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The Journal of Immunology, 1999, 162: 7241-7248.
Copyright © 1999 by The American Association of Immunologists

A Central Role for {alpha}ß T Cells in the Pathogenesis of Murine Lupus1

John P. Seery2,*, Eddie C. Y. Wang2,{dagger}, Victoria Cattell{ddagger}, Joseph M. Carroll3,*, Michael J. Owen{dagger} and Fiona M. Watt4,*

* Keratinocyte Laboratory and {dagger} Lymphocyte Molecular Biology Laboratory, Imperial Cancer Research Fund, London, United Kingdom; and {ddagger} Department of Histopathology, St Mary’s Hospital Medical School, Imperial College of Science, Technology and Medicine, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that female transgenic mice expressing IFN-{gamma} in the epidermis, under the control of the involucrin promoter, develop inflammatory skin disease and a form of murine lupus. To investigate the pathogenesis of this syndrome, we generated female IFN-{gamma} transgenic mice congenitally deficient in either {alpha}ß or {gamma}{delta} T cells. TCR{delta}-/- transgenics continued to produce antinuclear autoantibodies and to develop severe kidney lesions. In contrast, TCRß-/- IFN-{gamma} transgenic mice failed to produce antinucleosome, anti-dsDNA, or antihistone autoantibodies, and kidney disease was abolished. Both {alpha}ß- and {gamma}{delta}-deficient transgenics continued to develop IFN-{gamma}-associated skin disease, lymphadenopathy, and splenomegaly. The data show that the autoantibody-mediated pathology of murine lupus in IFN-{gamma} transgenic mice is completely {alpha}ß T cell dependent and that {gamma}{delta} T cells cannot drive autoantibody production. These results imply that production of antinuclear autoantibodies in IFN-{gamma} transgenic animals is Ag driven, and we identified clusters of apoptotic cells in the epidermis of the mice as a possible source of self Ags. Our findings emphasize the relevance of this murine lupus model to the human disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE)5 is a relatively common autoimmune disease characterized by autoantibody production against a variety of nuclear Ags and multiple end organ damage. Antinuclear Abs are thought to act directly in the pathogenesis of the disease, but the mechanisms underlying their production are ill defined (1). Affinity maturation of IgG antinuclear Abs implies a central role for autoantigen-specific CD4+ T cells in the pathogenesis of human SLE (2). In support of this contention, Desai-Mehta et al. (3) have recently isolated and characterized a subset of autoimmune Th cells from patients with SLE that strongly induce anti-dsDNA Ab production.

The precise role of T cell dependent mechanisms in murine lupus is controversial. Analysis of splenocyte somatic cell hybrids from MRL/lpr mice strongly implies that anti-dsDNA Abs arise in these animals by Ag driven clonal expansion and somatic mutation (4). The central role of CD4+ {alpha}ß T cells in murine lupus is further supported by the demonstration that the elimination or functional down-regulation of these cells results in significant disease amelioration (5, 6, 7, 8). However, recent studies of MRL/lpr mice deficient in {alpha}ß T cells showed no absolute requirement for this T cell subset in the development of lupus and suggest that the production of pathogenic antinuclear Abs can be driven by {gamma}{delta} T cells alone (9).

We have recently shown that female transgenic mice expressing IFN-{gamma} in the epidermis under the control of the involucrin promoter develop inflammatory skin disease, hypopigmentation, lymphadenopathy, and splenomegaly (10). In addition, female transgenic mice produce autoantibodies against dsDNA and histones and all have evidence of glomerular Ig deposition. Approximately one in four female transgenics goes on to develop a severe proliferative glomerulonephritis (11). Thus there are striking parallels with the human disease. To investigate whether T cell-dependent processes are involved in the pathogenesis of this lupus-like syndrome, we generated IFN-{gamma} transgenic mice deficient in either {alpha}ß or {gamma}{delta} T cells. Our data demonstrate that T cells play no role in IFN-{gamma}-induced skin inflammation and provide evidence that the production of antinuclear autoantibodies in this lupus model is an {alpha}ß T cell-dependent, Ag-driven process. Apoptotic keratinocytes in the hair follicles and interfollicular epidermis are identified as a possible source of Ag.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Transgenic mice expressing IFN-{gamma} in the epidermis have been described previously (10). Briefly, a transgene consisting of the cDNA for murine IFN-{gamma} under the control of the involucrin promoter was injected into fertilized oocytes from (CBA x C57/BL10)F1 mice. Three independent founder lines were generated: line 1205D contains two copies of the transgene, line 1205C contains 6 copies, and line 1212F contains 32 copies. Mice in all three founder lines had the same phenotype (10, 11). The 1205D line on the F1 background was used in all the experiments described in this study.

TCRß-/- (12) and TCR{delta}-/- mice (13), referred to as ß-/- and {delta}-/- mice respectively, were obtained from The Jackson Laboratories (Bar Harbor, ME). The ß-/- and {delta}-/- mice used in this study were on a C57BL/6 background. All mice were maintained and bred at the Imperial Cancer Research Fund animal facility.

Generation of IFN-{gamma} transgenic mice deficient in {alpha}ß or {gamma}{delta} T cells

Male mice heterozygous for the IFN-{gamma} transgene (founder line 1205D) were crossed with ß-/- or {delta}-/- females. From these litters males heterozygous for both the IFN-{gamma} transgene and the ß- or {delta}-chain deletion were crossed with ß-/- and {delta}-/- T cell-deficient females, respectively. From the resulting litters, female animals heterozygous for the IFN transgene and homozygous for either TCR deletion were obtained and used throughout this study. Age- and sex-matched littermates from this generation positive for the transgene and heterozygous for the relevant TCR deletion acted as controls. The available evidence suggests that T cell function is normal in animals heterozygous for either the ß- or {delta}-chain deletion (12, 13). As the males used to breed the test litters were heterozygous for the IFN-{gamma} transgene, ~50% of the females in each litter were not transgenic and were used to assess baseline autoantibody levels and renal histology. These animals, heterozygous for the relevant TCR deletion, are subsequently referred to as "littermates negative for the transgene," The term "control" is limited to animals positive for the IFN-{gamma} transgene and heterozygous for the relevant TCR deletion.

Genotyping of transgenics

The presence of the IFN-{gamma} transgene was detected using PCR on genomic DNA from ear or tail snips, as previously described (14). Genomic DNA was isolated by standard techniques (15). TCRß genotyping was performed by flow cytometry of PBLs stained with anti-mouse TCR-ß FITC-conjugated mAb (Becton Dickinson, Mountain View, CA) on a FACS Profile flow cytometer with CellQuest software (Becton Dickinson). TCR{delta} genotyping was conducted by PCR of genomic DNA using the following primers: TCR{delta}-F, 5'-AGATAATGAAAAACTACCAGAACC-3'; TCR{delta}-R, 5'-AATATGAAGTGACCAATTCTTACC-3' under the following PCR conditions: 1 cycle at 94°C for 5 min, 50°C for 30 s, and 72°C for 30 s; 30 cycles at 94°C for 20 s, 50°C for 30 s, and 72°C for 30 s; and 1 cycle at 94°C for 20 s, 50°C for 30 s, and 72°C for 10 min. DNA from heterozygous TCR{delta}+/- mice produced a 600-bp band, visualized by ethidium bromide in 1% agarose gels, which was absent in TCR{delta}-/- mice.

Histology

For light microscopy, kidney and skin tissue were fixed in formalin and paraffin embedded, and sections were stained with hematoxylin and eosin or periodic acid Schiff. All kidney sections were analyzed by an experienced renal pathologist (V.C.) blind to the T cell status of the animals. The severity of renal lesions observed was graded on the basis of degree of glomerular hypercellularity (equivocal, -/+; mild, +; moderate, ++; severe, +++), presence of polymorphonuclear leukocyte infiltrate, fibrin deposition, and crescent formation.

Immunofluorescence staining of kidney

For detection of glomerular IgG deposits, kidneys were snap frozen in an isopentane bath cooled in liquid nitrogen. Frozen sections embedded in OCT (Tissue Tek, Miles, Elkhart, IN) were cut at 5–8 µm thickness. Sections were air dried and blocked for 30 min with goat serum. Sections were then incubated for 45 min with Texas red conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) diluted at 1:100 in PBS containing 0.5% BSA (Sigma, Poole, Dorset, U.K.).

All Ab incubations were conducted at room temperature and were followed by thorough washing in PBS. Stained sections were mounted in Gelvatol (Monsanto, St. Louis, MO) and examined using a Zeiss Axiophot microscope.

Immunohistochemistry of lymph nodes and spleen

For immunohistochemistry, lymph nodes and spleens were snap frozen and sections cut onto glass slides and stored at -70°C. Before Ab staining, sections were fixed in acetone at -20°C for 5 min, and endogenous peroxidase activity blocked by incubation for 15 min at room temperature in 0.6% hydrogen peroxide. For double immunolabeling with anti-B220 and anti-CD3, sections were blocked with rabbit serum (Sigma) for 15 min at room temperature at 1:25 dilution before addition of primary Abs (see below; used as recommended by PharMingen, San Diego, CA). Germinal centers were stained with peanut agglutinin-biotin (1 µg/ml). Secondary reagents were combinations of the following: rabbit anti-rat IgG-alkaline phosphatase (AP) (Sigma) diluted 1:50; streptavidin-HRP (Dako, High Wycombe, U.K.) diluted 1:400; or streptavidin-AP (Dako) diluted 1:100. AP was visualized in blue using the Vector Blue AP Substrate kit III (Vector Laboratories, Peterborough, U.K.); HRP in red using 3-amino 9-ethyl carbazole (AEC) as described (16). Photographs of sections were taken with an Olympus LB x1 KDC System attached to a Leica microscope. Images were transferred onto Adobe Photoshop 5.0.2 using Kodak DCS Acquire (version 5.5.9) software.

Flow cytometry

Single cell suspensions were obtained by teasing spleens or lymph nodes and filtering the resultant cell mix through a sterile, glass wool-plugged pasteur pipette to remove stromal debris. Splenocytes or lymphocytes were incubated for 30 min on ice in DMEM supplemented with 5% FCS (E4-5), with combinations of the following preconjugated mAbs: anti-CD3-FITC, anti-B220-PE, anti-IgM-biotin, anti-IgD-FITC, anti-CD11c-biotin, anti-TCRß-biotin, and anti-{gamma}{delta}TCR-FITC (all from PharMingen), or anti-monocyte (F4/80)-FITC (Caltag, Burlingame, CA). Stained cells were washed twice with E4-5 before incubation with second-step reagents as above. Biotin conjugated Abs were visualized using streptavidin-Tricolor (Caltag). Twenty thousand events were collected per sample and analyzed on a FACSCalibur using Cell Quest software (Becton Dickinson, San Jose, CA).

Antihistone, anti-dsDNA, and antinucleosome ELISA

The levels of total Ig and IgG antihistone and anti-dsDNA Abs in serum were measured using a modification of previously described methods (17). Calf thymus histones (Sigma) were diluted in PBS to a concentration of 2.5 µg/ml, and 0.2 ml of this Ag solution was added to each well of an Immulon II microtiter plate (Dynatech, Alexandria, VA). After overnight incubation at 4°C, wells were coated with 0.4 ml gelatin (1 mg/ml in PBS) for at least 24 h at 4°C. After washing, 0.2 ml of serum samples diluted 1/100 to 1/1000 in 0.1% Tween 20, 1 mg/ml gelatin, and 0.5% BSA in PBS were added and incubated for 1.5 h at room temperature. After washing, total Ig bound was measured by adding HRP-conjugated rabbit anti-mouse Ig (Dako) or IgG was measured using HRP-conjugated goat anti-mouse IgG (Sigma). Both secondary Abs were used at a dilution of 1/4000 in 0.1% Tween 20 in PBS. After 1.5-h incubation at room temperature, the wells were washed and substrate solution added. The OD was then read with an automated spectrophotometer at 492 nm.

To measure anti-dsDNA Ab levels, wells were coated with dsDNA (Sigma). To attach dsDNA, microtiter wells were first coated with poly-L-lysine (Sigma) at 5.0 µg/ml in H2O for 1.5 h at 37°C. After washing, dsDNA was added at 5.0 µg/ml in PBS and incubated overnight at 4°C. After washing, serum samples diluted 1/100 to 1/1000 were added as described above.

Levels of IgG antinucleosome Abs were measured by ELISA as previously described (18). These assays were conducted by Dr. Sophie Koutouzov (Hopital Necker, Paris, France). Briefly, purified mononucleosomes prepared as previously described (19) were dissolved in PBS at 5 µg/ml, and 100 µl was added to Luxlon microtiter plates (CML, Nemours, France). Plates were incubated overnight at 4°C. Wells were washed with PBS-0.1% Tween 20, pH 7.4 (PBST) and postcoated for 2 h with 0.1 ml of PBS-10% FCS (pH 7.4). After washing, sera (1/100) diluted in PBST were added and reacted for 2 h. Bound Abs were detected with peroxidase-conjugated goat anti-mouse Fc antisera (Sigma). Binding was measured by adding ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) substrate (Southern Biotechnology Associates, Birmingham, AL), and OD was read at 405 nm by an automated spectrophotometer (Dynatech).

The antihistone and anti-dsDNA ELISA tests were run in triplicate and antinucleosome ELISA in duplicate. In addition, serum samples were checked for nonspecific binding to control wells lacking Ag. We have previously shown that this anti-dsDNA ELISA system shows no cross reactivity with ssDNA (11).

Fluorescent TUNEL staining of skin sections

TUNEL staining of paraffin sections of skin was conducted using a commercially available kit (Apoptosis Detection System, Fluorescein, Promega, Madison, WI). Formalin-fixed skin sections were deparaffinized and rehydrated sequentially in graded ethanol. Sections were then washed in 0.85% NaCl followed by PBS. Tissue sections were fixed in 4% methanol-free formaldehyde in PBS for 15 min at room temperature followed by extensive washing in PBS. Sections were subsequently incubated with Proteinase K (20 µg/ml in 100 mM Tris-HCl containing 50 mM EDTA, pH 8.0) for 8 min at room temperature. Sections were again formalin fixed, and after washing in PBS they were incubated for 10 min at room temperature with equilibration buffer (200 mM potassium cacodylate, 25 mM Tris-HCl, 0.2 mM DTT, 0.25 mg/ml BSA, and 2.5 mM cobalt chloride, pH 6.6). The reaction mixture (equilibration buffer containing 5 µM fluorescein-12-dUTP, 10 µM dATP, 100 µM EDTA, and 0.5 U/µl terminal transferase) was added and incubated at 37°C for 1 h. The reaction was stopped by immersing the slides in 2x SSC for 15 min at room temperature. Following extensive washing in PBS, sections were counterstained with propidium iodide solution (1 µg/ml in PBS). After washing in deionized water, stained sections were mounted in Gelvatol (Monsanto, St. Louis, MO) and examined using a Zeiss Axiophot microscope. Five noncontiguous skin sections 5 µm in thickness and 2–2.5 cm in length from each of two female IFN-{gamma} transgenic mice and age- and sex-matched littermate controls were examined for the presence of TUNEL-positive cells.

Statistics

Optical densities obtained with serum on ELISA from different groups of animals were compared using the Mann-Whitney U test. The significance of differences in the incidence of renal disease at dissection was assessed using Fisher’s exact test. The relationship between severity of renal disease and autoantibody levels was defined by calculating the Pearson product moment correlation coefficient.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Animals studied

All mice used were female and 5 mo of age at the time of study. Female IFN-{gamma} transgenic mice heterozygous for either the TCR ß-chain or TCR {delta}-chain deletion were used as controls since T cell function is essentially normal in these animals (12, 13). In addition, animals negative for IFN-{gamma}, referred to as littermates negative for the transgene, were used to assess baseline autoantibody levels and renal histology.

{gamma}{delta} T cells are not required for the production of antinuclear autoantibodies or the development of lupus nephritis in IFN-{gamma} transgenic mice

We have previously shown that the majority of female mice expressing IFN-{gamma} in the epidermis develop glomerular Ig deposits with light and electron microscopic evidence of glomerular disease. Approximately 25% of these animals develop a severe proliferative glomerulonephritis (11). Three of eight {delta}-/- IFN-{gamma} and three of eleven {delta}+/- IFN-{gamma} transgenic females examined developed the full IFN-{gamma}-associated lupus syndrome with IgG anti-dsDNA autoantibody production and proliferative glomerulonephritis on histology (Table IGo). Renal disease, when present, tended to be more severe in {delta}-/- IFN-{gamma} transgenics (Table IGo); however, the relatively low incidence of light microscopic renal lesions in these animals prevented statistical testing of this observation.


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  		Table I. Renal pathology in {gamma}{delta} T cell-deficient IFN-{gamma} transgenic mice1

 
Serum from the mice was screened for autoantibodies to dsDNA, histones and nucleosomes. There was no difference in IgG anti-dsDNA titers between {delta}+/- and {delta}-/- IFN-{gamma} transgenics (mean OD: {delta}-/-, 0.21; {delta}+/-, 0.16; p = NS) (Fig. 1Go). Levels of both IgG antinucleosome and IgG antihistone Abs were low in these litters. There appeared to be a tendency toward higher levels of antinucleosomal Ab production in {delta}+/- compared with {delta}-/- transgenics; however, this did not reach statistical significance (mean OD: {delta}+/-, 0.10; {delta}-/-, 0.05; p = NS) (data not shown). Transgenic animals from the litters produced levels of IgG antihistone autoantibodies comparable to littermates negative for the transgene (mean OD: {delta}-/-, 0.02; {delta}+/-, 0.01; littermates negative for the transgene: 0.02; p = NS) (Table IGo). The separation of antihistone and anti-dsDNA production has already been described in this model (11) and presumably relates to unidentified genetic factors.



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  		FIGURE 1. Anti-dsDNA IgG autoantibody levels in {gamma}{delta} T cell-deficient IFN-{gamma} transgenic mouse serum. Sera from 11 {delta}+/-, 8 {delta}-/- IFN-{gamma} transgenic mice, and 7 littermates negative for the transgene (LNT) were tested individually against dsDNA at 1/100 dilution. The OD value for each sample represents the mean of three measurements.

 
IFN-{gamma} transgenic mice deficient in {alpha}ß T cells do not produce IgG antihistone or anti-dsDNA autoantibodies and do not develop autoimmune kidney disease

We used ELISA assays to quantitate the levels of IgG anti-dsDNA, IgG antihistone and IgG antinucleosome autoantibodies in IFN-{gamma} transgenic ß+/- and ß-/- mice. The six ß+/- transgenics examined produced high levels of IgG antinuclear Abs (mean ODs: 0.29, 0.20, and 0.63 for IgG anti-dsDNA, antihistone, and antinucleosome autoantibodies, respectively) (Fig. 2Go). Compared with the ß+/- animals, levels of all three antinuclear Abs tested were significantly reduced in ß-/- IFN-{gamma} transgenics (mean ODs: 0.01, p < 0.003; 0.04, p < 0.004; and 0.02, p < 0.003 for IgG anti-dsDNA, IgG antihistone, and IgG antinucleosome autoantibodies, respectively). Indeed, serum from the 11 ß-/- transgenic mice tested showed antinuclear Ab levels no higher than age- and sex-matched littermates negative for the transgene (Fig. 2Go). In addition, levels of total Ig anti-dsDNA Abs (i.e., all Ig classes) in ß-/- IFN-{gamma} transgenic mice were comparable to those in matched littermates negative for the transgene (mean OD: 0.03 and 0.02, respectively, at 1/100 dilution, p = NS) (mean OD in ß+/- IFN-{gamma} transgenics: 0.21). Interestingly, serum from two littermates negative for the transgene showed low but significant levels of IgG antinucleosome autoantibodies (mean ODs: 0.28 and 0.30 at 1/100 dilution), suggesting a background susceptibility to autoantibody production as has previously been reported in C57BL mice (11, 20). Mice negative for the IFN-{gamma} transgene did not produce anti-dsDNA or antihistone Abs and never developed kidney disease (Ref. 11 and the present study).



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  		FIGURE 2. Anti-dsDNA (A), antihistone (B), and antinucleosome (C) IgG autoantibody levels in {alpha}ß T cell-deficient IFN-{gamma} transgenic mouse serum. Sera from 6 ß+/-, 11 ß-/- IFN-{gamma} transgenic mice, and 7 littermates negative for the transgene (LNT) were tested individually against dsDNA, histones, and nucleosomes at 1/100 dilution. The OD value for anti-dsDNA and antihistone autoantibodies represents the mean of three measurements. The OD value for antinucleosome autoantibodies represent the mean of two measurements.

 
Three of the six ß+/- female transgenics examined had proliferative glomerulonephritis. In contrast, renal histology was normal in the 15 ß-/- IFN-{gamma} transgenic animals examined (Table IIGo, p = 0.002). On direct immunofluorescence, both mesangial and capillary patterns of glomerular IgG deposition were observed in ß+/- IFN-{gamma} transgenics (Table IIGo). Glomerular deposition of IgG could not be demonstrated in any of 7 ß-/- IFN-{gamma} transgenic females tested (Table IIGo, p = 0.008). The 2 ß+/- IFN-{gamma} transgenic mice with nucleosome-restricted autoantibodies showed evidence of IgG deposition in the kidney but no definite evidence of glomerulonephritis on light microscopy (nos. 4 and 6, Table IIGo)


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  		Table II. Renal pathology and autoantibody levels in {alpha}ß T cell-deficient IFN-{gamma} transgenic mice

 
The severity of renal disease correlates with IgG anti-dsDNA autoantibody levels in IFN-{gamma} transgenic mice

Three groups of animals in the present study produced significant titers of anti-dsDNA Abs ({delta}+/-, {delta}-/-, and ß+/- IFN-{gamma} transgenics, n = 25), affording an opportunity to study the relationship between autoantibody levels and the severity of renal disease. Seven of the animals in these three groups developed moderate to severe glomerular hypercellularity. All seven had relatively high levels of anti-dsDNA Abs (nos. 4, 14, 16, and 19 in Table IGo and nos. 1, 2, and 5 in Table IIGo). There was a positive correlation between the degree of glomerular hypercellularity and anti-dsDNA Ab levels with a Pearson product moment correlation coefficient of 0.77 (p < 0.01). There was no correlation between the degree of glomerular hypercellularity and IgG antinucleosome levels (Pearson product moment correlation coefficient of 0.40, p = NS).

IFN-{gamma}-associated inflammatory skin disease, lymphadenopathy, and splenomegaly do not depend on the presence of {alpha}ß or {gamma}{delta} T cells

All IFN-{gamma} transgenic {delta}-/- and ß-/- mice developed the skin phenotype previously described in association with IFN-{gamma} overexpression in the epidermis (10). Early in the neonatal period T cell deficient transgenic animals exhibited marked hypopigmentation of the hair. A proportion of these mice developed hair loss, cutaneous erythema, and flaking, all of which were particularly marked around the limb joints. Histological examination of skin from both {delta}-/- and ß-/- IFN-{gamma} transgenics demonstrated a range in the severity of lesions from focal spongiosis in the epidermis with dermal inflammation to epidermal hyperplasia associated with hyperproliferation and a predominantly mononuclear, dermal inflammatory infiltrate as previously described (10).

On internal examination, gross peripheral lymphadenopathy and splenomegaly were obvious in the majority of IFN-{gamma} transgenics irrespective of T cell status. Detailed histological analysis of the lymph nodes and spleen from one of the ß-/- IFN-{gamma} transgenics showed a complete absence of T cell zones and germinal centers (Fig. 3Go). We have previously shown that superficial lymph nodes in IFN-{gamma} transgenics contain markedly elevated numbers of dendritic cells, consistent with migration of these APCs from the skin to draining nodes (10). This phenomenon persisted in ß-/- IFN-{gamma} transgenics. On FACS analysis of grossly enlarged lymph nodes from ß-/- mice, cells positive for F4/80 and CD11c comprised 4.9% of all nonerythrocyte cells present compared with 1.5% in lymph nodes from littermates negative for the transgene (data not shown).



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  		FIGURE 3. Absence of T cell regions and germinal centers in IFN-{gamma} transgenic, ß-/- mice. Spleens from IFN-{gamma} transgenic, ß+/- (A and C) or ß-/- (B and D) mice were stained for B (anti-B220) and T cells (anti-CD3) (A and B) or germinal centers (peanut agglutinin) (C and D). A, Distinct T cell regions (red, marked T) were seen within B cell regions (blue, marked B) in ß+/- spleens, but were absent in ß-/- spleens (B). C, Germinal centers were present in ß+/- spleens (arrow), but in D were absent from ß-/- spleens. (Scale bar: A–D = 500 µm).

 
There is evidence that congenital absence of T cells may result in impaired B cell development associated with loss of B cell CD23 expression (21). We found no evidence of such a phenomenon in ß-/- IFN-{gamma} transgenics. B cells from lymph nodes and spleen of ß-/-, ß+/- IFN-{gamma} transgenics and littermates negative for the transgene showed comparable levels of CD23 expression (Fig. 4Go).



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  		FIGURE 4. CD23 expression in IFN-{gamma} transgenic mice. CD23 cell surface expression on splenocytes (A) and lymph node cells (B) of IFN-{gamma} transgenic, ß+/-, or ß-/- mice and littermates negative for the transgene (WT) was investigated by FACS analysis using standard forward vs side scatter lymphocyte gates. There was a significantly greater proportion of both splenocytes (A) and lymph node cells (B) expressing CD23 in ß-/- compared with ß+/- mice. There were no differences in cell surface expression as estimated by the mean fluorescence channel (MFC) of CD23+ cells.

 
The skin of IFN-{gamma} transgenic mice contains foci of apoptotic cells

The dependency of antinuclear autoantibody production on intact {alpha}ß T cell function suggested that the generation of these Abs is an Ag-driven process and raises the question of the source of Ag. Apoptotic keratinocytes have been suggested as the source of self nuclear Ags in patients with SLE (22). In view of this, we examined hematoxylin and eosin-stained skin sections from IFN-{gamma} transgenic mice for the presence of apoptotic nuclei. Abnormal clusters of apoptotic cells were seen in the epidermis of these animals (Fig. 5Go, A and B). These clusters were particularly common in the hair follicles (Fig. 5GoA). Apoptotic nuclei were observed in the skin of mice expressing the IFN-{gamma} transgene, regardless of the T cell status of the animals. TUNEL-positive cells were present in the hair follicles and interfollicular epidermis in IFN-{gamma} transgenic mice. The distribution of these positive cells was patchy (the number of positive cells in IFN-{gamma} transgenic interfollicular epidermis varied from 0 to 3 per section, compared with 0 in all sections fields examined from nontransgenic littermates). In addition, regions of the dermis in these animals contained large quantities of TUNEL-positive material (Fig. 5Go, C and D), which often had the appearance of having been phagocytosed (Fig. 5GoD). No TUNEL-positive cells or material was seen in the interfollicular epidermis or dermis of littermates negative for the transgene (Fig. 5GoF). Occasional hair follicles from transgene-negative animals contained TUNEL-positive cells, but this is not unexpected as apoptosis may play a central role in hair follicle regression (catagen) (data not shown) (23).



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  		FIGURE 5. Apoptotic nuclei and TUNEL-positive cells in the skin of IFN-{gamma} transgenic mice (A and B). Shown are hematoxylin and eosin-stained sections of hair follicle (A) and interfollicular epidermis (B) in a transgenic mouse. Arrows indicate apoptotic nuclei. C–F, TUNEL labeling of IFN-{gamma} transgenic (C–E) and littermate nontransgenic (F) skin. Note the TUNEL-positive material in the hair follicle, interfollicular epidermis, and dermis of IFN-{gamma} transgenic skin (C, arrows). Much of the TUNEL-positive material in the dermis appeared to have been phagocytosed (D, arrow). TUNEL-positive interfollicular basal cell is shown in E. No TUNEL-positive cells or material are present in skin from littermate negative for the transgene (F). (Scale bars: A, B, D, and E = 180 µm; C and F = 50 µm).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Targeting an IFN-{gamma} transgene to the suprabasal epidermal layers via the involucrin promoter leads to a lupus-like syndrome in transgenic mice. The limited range of autoantibodies detected in these mice suggested that specific, perhaps Ag-driven, rather than polyclonal B cell activation is involved in their generation (11). We have shown that the production of pathogenic IgG antinuclear autoantibodies and end organ disease in this model are critically dependent on the presence of {alpha}ß T cells. This observation is consistent with Ag-driven, {alpha}ß T cell activation of specific autoantibody producing B cells.

An alternative explanation is that B cell development is abnormal in {alpha}ß T cell-deficient animals since it has been shown that in thymectomized mice complete absence of T cells results in impaired B cell development and defective T-B cell interactions (21, 24). There is evidence that this phenomenon is secondary to a marked reduction in B cell CD23 expression (21). However, this mechanism is unlikely to be playing a role in ß-/- IFN-{gamma} transgenic mice for a number of reasons. ß-/- IFN-{gamma} transgenics are not completely T cell deficient, and the available evidence suggests that {gamma}{delta} T cells can substitute for {alpha}ß T cells in driving B cell development (25). In support of this contention, B cell CD23 expression in ß-/- IFN-{gamma} transgenics was comparable to that in ß+/- IFN-{gamma} transgenics (Fig. 4Go).

IFN-{gamma} transgenic mice mirror many of the clinical and pathological findings of human SLE. Greatly increased susceptibility to disease in females, variable penetrance of glomerulonephritis, and the correlation of disease severity with anti-dsDNA Ab titers are central features of the naturally occurring human disease (1, 26). Furthermore, IFN-{gamma}-associated skin disease in the mice displays many abnormalities characteristic of acute cutaneous lupus erythematosus (ACLE) in humans: keratinocyte MHC and ICAM-1 expression, loss of epidermal dendritic cells, dermal mononuclear infiltrate, hydropic degeneration of basal cells, and alopecia associated with apoptotic bodies in the hair follicles (Refs. 10, 27, and 28; Fig. 5Go). Our demonstration of an absolute requirement for {alpha}ß T cells in the pathogenesis of IFN-{gamma}-induced murine lupus further emphasizes the relevance of this model to SLE as the available evidence implies a central role for this T cell subset in the human disease.

Our findings are also in keeping with the results of several lines of investigation demonstrating an essential role for CD4+ {alpha}ß T cells in the pathogenesis of lupus in murine models of the disease (5, 6, 7). However, our results contrast with the data of Peng et al. (29), who showed that MRL/lpr mice deficient in {alpha}ß T cells develop murine lupus with antinuclear autoantibodies and immune complex renal disease. They postulated that {gamma}{delta} T cells could substitute for {alpha}ß T cells in autoantibody generation and mediation of end organ damage. We found no evidence of such a phenomenon, as ß-/- IFN-{gamma} transgenic mice developed none of the extra-cutaneous features of murine lupus.

The contrasting findings may reflect differences in the two murine lupus models. For example, B cell function is known to be intrinsically abnormal in MRL/lpr mice with polyclonal activation possibly rendering Ab secretion less dependent on T cell help (30, 31). It has been argued that pathogenic antinuclear Abs arise by a two-stage process in the MRL/lpr model. In the first stage, T cell-independent polyclonal B cell activation initially results in production of low affinity IgM autoantibodies. In the second stage, an {alpha}ß T cell dependent process results in a class switch to high affinity IgG autoantibody production (3, 32). We could find no evidence of a T cell-independent stage in IFN-{gamma} transgenic mice, as we could not demonstrate autoantibodies of any class in {alpha}ß T cell-deficient transgenics up to an age of 6 mo, at which time TCR-{alpha}ß-deficient MRL/lpr mice have already developed them (29).

It has also been shown that mice congenitally deficient in {alpha}ß T cells due to a deletion mutation in the TCR{alpha} subunit spontaneously develop a lupus-like syndrome (32). However, it should be noted that the presence of {alpha}-/ß+ T cells in the periphery in these animals may act as an alternative source of T cell help (33). This T cell subset is not present in ß-/- mice, and the two lines of mice are, therefore, not directly comparable.

The presence of anti-dsDNA Abs seems to be a prerequisite for the development of severe renal disease in IFN-{gamma} transgenic mice. The importance of these autoantibodies in the pathogenesis of renal disease in IFN-{gamma} transgenics is supported by the correlation between the severity of glomerular hypercellularity and IgG anti-dsDNA Ab levels in the three groups of animals with intact {alpha}ß T cell function (ß+/-, {delta}+/-, {delta}-/-). It seems likely that loss of these autoantibodies is a key factor in the absence of renal disease in ß-/- IFN-{gamma} transgenics. Nevertheless, there is strong evidence that antinucleosome Abs are capable of inducing lupus nephritis (34, 35) and nucleosome-specific Th cells have been shown to play a key role in triggering nephritis in murine lupus (36). The mechanism by which antinucleosome Abs cause glomerulonephritis is controversial but may involve deposition of nucleosome/antinucleosome Ab complexes in the glomerular basement membrane relatively early in the disease (37). Therefore, loss of this reactivity in ß-/- transgenics may be important in the abolition of renal disease. However, it is of interest to note that the two ß+/- IFN-{gamma} transgenics with IgG antinucleosome restricted autoantibodies did not show definite histological evidence of kidney disease, although it is possible that lesions might have developed with age (nos. 4 and 6, Table IIGo). The development of proliferative glomerulonephritis in the absence of IgG antihistone Abs (e.g., mice nos. 4, 7, 11, 16, and 19, Table IGo) demonstrates that antihistone autoantibodies do not play an essential role in the pathogenesis of lupus nephritis.

It has been reported that deficiency of {gamma}{delta} T cells in MRL/lpr mice results in a rise in anti-dsDNA titers and worsening of renal disease (29). In the present study there also appeared to be a trend toward more severe renal disease in {delta}-/- IFN-{gamma} transgenics (Table IGo). In contrast to {delta}-/- MRL/lpr mice, this phenomenon was observed in the absence of any significant rise in anti-dsDNA levels, thus raising questions about the mechanisms involved. There is evidence that CD4+ T cells play a role in triggering glomerular hyperproliferation and crescent formation in animal models of glomerulonephritis (38). In addition, several studies suggest that {gamma}{delta} T cells antagonize {alpha}ß T cell function, resulting in reduced tissue injury in both autoimmune and pathogen-induced inflammation (39, 40, 41, 42). Therefore, it is conceivable that the possible worsening of renal disease in {delta}-/- IFN-{gamma} transgenic mice results from loss of a direct inhibitory effect of {gamma}{delta} T cells on glomerular damage.

The occurrence of IFN-{gamma}-associated inflammatory skin disease, lymphadenopathy, and splenomegaly in the absence of systemic complications of the disease indicates that these processes are separable in our lupus model. Although uncommon, ACLE can occur in the absence of systemic autoimmune disease in humans (28), indicating a further parallel between the transgenic model and the human disease.

There is evidence that germinal centers can form in ß-/- mice in response to infectious agents (43). We could not demonstrate germinal centers in lymph nodes or spleen from ß-/- transgenics, although we did not carry out immunizations in our mice, and T cell zones were completely absent. We have previously shown that lymph node enlargement in IFN-{gamma} transgenics is associated with a marked increase in dendritic cell content (10). This phenomenon persisted in ß-/- transgenics, which is at least consistent with IFN-{gamma}-induced migration of dendritic cells from the skin to the draining lymph nodes independent of T cell function.

The observation that exposure of the skin of patients with SLE to UV light can produce marked exacerbation of the systemic disease implicates the epidermis as the source of self Ag in the generation of pathogenic antinuclear Abs (44). The nature of the APCs and Ags involved in this process remains to be determined. As in ACLE, keratinocytes in IFN-{gamma} transgenics express both MHC class II and ICAM-1 (10). However, we think it is unlikely that keratinocytes act directly as APCs in these animals as they do not express the costimulatory molecule B7 and T cell infiltration of the epidermis is not a major feature of the skin disease (10). Furthermore, it has been shown that Ag presentation by keratinocytes in vitro induces tolerance in Ag-specific T cells (45). Immunohistochemical data suggest that migration of Langerhans cells from the epidermis to draining lymph nodes is a major feature of the inflammatory skin disease in IFN-{gamma} transgenics (10). It is reasonable to suggest that, in IFN-{gamma} transgenic mice, autoantigens from the skin are taken up by Langerhans cells and presented to Ag-specific autoreactive {alpha}ß T cells in the draining lymph nodes with consequent stimulation of antinuclear Ab producing B cells. The demonstration of apoptotic cells in the epidermis and TUNEL-positive material in the dermis of IFN-{gamma} transgenics suggests a possible source of autoantigen.

IFN-{gamma} is known to induce keratinocyte apoptosis, possibly via facilitation of Fas-Fas ligand interactions (46). Apoptotic cells in epithelia are known to be rapidly phagocytosed by macrophages, and we have previously shown that the majority of infiltrating cells in the dermis of IFN-{gamma} transgenics are of the macrophage lineage (10, 47). Therefore, the demonstration of large quantities of TUNEL-positive material in the dermis may reflect phagocytosis of apoptotic material from the overlying epidermis. That components of apoptotic cells can act as Ags in the generation of antinuclear Abs is supported by several lines of evidence. Systemic injection of apoptotic nuclei has been shown to result in antinuclear autoantibody production in mice (48). Furthermore, Ags contained in surface blebs of apoptotic keratinocytes have been implicated as the source of self nuclear Ags in patients with SLE-complicating C1q deficiency (49). Keratinocyte Fas expression and apoptotic cells can be demonstrated in lesional skin from patients with SLE, occurring most commonly in the hair follicles (27). Nucleosomes are released from apoptotic cells, and it has been shown that nucleosome-restricted Abs are the first to emerge during the course of murine lupus, suggesting that the nucleosome may be the initial driving immunogen in the lupus autoimmune response (18, 50). The demonstration of nucleosome restricted Abs in IFN-{gamma} transgenics is therefore at least in keeping with the concept of apoptotic cells as a source of self Ag in these animals.

Our findings are consistent with a specific {alpha}ß T cell-mediated, Ag-driven process giving rise to pathogenic IgG antinucleosome, anti-dsDNA, and antihistone autoantibodies in IFN-{gamma} transgenic mice. This fact, combined with a clear candidate anatomical location for the process, may allow identification of the Ags involved in generating this autoimmune response. The IFN-{gamma} transgenic lupus model mirrors many features of the naturally occurring human disease both in terms of clinical findings and pathogenesis. This model may, therefore, prove valuable for studying the effects of both immunotherapies and anti-apoptotic therapies aimed at SLE.


   Acknowledgments
 
We thank Dr. Sophie Koutouzov (Hopital Necker, Paris, France) for carrying out the antinucleosome ELISA and for helpful comments. We thank the Imperial Cancer Research Fund Histopathology Unit and Transgenic Facility for excellent technical assistance, and Dr. Ernst Kriehuber (Department of Dermatology, Vienna General Hospital) for useful discussions and ideas.


   Footnotes
 
1 J.P.S. was supported by funds from a European Union Biomed Network. E.C.Y.W. was funded by the Beit Memorial Foundation for Medical Research. Back

2 J.P.S. and E.C.Y.W. contributed equally to this work. Back

3 Current address: Genetics Institute, Andover, MA 01810. Back

4 Address correspondence and reprint requests to Dr. Fiona M. Watt, Keratinocyte Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, U.K. E-mail address: Back

5 Abbreviations used in this paper: SLE, systemic lupus erythematosus; ACLE, acute cutaneous lupus erythematosus. Back

Received for publication November 10, 1998. Accepted for publication April 7, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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