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Costimulation by B7-1 and B7-2 Is Required for Autoimmune Disease in MRL-Fas^lpr Mice¹

Koji Kinoshita^*, Greg Tesch^*, Andreas Schwarting^*, Ruth Maron, Arlene H. Sharpe and Vicki Rubin Kelley^2,*

^* Laboratory of Molecular Autoimmune Disease, Renal Division, Department of Medicine, Center for Neurological Disease, and Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Autoimmune lupus nephritis is dependent on infiltrating autoreactive leukocytes and Igs. B7 costimulatory molecules (B7-1 and B7-2) provide signals essential for T cell activation and Ig class switching. In MRL-Fas^lpr mice, a model of human lupus, although multiple tissues are targeted for autoimmune injury, nephritis is fatal. We identified intrarenal B7-1 and B7-2 expression, restricted to kidney-infiltrating leukocytes, before and increasing with progressive nephritis in MRL-Fas^lpr mice. Thus, we hypothesized that the B7 pathway is required for autoimmune disease in MRL-Fas^lpr mice. To investigate the role of B7 costimulatory molecules in this autoimmune disease, we generated a MRL-Fas^lpr strain deficient in B7-1 and B7-2. Strikingly, MRL-Fas^lpr mice lacking both B7 costimulators do not develop kidney (glomerular, tubular, interstitial, vascular) pathology, or proteinuria, and survive far longer. Intrarenal downstream effector transcripts (IFN-, IL-12, monocyte chemoattractant protein-1, CSF-1) linked to nephritis remained at normal levels compared with wild-type mice. Skin lesions and lymphoid enlargement characteristic of MRL-Fas^lpr mice were diminished in B7-1/B7-2-deficient MRL-Fas^lpr mice. B7-1/B7-2-deficient MRL-Fas^lpr mice did not develop leukocytic infiltrates, elevated serum IgG and isotypes (G1,G2b,G3), autoantibodies, and intrarenal IgG deposits. Our findings demonstrate that B7-1 and B7-2 costimulatory pathways are critical to the pathogenesis of autoimmune lupus.

	Introduction

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MRL-Fas^lprmice develop a systemic autoimmune disease sharing features with human lupus (1). Although pathology is evident in the skin, lungs, salivary and mandibular glands, and joints, kidney disease is the usual cause of death (2). Massive lymphadenopathy and splenomegaly result from the trafficking and accumulation of T cells within these lymphoid tissues, and T cell infiltrates are prominent in most vital tissues. Furthermore, there are notably high levels of circulating Igs, including a multitude of autoantibodies. The rapid tempo (50% mortality at 5.5 mo of age) and the predictability of these pathologic hallmarks makes this an attractive model in which to study the pathogenesis of autoimmune disease.

Kidney disease in the MRL-Fas^lpr mouse is complex (3, 4). Renal pathology involves glomerular, tubular, interstitial, and vascular components. Each component is infiltrated by leukocytes, including T cells and macrophages. The T cells that accumulate in the kidney include CD4, CD8, and CD4^-, CD8^-, B220 double negative (DN)³ T cells. Based on evidence using "knockouts" that deplete these T cell populations, it is clear that T cells are required for autoimmune disease (5, 6, 7). The T cell-mediated mechanism responsible for inciting kidney disease requires IFN- (8). IFN-, released from activated T cells within the kidney, triggers a cascade of cytokines (IL-12, CSF-1, TNF-) culminating in tissue destruction (8, 9, 10, 11, 12). Thus, identifying the signals responsible for T cell activation and clonal expansion within tissues, such as the kidney, offers potential therapeutic targets for combating autoimmune tissue destruction.

The B7 family of costimulatory molecules, B7-1 (CD80) and B7-2 (CD86), regulates T cell activation, differentiation, and peripheral tolerance. The B7-1 and B7-2 costimulators have dual specificity for their ligands CD28 and CTL-associated molecule-4 (CTLA-4). The B7-CD28 interactions promote T cell growth, survival, and differentiation, while B7-CTLA-4 interactions provide a down-regulatory signal for T cell activation with the potential for regulating autoreactive T cells in the periphery (13). The kinetics of B7-1 and B7-2 expression are distinct: B7-2 is constitutively expressed at low levels on APC (i.e., macrophages, dendritic cells, and B cells) and is rapidly up-regulated on T cells and APCs in response to cytokines, activation signals, and infections. B7-1 expression is induced on activated APCs and T cells and is up-regulated later than B7-2. B7-1 and B7-2 have critical overlapping functions in regulating T cell cytokines and B cell Igs (14, 15). This overlap in function suggests that blocking both B7 costimulators (B7-1 and B7-2) may be a potential therapeutic strategy for progressive autoimmune diseases.

Blockade of the B7 pathway using receptor antagonists and Abs has provided insight into the importance of these costimulatory molecules in autoimmune disease (16, 17, 18, 19). B7 costimulation may contribute to the development of systemic autoimmune disease in the MRL-Fas^lpr mouse in several ways. B7 costimulation may be critical for activation of self-reactive T cells and the production of pathogenic cytokines and chemokines. B7 costimulation may also be essential for generating autoantibodies because B7 costimulators have an obligatory role in Ig class switching and germinal center formation (15). In this report, we investigate the importance of B7 costimulation in the pathogenesis of autoimmune disease in MRL-Fas^lpr mice through the generation and analysis of a B7-1/B7-2-deficient (-/-) MRL-Fas^lpr mouse strain. We investigated whether 1) B7-1/B7-2 is necessary for progressive, fatal autoimmune disease; 2) B7-1/B7-2 molecules mediate pathology in the kidney, lung, liver, skin, lymph nodes, and spleen; 3) B7-1/B7-2 expression is required for T cell infiltration into the kidney and the other tissues targeted for destruction; and 4) B7-1/B7-2 molecules are required for the production of nephritogenic Igs.

We now report that B7 costimulatory molecules provide essential and proximal signals that are required for the development of autoimmune disease in MRL-Fas^lpr mice. B7-1 and B7-2 expressed by kidney-infiltrating leukocytes are detected before overt pathology and increase with progessive nephritis. This intrarenal B7 expression is critical for the activation of self-reactive T cells because B7-1/B7-2-deficient mice were entirely protected from fatal kidney disease. In the absence of B7 molecules, leukocytes did not accumulate in kidney and lungs and were reduced in lymphoid tissues. In addition, B7-1/B7-2-deficient MRL-Fas^lpr mice did not develop elevated IgG isotypes, nor autoantibodies characteristic of MRL-Fas^lpr mice. Thus, the B7 costimulatory pathways are critical in the pathogenesis of autoimmune lupus.

	Materials and Methods

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Mice

MRL/MpJ-Fas^lpr/Fas^lpr (MRL-Fas^lpr), MRL/MpJ-++ (MRL-++), and C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B7-1/B7-2-deficient mice (129/SvS4Jae) were constructed as previously reported and maintained in our pathogen-free animal facility (15).

Generating B7-1/B7-2-deficient MRL-Fas^lpr mice

We constructed a B7-1/B7-2-deficient MRL-Fas^lpr strain using a backcross-intercross breeding scheme (8). MRL-Fas^lpr mice were mated with B7-1/B7-2-deficient mice to yield heterozygous F₁ offspring. We intercrossed F₁ mice and screened the progeny for the Fas^lpr (+/+) mutation and B7-1/B7-2 genes the progeny using tail genomic DNA amplified by PCR and identified with specific oligonucleotide primers. Specifically, we extracted DNA from mouse tails using the QIAmp tissue kit (Qiagen, Hilden, Germany). Tail DNA was assessed with primers recognizing the B7.1 gene (antisense, 5'-TGT GCC CCG GTC TGA AAG GAC-3'; sense, 5'-TGA ACA ACT GTC CAA GTC AGT-3') and B7.2 gene (antisense, 5'-CGA TCA CTG ACA GTT CTG TTA-3'; sense, 5'-ACA TAA GCC TGA GTG AGC TG-3') (15). The Fas^lpr mutation was identified as previously reported (20). The progeny were backcrossed with the MRL-Fas^lpr strain. After three generations of backcross-intercross matings, we generated B7-1/B7-2^-/- MRL-Fas^lpr and B7-1/B7-2^+/- MRL-Fas^lpr/lpr strains (94% MRL background). We analyzed B7-1/B7-2-deficient and intact MRL-Fas^lpr strains at three generations of backcross-intercross matings, because we have established that the tempo and expression of autoimmune disease and survival is similar to the wild-type MRL-Fas^lpr strain by the third backcross generation (8, 47).⁴ This was verified by comparing B7-1/B7-2^+/+ wild-type and B7-1B7-2^+/- intact MRL-Fas^lpr mice on the third backcross generation for survival, kidney disease, and autoantibodies (see below). The B7-1/B7-2-deficient and intact MRL-Fas^lpr of the third generation are referred to hereafter as B7-1/B7-2^-/- MRL-Fas^lpr and B7-1/B7-2^+/- MRL-Fas^lpr, respectively.

Proteinuria

We assessed urinary protein levels monthly using albumin reagent strips (Albustix; Bayer Diagnostic Division, Elkhart, IN), and graded them semiquantitatively (0, none; 1, 30–100 mg/dl; 2, 100–300 mg/dl; 3, 300-1000 mg/dl; 4, >1000 mg/dl). Each monthly value was determined by sampling and measuring urine on sequential days. In the event that these values differed (>5%), we repeated this process.

Gross pathology

We scored gross skin pathology in B7-1/B7-2^-/- and B7-1/B7-2^+/- MRL-Fas^lpr mice monthly. We evaluated lymphadenopathy comparing B7-1/B7-2^-/- and B7-1/B7-2^+/- MRL-Fas^lpr mice monthly using a scale of 0–3 assessing the number and size of palpable nodes (0, none; 1, one, small; 2, two, small to moderate; 3, three or more, moderate to large). Skin lesions, which consist of alopecia and scab formation, were scored from 0 to 3 based on the number of lesions and area (0, none; 1, one, <0.5 cm; 2, two or more, <0.5 cm; 3, multiple, >0.5 cm). Spleen enlargement was assessed at the time of sacrifice or death. We compared the spleen weights in the B7-1/B7-2^-/- and B7-1/B7-2^+/- MRL-Fas^lpr mice.

Histopathology

Kidneys were either snap-frozen in OCT compound (Miles Scientific, Naperville, IL) for cryostat sectioning or fixed in 10% neutral-buffered formalin. Formalin-fixed tissue was embedded in paraffin, and 4-µm sections were stained with hematoxylin and periodic acid Schiffs and evaluated by light microscopy (21). We evaluated the glomerular, tubular, interstitial, and perivascular pathology morphometically. The glomeruli were assessed by counting 50 glomerular cross-sections (gcs) per kidney and scoring each glomerulus on a semiquantitative scale: 0, normal (35–40 cells/gcs); 1, mild (few lesions with slight proliferative changes and hypercellularity) (41–50 cells/gcs); 2, moderate (moderate hypercellularity) (51–60 cells/gcs) segmental and/or diffuse proliferative changes, hyalinosis, and moderate exudate; 3, severe hypercellularity (>60 cell/gcs) with segmental or global sclerosis and/or severe necrosis, crescent formation, and heavy exudation. We evaluated tubular pathology by counting the percentage tubules that were damaged (dilation and/or atrophy and/or necrosis in 200 randomly selected tubules (magnification, x400). We evaluated the interstitial pathology by counting the number of infiltrating cells in 20 random interstitial fields (magnification, x400). The perivascular cell accumulation was determined by scoring the number of cell layers surrounding 10 random inter and intralobular arteries (score: 0, none; 1, <5 layers surrounding more than half of the vessel; 2, 5–10 layers surrounding less than half of the vessel; 3, >10 layers surround less than half the vessel). Scoring was evaluated using coded slides.

Lungs were fixed in formalin, sectioned (4 µm), stained with hematoxylin and eosin, and evaluated by light microscopy (22). The perivascular leukocyte infiltration was determined using a morphometric analysis. We measured the leukocyte infiltrates surrounding 10 random vessels (score: 0, none; 1, less than three layers surrounding <50%; 2, three to six cell layers surrounding >50%; 3, more than six layers). Peribronchiolar leukocyte infiltration was determined by semiquantitative scoring the cells surrounding 10 random bronchi (score: 0, none; 1, less than three layers surrounding >50% bronchi; 2, three to six cell layers surrounding >50% bronchi; 3, more than six layers surrounding >50% bronchi).

Identifying B7-1/B7-2 in kidney sections

To evaluate the presence of B7-1 and B7-2, cryostat sectioned kidneys (4 µm) were fixed in ice-cold acetone for 10 min. We detected B7-1 and B7-2 in kidney sections by an indirect immunoperoxidase procedure using hamster anti-mouse B7-1 Ab (PharMingen, San Diego, CA) (5 µg/ml) and rat anti-mouse B7-2 Ab (PharMingen) (5 µg/ml) as previously described (23). The specificity of the B7-1 and B7-2 Abs has been previously established (24). As negative controls we used B7-1/B7-2^-/- kidneys and nonreactive Abs consisting of polyclonal hamster IgG (PharMingen) for B7-1 and rat anti-mouse IgG2a (PharMingen) for B7-2.

Identifying kidney leukocytic infiltrates

Cryostat-sectioned kidneys were stained for the presence of 1) macrophages with F4/80 hybridoma culture supernatant (HB198; American Type Culture Collection, Manassas, VA); 2) T cells with Abs to CD4, CD8, and B220 Ags (PharMingen); and 3) B cells with Abs to CD21/35 rat anti-mouse mAb (PharMingen) using immunoperoxidase procedures. We replaced the primary Ab with normal rat IgG as a specificity control. Macrophages and T cells within the kidney were enumerated and reported as cells/glomerulus or cells/interstitial field as previously described (8).

Identifying B7-1/B7-2 and cytokine/chemokine transcripts in the kidney

Total RNA was extracted from the snap-frozen renal cortex of half a kidney using RNAzol B (Tel-Test, Friendswood, TX). A reverse transcription (RT) reaction was performed on this RNA using oligo(dT) and the Superscript II DNA preamplification kit (Life Technologies, Grand Island, NY). The resulting RT product was used as a cDNA template for PCR with analysis. B7-1/B7-2 transcripts were measured by semiquantitative RT-PCR (15). Similarly, IFN-, macrophage chemoattractant protein (MCP)-1, IL-12, and CSF-1 expression were detected as 400-, 350-, 350-, and 245-bp PCR products, respectively (8, 11, 25, 26). GAPDH expression, the housekeeping gene, was detected as a 500-bp product resulting from PCR using specific oligonucleotide primers.

B7-1/B7-2 in tubular epithelial cell (TEC)

To determine whether TEC express B7-1/B7-2, TEC were isolated from MRL-Fas^lpr and MRL-++ mice as previously described (27). TEC were grown on collagen-coated plates, cultured for 5–10 passages, and stimulated with IFN- (50 U/ml) or LPS (5 µg/ml) for 24 h. Total RNA was isolated from stimulated and unstimulated TEC, and B7-1/B7-2 transcripts were measured by semiquantitative RT-PCR according to previously reported methods (15).

Serum Igs

To assess the serum Igs, we collected serum from B7-1/B7-2-deficient and intact MRL-Fas^lpr mice at 5 mo of age (n = 8/group). We used an ELISA to measure total IgG and IgM and IgG isotypes including IgG1, IgG2a, IgG2b, and IgG3. Plates coated with goat anti-mouse Ig Ab (Southern Biotechnology Associates, Birmingham, AL) were developed with alkaline phosphatase-conjugated isotype-specific anti-Ig Abs (Southern Biotechnology Associates). We diluted the sera from 1:100 to 1:72,900 and analyzed Ig isotype concentrations using standard curves generated with Mouse Standard Panel (Southern Biotechnology Associates).

Serum antinuclear Abs (ANA)

ANA were detected using the Hep2 cell ANA kit (The Binding Site, Birmingham, U.K.). Briefly, serum samples (1:40 dilution) were incubated with the Hep2 substrate, washed, and incubated with fluorescein-conjugated anti-mouse IgG (Cappel, PA) (1: 100 dilution). ANA were analyzed using a fluorescence microscope and scored for intensity on a scale of 0–4 (0, none; 1, weak; 2, moderate; 3, strong; 4, very strong).

IgG deposits within renal glomeruli

Kidney cryostat cross-sections (4 µ thick) were incubated with fluorescein anti-mouse IgG (ICN Biomedical, Costa Mesa, CA) and titered at dilutions of 1:1000, 1:5000, and 1:25000. Slides were analyzed using a fluorescence microscope at the lowest positive dilution (1:1000). The fluorescence intensity within the glomerular capillary walls were scored on a scale of 0–4 (0, none; 1, weak; 2, moderate; 3, strong). At least 10 glomeruli per section were analyzed. Values are recorded as mean/group ± SD.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
B7-1/B7-2 is expressed before renal injury in wild-type MRL-Fas^lpr kidneys and rises with advancing disease

To determine whether B7-1 and B7-2 were up-regulated in wild-type MRL-Fas^lpr kidneys, we evaluated MRL-Fas^lpr mice during progressive renal injury (2, 4, and 6 mo of age). B7-1 and B7-2 transcripts were expressed before nephritis in MRL-Fas^lpr mice. In contrast, neither B7-1 nor B7-2 were detectable in age- and sex-matched normal C57BL/6 kidneys (Fig. 1A). B7-1 and B7-2 transcripts continued to increase in MRL-Fas^lpr mice with advancing age (Fig. 1A). Because the severity of renal pathology and loss of function in MRL-Fas^lpr mice progressively increases from 2 to 6 mo of age, we conclude that B7-1 and B7-2 transcripts increased in proportion to the severity of kidney disease (28). In addition, B7-1 and B7-2 proteins increased within the MRL-Fas^lpr kidneys at 4 and 6 mo of age, paralleling the rise in B7-1 and B7-2 transcripts (Fig. 1B). In contrast, normal C57BL/6 kidneys did not express B7-1/B7-2 protein (Fig. 1B). Using immunostaining, we localized B7-1 and B7-2 protein expression to glomerular (intra and peri), interstitial, and perivascular areas (Fig. 1C). B7-1 and B7-2 proteins were notably absent in kidney intrinsic cells, including TEC in MRL-Fas^lpr mice (immunostaining). Furthermore, B7-1/B7-2 transcripts were not detected in isolated unstimulated or stimulated (LPS or IFN-) TEC derived from MRL-Fas^lpr and congenic MRL-++ kidneys using RT-PCR (Table I). It should be noted that B7-1 and B7-2 were not detected within the kidneys of the B7-1/B7-2^-/- MRL-Fas^lpr kidneys (negative control), nor using control Abs for B7-1 and B7-2, hamster anti-IgG and rat anti-mouse IgG2a, respectively. Thus, we concluded that the B7-1 and B7-2 in MRL-Fas^lpr kidneys is restricted to kidney-infiltrating leukocytes.

View larger version (70K): [in this window] [in a new window]   FIGURE 1. B7-1 and B7-2 costimulatory molecules are up-regulated during the progressive kidney disease in MRL-Faslpr mice. A, B7-1 and B7-2 transcripts were increased in MRL-Faslpr mice at 2, 4, and 6 mo of age and were increased as compared with C57BL/6 strain as determined by RT-PCR (*, p < 0.001; **, p < 0.05; n = 3/group). Values are mean ± SD. B, B7-1 and B7-2 protein increased within the kidneys of MRL-Faslpr mice from 2, 4, and 6 mo of age and were increased in comparison to the C57BL/6 strain as evaluated by immunostaining (grade: 0 (none) to 3 (maximum)) (*, p < 0.001; **, p < 0.005; n = 3/group). Values are mean ± SD. C, B7-1 and B7-2 expression are most prominent in the areas surrounding glomeruli (long thick arrow), and it also was detected in the interstitium, and within glomeruli (long thin arrow) and is notably absent in TEC (arrowhead) in MRL-Faslpr kidneys (magnification, x400).

Survival in the B7-1/B7-2^-/- MRL-Fas^lpr female and male mice was dramatically extended as compared with B7-1/B7-2^+/- MRL-Fas^lpr mice (Fig. 2). The 50% mortality in the B7-1/B7-2^+/- MRL-Fas^lpr strain was 8 mo of age. In comparison, only 9% of age- and sex-matched B7-1/B7-2^-/- MRL-Fas^lpr strain were dead at this age (n = 43 and 21, respectively, p < 0.01). Similarly, the majority (72%) of B7-1/B7-2^+/- MRL-Fas^lpr mice were dead at 12 mo of age, as compared with a minority (28%) of the B7-1/B7-2^-/- MRL-Fas^lpr strain. In addition, we followed survival in a smaller group (n = 14) of B7-1/B7-2^+/+ MRL-Fas^lpr mice (third generation); survival in this group was similar to the B7-1/B7-2^+/- MRL-Fas^lpr strain (50% mortality 7.5 mo of age, data not shown). It should be noted that the wild-type MRL-Fas^lpr and 129/Sv strain 50% mortality is 5.5 mo and >20 mo of age, repectively (29, 30). Therefore, survival in the B7-1/B7-2^+/- MRL-Fas^lpr strain (third generation) is similar to the wild-type MRL-Fas^lpr strain.

View larger version (12K): [in this window] [in a new window]   FIGURE 2. B7-1/B7-2-/- MRL-Faslpr mice survive longer than B7-1/B7-2+/- MRL-Faslpr mice. At 8 mo of age, 60% of B7-1/B7-2+/- MRL-Faslpr strain (n = 43) have not survived. By comparison, nearly all (91%) of the B7-1/B7-2-/- MRL-Faslpr mice (n = 21) remained alive (p > 0.05).

B7-1/B7-2^-/- MRL-Fas^lpr mice were protected from proteinuria (Fig. 3A). The levels of urinary proteins in B7-1/B7-2^+/- MRL-Fas^lpr mice (n = 40–47/group) increased incrementally 2-fold every 2 mo between 2 and 8 mo of age (p < 0.001). By comparison, urinary proteins did not increase in the age- and sex-matched B7-1/B7-2^-/- MRL-Fas^lpr strain (n = 31–46/group) during this time frame and remained similar to normal C57BL/6 mice (n = 6/group) (Fig. 3A). In addition, the urinary protein levels in the mice just before death in the B7-1/B7-2^-/- group remained low (1.2 ± 0.5, n = 9), while these levels in the B7-1/B7-2^+/- MRL-Fas^lpr strain were substantially elevated (2.6 ± 0.7, n = 30, p < 0.001). Furthermore, the urinary protein levels in the B7-1/B7-2^-/- MRL-Fas^lpr mice that remained alive at 12–13 mo of age did not increase (1.2 ± 0.5, n = 12) as compared with earlier time points (Fig. 3A). Thus, the B7-1/B7-2^-/- MRL-Fas^lpr strain is protected from proteinuria.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Urinary proteins, skin lesions, lymphadenopathy, and splenomegaly are reduced in B7-1/B7-2-/- MRL-Faslpr mice. Urinary protein levels did not increase in B7-1/B7-2-/- MRL-Faslpr mice (n = 31–46) and remained similar to C57BL/6 normal mice (n = 6). By comparison, urinary protein levels increased progressively from 2 to 8 mo of age in the B7-1/B7-2+/- MRL-Faslpr strain (n = 40–47; *, p < 0.01). Similarly, the incidence and severity of skin lesions were two times less in B7-1/B7-2-/- (n = 31–46) vs B7-1/B7-2+/- (n = 40–47) MRL-Faslpr mice at 4, 6, and 8 mo of age (*, p < 0.05) but not normal mice (#, p < 0.001). In addition, lymphadenopathy was similarly 2-fold less in B7-1/B7-2-/- (n = 31–46) vs B7-1/B7-2+/- (n = 40–47) MRL-Faslpr mice at 4, 6, and 8 mo of age (**, p < 0.05), but was greater than normal C57BL/6 (p < 0.005). Finally, splenomegaly, determined by the spleen weight, was less in B7-1/B7-2-/- (n = 31–46) vs B7-1/B7-2+/- (n = 40–47) MRL-Faslpr strain at 5 mo of age (**, p < 0.05). However, the B7-1/B7-2-/- MRL-Faslpr spleens were larger than normal spleens (C57BL/6; #, p < 0.001; n = 4). Values are mean ± SD.

Skin lesions, lymphadenopathy, and splenomegaly are gross pathologic features characteristic of systemic autoimmune disease in MRL-Fas^lpr mice. Gross pathologic skin lesions were reduced 2-fold in B7-1/B7-2^-/- MRL-Fas^lpr mice (n = 31–46/group) as compared with B7-1/B7-2^+/- MRL-Fas^lpr mice (n = 40–47) evaluated at 4, 6, and 8 mo of age (p < 0.001) (Fig. 3B). Similarly, lymphadenopathy was 2-fold less in B7-1/B7-2^-/- MRL-Fas^lpr mice (n = 40–47/group) as compared with B7-1/B7-2^+/- MRL-Fas^lpr mice (n = 31–46) at 6 or 8 mo of age, respectively (p < 0.005) (Fig. 3C). And the spleen size was 2-fold smaller in B7-1/B7-2^-/- MRL-Fas^lpr mice (n = 8) as compared with the B7-1/B7-2^+/- MRL-Fas^lpr mice (n = 8; p < 0.05). However, B7-1/B7-2^-/- MRL-Fas^lpr mice were not totally protected from an increase in lymphocytes accumulating in the lymph nodes and spleens (histologic and flow cytometric analysis, data not shown), because these lymphoid tissues were larger than normal (C57BL/6 strain) (n = 8; p < 0.01; Fig. 3D).

B7-1/B7-2-deficient MRL-Fas^lpr are protected from renal disease

Renal disease including glomerular, tubular, and vascular pathology was entirely prevented in B7-1/B7-2^-/- MRL-Fas^lpr mice. We compared renal disease in groups of B7-1/B7-2^-/- and B7-1/B7-2^+/- MRL-Fas^lpr strains at 5 mo of age (Figs. 4A and 5). B7-1/B7-2^+/- MRL-Fas^lpr mice at 5 mo of age had 1) glomerular pathology consisting of hypercellularity, hyalinosis, and moderate exudates; 2) tubular pathology consisting of dilation, atrophy, and/or necrosis in 20% of tubules; 3) leukocytes in the interstitium (80 cells/field); and 4) leukocytes in the surrounding vessels (5–10 layers surrounding majority of vessel). By comparison, renal glomeruli, tubules, and the interstitium and vasculature in the B7-1/B7-2^-/- MRL-Fas^lpr mice remained similar to age- and sex-matched C57BL/6 mice with normal kidneys (p < 0.001; n = 8/group; Fig. 4A). Because kidney disease in MRL-Fas^lpr mice consists of an infiltration of leukocytes including macrophages (F/4/80⁺) and CD4, CD8, DN (B220⁺, CD 21/35^-) T cells, we probed for the presence of these leukocytes in these B7-1/B7-2-deficient MRL-Fas^lpr kidneys. The numbers of macrophages and T cells (CD4, CD8, and DN) in the B7-1/B7-2^+/- MRL-Fas^lpr kidney increased dramatically in the glomerular, interstitial, and perivascular areas (Fig. 4B). Strikingly, macrophages and these T cell populations did not increase within the entire kidney in B7-1/B7-2^-/- mice and remained similar to the normal C57BL/6 kidneys (p > 0.05; n = 8; Fig. 4B).

View larger version (40K): [in this window] [in a new window]   FIGURE 4. In the absence of B7-1 and B7-2, kidney disease is prevented in MRL-Faslpr mice. A, Renal pathology was evaluated by periodic acid Schiffs staining. The glomerular, tubular, interstitial, and vascular pathology were prominent in B7-1/B7-2+/- MRL-Faslpr mice at 5 mo of age (n = 8). By comparison, B7-1/B7-2-/- MRL-Faslpr mice remained similar to C57BL/6 mice (n = 4) with normal kidneys (*, p < 0.0005; **, p < 0.05; ***, p < 0.001). Protection was enduring because renal disease was not evident in the B7-1/B7-2-/- MRL-Faslpr mice at 13 mo of age; these kidneys could not be distinguished from normal C57BL/6 kidneys (n = 4/group; **, p > 0.05). Thus, the MRL-Faslpr lacking B7-1/B7-2 were spared from kidney pathology. B, B7-1/B7-2-/- MRL-Faslpr mice were protected from kidney-infiltrating macrophages and T cells. Macrophages and CD4, CD8, and DN T cells (B220+) infiltrate the periglomerular, interstitial, and perivascular areas in B7-1/B7-2+/- MRL-Faslpr kidneys. In contrast, these leukocytes did not infiltrate the MRL-Faslpr kidneys lacking B7-1/B7-2 (***, p < 0.001; n = 8/group at 5 mo of age) and remained similar to C57BL/6 kidneys (n = 4). Values are mean ± SD.

Intrarenal cytokines/chemokines that are up-regulated and promote kidney disease in MRL-Fas^lpr kidneys are not increased in B7-1/B7-2^-/- MRL-Fas^lpr mice

We previously established that CSF-1, IFN-, MCP-1, and IL-12 are up-regulated in the kidney in advance of injury and increase with progressive renal damage in MRL-Fas^lpr mice (8, 11, 31, 32).

In addition, we have established that provision of CSF-1, IFN-, or IL-12 via gene transfer fosters an influx of distinct leukocytic phenotypes that incite kidney injury (11, 32, 33), while removing IFN-R substantially dimishes kidney injury in MRL-Fas^lpr mice (8). In this study, CSF-1, IFN-, IL-12, and MCP-1 are up-regulated in the B7-1/B7-2 intact MRL-Fas^lpr kidney at 5 mo of age (Fig. 6, A–D). In contrast, CSF-1, IFN-, IL-12, and MCP-1 are not up-regulated in B7-1/B7-2^-/- MRL-Fas^lpr mice and remain similar to age- and sex-matched normal C57BL/6 kidneys (Fig. 6, A–D). Taken together, we conclude the B7-1/B7-2^-/- MRL-Fas^lpr mice are protected from kidney disease mediated by nephritogenic cytokines.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. Nephritogenic cytokine transcripts including IFN-, IL-12, MCP-1, and CSF transcripts are not up-regulated in B7-1/B7-2-/- kidneys as compared with B7-1/B7-2+/- MRL-Faslpr kidneys. A, IFN- is reduced in B7-1/B7-2-/- MRL-Faslpr kidney as compared with B7-1/B7-2+/- MRL-Faslpr kidney measured by semiquantitative RT-PCR (n = 8/group; 5 mo of age; *, p < 0.005). B, IL-12 is reduced in B7-1/B7-2-/- MRL-Faslpr kidney as compared with B7-1/B7-2+/- MRL-Faslpr kidney measured by semiquantitative RT-PCR (n = 8/group; 5 mo of age; **, p < 0.01). C, MCP-1 is reduced in B7-1/B7-2-/- MRL-Faslpr kidney as compared with B7-1/B7-2+/- MRL-Faslpr kidney measured by semiquantitative RT-PCR (n = 8/group; 5 mo of age; ***, p < 0.001). D, CSF-1 is reduced in B7-1/B7-2-/- MRL-Faslpr kidney as compared with B7-1/B7-2+/- MRL-Faslpr kidney measured by semiquantitative RT-PCR (n = 8/group; 5 mo of age; **, p < 0.01). The transcript level of these nephritogenic cytokines/chemokines remained similar to normal age- and sex-matched (C57BL/6, n = 3) kidneys. Values are mean ± SD.

In addition to the kidney, the lungs in MRL-Fas^lpr have a progressive influx of leukocytes surrounding the vasculature. Similar to the kidney, B7-1/B7-2^-/- MRL-Fas^lpr mice did not have an accumulation of leukocytes in the perivascular, nor peribronchiolar areas at 5 mo of age as compared with B7-1/B7-2^+/- MRL-Fas^lpr mice (n = 8/group; _*, p < 0.001) and remained similar to age- and sex-matched MRL-++ mice with normal lungs (Fig. 7).Thus, the B7-1/B7-2 lungs are spared from leukocytic infiltrates.

View larger version (16K): [in this window] [in a new window]   FIGURE 7. Pulmonary leukocyte infiltration is prevented in B7-1/B7-2-/- MRL-Faslpr mice. Pulmonary infiltration in B7-1/B7-2-/- MRL-Faslpr mice (n = 8) at 5 mo of age was reduced compared with that in B7-1/B7-2+/- MRL-Faslpr mice (n = 8) and remained similar to MRL-++ mice (n = 4) with normal lung pathology (*, p < 0.001).

To determine whether B7-1/B7-2 are required for the increase in serum Igs, which is notable in the MRL-Fas^lpr serum, we evaluated serum of B7-1/B7-2^-/- MRL-Fas^lpr mice and B7-1/B7-2^+/- MRL-Fas^lpr mice at 5 mo of age (Fig. 8). Total IgG, IgG1, IgG2b, IgG2a, and IgG3, and IgM were substantially elevated in the serum of B7-1/B7-2^+/- MRL-Fas^lpr mice and were similar to age- and sex-matched wild-type MRL-Fas^lpr mice (n = 8/group; Fig. 8). In contrast, total IgG and IgG1, IgG2b, and IgG3 in B7-1/B7-2^-/- MRL-Fas^lpr mice remained similar to C57BL/6 normal serum levels (n = 8/group; _*, p < 0.01 and _**, p < 0.005; Fig. 8). Notably, the levels of IgG2a in B7-1/B7-2^-/- and B7-1/B7-2^+/- MRL-Fas^lpr mice were similar, and serum IgM was augmented in MRL-Fas^lpr mice lacking both B7-1 and B7-2 (p < 0.01; Fig. 8).

View larger version (42K): [in this window] [in a new window]   FIGURE 8. The increase in serum total IgG, IgG1, IgG2b, and IgG3 is prevented in B7-1/B7-2-/- MRL-Faslpr mice. B7-1/B7-2+/- MRL-Faslpr have an increase in total IgG, IgG1, IgG2a, IgG2b, IgG3, and IgM, similar to the wild-type MRL-Faslpr strain, as compared with normal C57BL/6 mice (n = 8/group; *, p < 0.01; **, p < 0.005). Of note, the level of IgG2a in the B7-1/B7-2-/- MRL-Faslpr mice was not reduced, while the level of IgM was higher in the B7-1/B7-2-/- MRL-Faslpr strain as compared with the B7-1/B7-2+/- MRL-Faslpr or wild-type MRL-Faslpr strains (*, p < 0.01). Ig levels measured by ELISA. Values are mean ± SD. Wild type*, MRL-Faslpr mice that have not been crossed with another strain.

Elevated ANA are characteristic of wild-type MRL-Fas^lpr mice (5 mo of age) and are not detected in normal C3H/FeJ mice (n = 4; p < 0.025; Fig. 9). The B7-1/B7-2^+/- MRL-Fas^lpr mice have a increase in serum ANA at 3 and 5 mo of age (n = 5 and 6, respectively) as compared with normal serum levels (C3H/FeJ mice; n = 3; p < 0.01). Serum ANA levels in B7-1/B7-2^+/- MRL-Fas^lpr mice at 5 mo of age are similar to wild-type MRL-Fas^lpr mice (n = 4; Fig. 9). In contrast, the B7-1/B7-2^-/- MRL-Fas^lpr strain at 3 and 5 mo of age (n = 5 and 6, respectively) has 4-fold less serum ANA than the B7-1/B7-2^+/- MRL-Fas^lpr strain (n = 6/group; p < 0.01; Fig. 9). B7-1/B7-2^-/- MRL-Fas^lpr serum ANA do not remain totally normal but rise modestly by 5 mo of age as compared with age- and sex-matched C3H-FeJ strain (p < 0.01; Fig. 9).

View larger version (16K): [in this window] [in a new window]   FIGURE 9. ANA are prevented in the B7-1/B7-2-/- MRL-Faslpr mice. ANA in the B7-1/B7-2-/- MRL-Faslpr serum at 3 and 5 mo of age was prevented from rising as compared with B7-1/B7-2-/- MRL-Faslpr mice (n = 5 and 6, respectively; *, p < 0.01). By comparison, the B7-1/B7-2 intact MRL-Faslpr and the age- and sex-matched wild-type MRL-Faslpr mice had similar levels of serum ANA. It should be noted that there was a modest increase in serum ANA in B7-1/B7-2-/- MRL-Faslpr mice at 5, but not 3, mo of age, as compared with normal mice (n = 6 and 4, respectively; p < 0.01). Fluorescence was scored on a scale of 0 (none) to 4 (very high). Values are mean ± SD; *, p < 0.01; **, p < 0.025; (wild type)*, MRL-Faslpr mice that have not been crossed with another strain. The Mann-Whitney U test was used for statistical analysis.

IgG deposits in glomeruli of the wild-type MRL-Fas^lpr strain are notable at 5 mo of age as compared with normal glomeruli (C3H/FeJ) and reflects the extent of glomerular damage (n = 3/strain; p < 0.05). IgG deposits in the glomeruli of wild-type MRL-Fas^lpr mice and B7-1/B7-2^+/- MRL-Fas^lpr mice 5 mo of age were similar (n = 6 and 3, respectively; Fig. 10). In contrast, the IgG deposits in the B7-1/B7-2^-/- MRL-Fas^lpr glomeruli did not increase (n = 6; p < 0.01; Fig. 10) and remained similar to normal age- and sex-matched C57BL/6 glomeruli (n = 3).

View larger version (17K): [in this window] [in a new window]   FIGURE 10. Intrarenal deposition of IgG is prevented in B7-1/ B7-2-/- MRL-Faslpr mice. There is a reduction in IgG in the B7-1/B7-2-/- MRL-Faslpr glomeruli at 5 mo of age as compared with B7-1/B7-2+/- MRL-Faslpr glomeruli (n = 6/group, p < 0.01). The amount of IgG in the kidneys of B7-1/B7-2-/- MRL-Faslpr glomeruli is similar to age- and sex-matched normal mice (C57BL/6; n = 3). The amount of IgG in the glomeruli of B7-1/B7-2+/- MRL-Faslpr mice is similar to the wild-type, standard MRL-Faslpr strain. IgG in the kidneys was detected using fluorescein-conjugated anti-mouse IgG. Fluorescence intensity was scored on a scale of 0–4 (0, none; 1, weak; 2, moderate; 3, strong). Values are mean ± SD; *, p < 0.01; **, p < 0.05; (wild type)*, MRL-Faslpr that have not been crossed with another strain. The Mann-Whitney U test was used for statistical analysis.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

B7 costimulatory molecules are responsible for the accumulation of leukocytes in tissues targeted for autoimmune destruction in MRL-Fas^lpr mice. Massive accumulation of leukocytes (CD4, CD8, DN T cells, and macrophages) within multiple tissues is a hallmark of autoimmune disease in the MRL-Fas^lpr strain (34, 35). Leukocytic infiltrates are prominent within the kidney and lung and are responsible for lymphadenopathy and splenomegaly. In the absence of B7 costimulatory molecules, leukocytes do not accumulate in the kidney and lungs and are dramatically reduced in lymphoid tissues. There are many possible explanations linked to the requirement for B7-1 and B7-2 in T cell activation. The absence of B7 molecules may limit T cell expansion, homing to tissues, T cell adherance, T cell restimulation in target organs, T cell proliferation, and leukocyte recruitment. For example, IFN- released from activated T cells induces chemokines production by parenchymal cells (36, 37, 38). It is possible that the leukocytes are not recruited into the kidney, or other tissues, because intrarenal chemokines including MCP-1 are not expressed. In fact, we report that MCP-1 expression is not up-regulated in the B7-1/B7-2^-/- MRL-Fas^lpr kidneys. Thus, chemokines may not be expressed because T cells generating IFN- are not in the kidney. In support of this concept, IFN-, which is abundant in the B7-1/B7-2^+/- MRL-Fas^lpr kidney, is barely detected in B7-1/B7-2^-/- MRL-Fas^lpr kidneys. Another possibility is that T cells that enter the kidney do not bind to parenchymal cells and therefore fail to enter or remain within these tissues. Because activated, but not naive, T cells express functional selectin and integrin ligands, in the absence of B7-mediated activation T cells may not be sufficiently adherent to enter the kidney (39, 40). Alternatively, it is feasible that leukocytes that traffic to the kidney do not accumulate because they are not reactivated within the kidney, and consequently they do not expand locally. In this regard, we have determined that IL-12 released into the kidney via a gene transfer system fosters the local expansion of IFN--secreting T cells (32). Because IL-12 expression is not up-regulated in B7-1/B7-2^-/- MRL-Fas^lpr mice, this proximal initiation signal required for local T cell expansion is lacking. In addition, we have determined that T cell priming in B7-1/B7-2-deficient MRL-Fas^lpr mice does occur, but requires a higher concentration of Ag to elicit cytokine production and proliferation (trinitrophenyl-keyhole limpet hemocyanin immunization, data not shown) and is consistent with the antigenic response reported in B7-1/B7-2-deficient normal strains (13, 14, 15). These findings suggest that the B7 pathway optimizes a suboptimal immune response. Clearly, additional studies are required to pinpoint the B7-dependent mechanism(s) regulating leukocytic accumulation. Nevertheless, our finding suggest that the B7 pathway is essential in driving autoimmune and other diseases mediated by leukocytes.

The impact of B7-1/B7-2 may not be limited to the initiation phase of autoimmune disease. B7-1/B7-2 exert effects at more distal stages during the progression of kidney disease. We have determined that 1) IL-12 gene transfer into the kidney incites renal disease in MRL-Fas^lpr mice (32); 2) B7-1/B7-2-deficient MRL-Fas^lpr fail to up-regulate IL-12 within the kidney (this report); and 3) IL-12 gene transfer into the kidney in B7-1/B7-2-deficient MRL-Fas^lpr mice does not elicit kidney disease (our unpublished observation). Thus, even if there was sufficient IL-12 within the kidney to incite renal disease, in the absence of B7 costimulatory molecules, these mice remain protected. This indicates a role for B7 in initiation and effector phases of autoimmune lupus. This is consistent with the dependence of IL-12 signaling via B7-1 and B7-2 to reverse established Ag-specific tolerance in contact sensitivity (41). In addition, studies in experimental autoimmune encephilitis; a model of autoimmune disease that is organ specific, indicates that B7 costimulation is critical not only in the initial activation and expansion of autoreactive T cells, but in the effector phase of T cell activation within the CNS. Taken together, we suggest that B7 costimulation is instrumental at multiple stages in the cascade of events culminating in systemic autoimmune tissue destruction. This suggests that blockade of the B7 costimulatory pathway will combat autoimmune tissue destruction in patients with well established disease.

Kidney disease is not exclusively dependent on T cell-stimulated cytokines; Ab-mediated mechanisms contribute to glomerulonephritis. Total IgG, IgG1, IgG2b, and autoantibodies, but not IgG2a or IgM, remain at normal values in B7-1/B7-2-deficient MRL-Fas^lpr mice that are protected from autoimmune kidney disease. This is consistent with the concept that pathogenic autoantibody production by B cells in murine lupus requires autoreactive T cell help. For example, MRL-Fas^lpr mice deficient in CD4 have diminished IgG and autoantibodies and are protected from kidney disease (7, 42). In addition, it is established that B7 is required for Ag-specific IgG responses. In mice lacking B7-1 and B7-2, there is a failure to switch Ig isotypes and form germinal centers in response to Ag-specific immunization (15). It is particularly, relevant that glomeruli in B7-1/B7-2-deficient MRL-Fas^lpr mice are spared from Ig deposits. In our study, we determined that IgG3, but not IgG2a, isotype levels remained normal in the B7-1/B7-2-deficient MRL-Fas^lpr strain. Of note, although IgG2a is the dominant subclass in the MRL-Fas^lpr serum throughout the life span, circulating and deposited IgG3, but not IgG2a, correlates with glomerular disease and is instrumental in glomerulonephritis (43, 44, 45). Our finding lends further credence to the concept that glomerular IgG3, and not IgG2a, is pathogenic in lupus nephritis. Taken together, we attribute the total, enduring protection from kidney disease in the MRL-Fas^lpr strain lacking both B7-1and B7-2 to the intrarenal absence of leukocyte infiltrates and Igs.

We suggest that dual blockade of B7-1 and B7-2 molecules is required to confer enduring protection from autoantibody production and fatal autoimmune kidney disease. The necessity to block both B7 costimulatory molecules is based on comparing the B7-1/B7-2-deficient MRL-Fas^lpr with the individual B7-1- and B7-2-deficient MRL-Fas^lpr strains reported by Liang et al.: 1) B7-1^-/- or B7-2^-/- MRL-Fas^lpr mice develop increased autoantibodies characteristic of the MRL-Fas^lpr strain (46); 2) B7-1^-/- MRL-Fas^lpr mice develop renal pathology that is more severe than in MRL-Fas^lpr mice (46); and 3) B7-2^-/- MRL-Fas^lpr mice are only partially protected from renal pathology (46). In contrast, MRL-Fas^lpr mice lacking B7-1 together with B7-2 remain protected from an increase in autoantibodies and fatal kidney disease. These findings suggest that B7-1 compensates for costimulation in the absence of B7-2 and vice versa. Together, these studies indicate that B7-1 and B7-2 have some overlapping functions, a finding noted in the organ-specific autoimmune disease, experimental autoimmune encephilitis. However, if the B7-1 and B7-2 functions were strictly overlapping, the individual B7-1- and B7-2-deficient MRL-Fas^lpr strains should have the same phenotype. Because the phenotypes differ, this indicates that B7-1 and B7-2 also have distinct functions (46). In addition to kidney disease, we have determined that other tissues targeted for autoimmune disease including the lung, liver (data not shown), and lymphoid tissues are protected in the absence of B7-1 and B7-2 in MRL-Fas^lpr mice. Further studies are needed to compare the individual roles of B7-1 and B7-2 costimulators in all facets of MRL-Fas^lpr autoimmune disease.

It is also worth noting that treating MRL-Fas^lpr mice with both anti-B7-1 and anti-B7-2 Abs rather than either Ab alone was required to diminish the rise in autoantibodies characteristic of this strain (46). By comparison, autoantibodies (ANA, dsDNA) were normal in the B7-1/B7-2-deficient MRL-Fas^lpr strain. Thus, provision of anti-B7-1 together with anti-B7-2 may offer a therapeutic approach for treating autoantibody-mediated disease.

While our studies point to a central role for B7-1 and B7-2 in the pathogenesis of systemic autoimmune disease, our data suggests that there are B7-dependent and -independent mechanisms leading to autoimmune tissue destruction. For example, skin lesions and the spleen and lymph nodes in the B7-1/B7-2^-/- MRL-Fas^lpr strain were only partially reduced (fewer CD4, CD8, and macrophages in the spleen, data not shown), but not entirely prevented. The challenge remains to identify the B7-independent mechanisms that complement B7-mediated events to construct a therapeutic strategy to halt autoimmune disease that is not solely obviated by targeting the B7-1/B7-2 pathway. Nevertheless, our data suggest that blockade of B7-1 together with B7-2 will be a powerful therapeutic approach for treating autoimmune kidney, lung, and other forms of autoimmune disease.

View larger version (162K): [in this window] [in a new window]   FIGURE 5. Photomicrographs of B7-1/B7-2-/- MRL-Faslpr mice that are protected from kidney disease. B7-1/B7-2+/- MRL-Faslpr mice had sclerotic glomeruli (short, wide arrow), interstitial leukocytic infiltrates (long arrow), and tubular atrophy and casts (star). By comparison, the kidneys of the B7-1/B7-2-/- MRL-Faslpr mice remained normal. Magnification, x400, periodic acid Schiffs staining.

	Acknowledgments

	Footnotes

 
¹ This work was supported in part by the National Institutes of Health Grant DK 36149 (to V.R.K.) and DK52369 (to V.R.K.) and AI 38310 (to A.H.S.).

² Address correspondence and reprint requests to Dr. Vicki Rubin Kelley, Brigham and Women’s Hospital, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115.

³ Abbreviations used in this paper: DN, double negative; gcs, glomerular cross-section; MCP, macrophage chemoattractant protein; TEC, tubular epithelial cell; ANA, antinuclear Ab.

⁴ G. Tesch. Submitted for publication.

Received for publication October 8, 1999. Accepted for publication March 16, 2000.

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