complexes with thymocytes which may be maturationlinked and possibly related to emigration (1). A novel set of MTS mAb has been previously described which have specificity for thymic epithelial cells including pan epithelium (MTS1), subcapsular and medullary epithelium (MTS10), and cortical epithelium (MTS44). The biochemical nature of the molecules that are the target of such MTS mAb’s has not been determined (1, 6). Therefore, a decrease of MTS staining implies two possibilities: absence of epithelium and change of molecules on epithelium. Several older reports have also characterized thymic tissue abnormalities in NZB mice (35–37). In addition, staining of the thymic epithelium by anti-keratin antibody demonstrates many epithelial cell free regions in the thymus of NZB mice (6). Although thymic explants from BALB/c or other normal mice generally produce thymic epithelial cells in culture, thymic tissues from NZB mice generate only fibroblast growth (35). This

could be due to a programmed defect in the proliferative ability of NZB thymic epithelium or to a suboptimal and/or defect in the microenvironment. NZB thymus also shows a decrease in the large medullary epithelium while abnormalities within the cortical thymic epithelia increase with age (6). Moreover, normal mice possess thymic epithelial cells which show increased expression of the KL1 marker following in vivo administration of hydrocortisone; this increase was not observed in NZB mice (38). In addition, our findings indicate that not all backcross mice with abnormalities of thymic microenvironment (83.3%) express the disease. Those data suggest that non-thymus-derived and/or non-MHC-locus-related immune system(s) may have some role in the regulation of disease. However, the abnormalities of thymic microenvironment may be a necessary condition for pathogenesis of SLE. Considerable evidence indicates that the development of disease in B/W F1 mice is genetically determined and that MHC and non-MHC genes from both the NZB and NZW parental strains contribute the full expression of disease (reviewed in (17). At least 12 non-MHC genes have been mapped in this model of lupus to be linked with lupus nephritis and/or IgG autoantibody production (17). Studies are attempting to correlate the different disease loci with subphenotypes in order to gain insight into the etiologic gene at each locus and the mechanism by which disease susceptibility is enhanced. In the present work, we focused on disease-susceptibility loci from NZB and the possibility that their mechanism was mediated through the genetically determined thymic abnormalities previously described in this strain. In B/W mice, this trait appeared to be mostly dominant in its expression. Interestingly, markers on distal chromosome 4 showed a trend for linkage with thymic medullary abnormalities in the current backcross study. This chromosomal interval colocalizes with a locus named Nba1 for New Zealand black autoimmunity 1, which had been mapped in a previous (NZB 3 NZW)F1 3 NZW backcross and was the major dominant non-MHC contribution from NZB mice to nephritis (11, 12). The trend for linkage with thymic abnormalities was impressive considering the small number of mice studied, especially in the negative phenotype group. We also noted trends for linkage with markers on distal chromosome 1. An NZB locus named Nba2 has been mapped to this chromosomal position as a mostly recessive contribution in several different crosses of NZB mice with nonautoimmune strains (17, 19). Nba2 has been shown to contribute to both nephritis and IgG autoantibody production. Its contribution to these traits was not found in previous analyses of (NZB 3 NZW)F1 3 NZW backcross mice (11, 12, 17) probably because of separate contributions from NZW at this chromosomal position (reviewed in (17)). This is consistent with the results from

NZB, THYMIC MICROENVIRONMENT

the current analysis. The trend for linkage with thymic abnormalities in the present study may relate to the fact that NZW loci do not contribute to this trait and therefore do not obscure the NZB contribution at this position. Because of the small number of markers mapped and the relatively small number of mice analyzed, we strongly believe that the trends for linkage in the current study indicate loci that contribute to thymic abnormalities and do not represent false-positive mappings. If this is correct, our results also suggest that the thymic abnormalities in NZB mice represent a complex genetic trait, with contributions from multiple loci. This conclusion will certainly be clearer when a genome-wide screen is completed in many more backcross mice. The mechanism(s) by which thymic abnormalities may contribute to murine lupus is unknown. It seems likely that T cell development is involved. However, there are at least two sets of T cell clones maturing in the thymus—an autoreactive set of clones and regulatory clones; differences in the quality and/or quantity may dictate clinical disease. Similarly, it is possible that the primary cells that regulate autoreactivity are TCR1 NK cells which have specificity for non-MHC ligands such as CD1 and express KIRs. Thus, abnormalities in the expression of these non-MHC molecules may lead to inappropriate deletion of autoreactive T cells. Finally, it will be important to define the biochemical nature of the thymic tissue abnormalities in NZB and B/W mice as well as to study larger numbers of animals in order to fully define the genetic contributions. Clearly, the answers to these questions may contribute to our understanding of how abnormal thymic architecture translates into immunopathology.

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Received August 6, 1998; accepted with revision November 13, 1998

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