Immunoregulatory Role of CD1d in the Hydrocarbon Oil-Induced Model of Lupus Nephritis

Mice

CD1d° 129 × C57BL/6 mice (25) were crossed onto the BALB/cJ background (The Jackson Laboratory, Bar Harbor, ME) for nine generations. At each backcross, heterozygous (CD1d^+/−) mice were identified by Southern blotting (25) or by PCR using the neo (sense, 5′-CTTGGGTGGAGAGGCTATTC-3′; antisense, 5′-AGGTGAGATGACAGGAGATC-3′) and CD1d primers (sense, 5′-AATTACACCTTCCGCTGCC-3′; antisense, 5′-CTTCGTGAAGCTGATGGTGG-3′) under the following conditions: 94°C for 30 s, 56°C for 1 min, and 72°C for 1 min for 35 cycles. The N9 CD1d^+/− BALB/c mice were intercrossed to establish CD1d° BALB/c mice. The CD1d° phenotype was further confirmed by flow cytometry of PBLs stained with a conjugated anti-CD1 mAb, 1B1 (BD PharMingen, San Diego, CA).

Establishment of pristane-induced lupus

BALB/cJ mice were inoculated once with 0.5 ml of pristane (2,6,10,14-tetramethyl-pentadecane; Sigma-Aldrich, St. Louis, MO) (23) Mice were bled before and at 3 and 6 mo after pristane inoculation, and sera were frozen for analysis of autoantibodies. All mice were monitored for proteinuria once a month and were sacrificed at 10 mo of age to harvest kidneys.

Assessment of lupus disease

Kidney disease was assessed in CD1d° and wild-type littermates, as described previously (2, 7). Proteinuria was measured on a 0–4+ scale using a colorimetric assay strip for albumin (Albustix; Bayer, Elkhart, IN), where 0 = absent, 1+ ≤ 30 (mild), 2+ = 100 (moderate), 3+ = 300, and 4+ ≥ 2000 mg/dl (severe). Blood urea nitrogen (BUN) levels were measured by impregnating Azostix (Bayer) with a drop of fresh blood and using the following scale: 1+ (normal) = 5–15, 2+ (mild) = 15–26, and 3+ (severe) elevation ≥ 30 mg/dl.

Renal histology

Paraffin sections of kidneys fixed in 4% paraformaldehyde were stained with H&E, periodic acid-Schiff, and Masson’s trichrome. Stained sections were scored for the following features on a 0–3 scale by three of us (R.R.S., J.-Q.Y., S.R.K.) in a blind fashion, as described previously (22): 1) Glomerular activity score (GAS) that included glomerular proliferation, karyorrhexis, fibrinoid necrosis, cellular crescents, inflammatory cells, and hyaline deposits; 2) tubulointerstitial activity score (TIAS) that included interstitial inflammation, tubular cell pyknosis, nuclear activation, cell necrosis and cell flattening, and epithelial cells or macrophages in tubular lumens; 3) chronic lesions score (CLS) that included glomerular scars, glomerulosclerosis, fibrous crescents, tubular atrophy, and interstitial fibrosis; and 4) vascular lesion score that included arterial/arteriolar lesions. The raw scores assigned by various readers were averaged to obtain a mean score for each of the individual features. The mean scores for individual features were summed to obtain the four main scores (GAS, TIAS, CLS, and vascular lesion score) and then all four scores were summed to determine a composite kidney biopsy score (KBS).

Renal immunostaining

Frozen kidney sections were fixed with methanol and acetone (1:1) for 5 min. Slides were washed and incubated with biotinylated rat anti-mouse-Thy1.2, -CD4, -CD8, -CD11b and -B220 mAbs or control rat IgG (BD PharMingen). Sections were then stained using Vectastain ABC-AP kit and Vector red alkaline phosphatase substrate kit I (Vector Laboratories, Burlingame, CA) following the manufacturer’s instructions. For immunofluorescence, sections were stained with FITC-conjugated goat anti-mouse IgG (Sigma-Aldrich). Slides were read by three of us in a blind fashion.

Autoantibodies against nuclear and cytoplasmic Ags

Abs against cellular proteins were analyzed by immunoprecipitation of ³⁵S-radiolabeled K562 cell extract using 4 μl of serum/sample (23). Specificity was confirmed using reference sera containing anti-nRNP, Sm, Su, or ribosomal P Abs. For anti-ribosomal P peptide ELISA, a carboxyl-terminal 22-aa peptide carrying the major epitope of ribosomal PO recognized by human and murine autoimmune sera was synthesized by F-moc chemistry using a Rainin Symphony/Multiplex peptide synthesizer and purified by reversed-phase HPLC. Microtiter plate wells (Maxisorp Immunoplate; Nunc, Naperville, IL) were coated with 50 μl of peptide (2 μg/ml) in 20 mM Tris-HCl (pH 8) at 4°C for 16 h. The wells were washed once with NET/Nonidet P-40 (150 mM NaCl, 2 mM EDTA, 20 mM Tris (pH 7.5), and 0.3% Nonidet P-40) and blocked with 0.5% BSA in NET/Nonidet P-40 for 1 h at 22°C. They were then incubated with 100 μl of 1/500 mouse serum in blocking buffer for 2 h. Wells were washed, incubated with 100 μl of 1/1000 alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) for 2 h. Plates were developed with p-nitrophenyl phosphate substrate and OD was determined at 405 nm using an ELISA plate reader (Molecular Devices, Menlo Park, CA). Anti-DNA Ab were measured by ELISA, as previously described (6, 7). Anti-DNA Ab titers are expressed as units per milliliter using a reference-positive standard of pooled serum from MRL-lpr/lpr mice.

Flow cytometry

For liver NKT cells, liver was perfused with PBS via the portal vein until opaque and pressed through a 70-μm cell strainer (BD Biosciences, Mountain View, CA). Hepatocytes were pelleted by centrifugation at 30 × g for 3 min. The remaining liver cells in the supernatant were pelleted at 300 × g for 5 min and then resuspended in a 40% isotonic Percoll solution (Amersham Pharmacia Biotech, Piscataway, NJ). This suspension was underlaid with a 60% isotonic Percoll solution. After centrifugation for 20 min at 1500 × g, mononuclear cells were isolated at the 40/60% interface, and then washed once with RPMI 1640 medium (Life Technologies, Grand Island, NY) with 5% FCS (HyClone Laboratories, Logan, UT). The cells were stained with murine CD1d/α-GalCer tetramers that were generated as described elsewhere (18) or with human CD1d/α-GalCer tetramers that also recognize murine NKT cells (17). Stained cells were analyzed by flow cytometry. Spleen or thymus cells were incubated with anti-CD16/32 (2.4G2; BD PharMingen) to block FcRγII/III, followed by staining with various conjugated mAbs (all BD PharMingen), as indicated in the figure legends. Stained cells were analyzed using a BD Biosciences FACSCalibur flow cytometer and CellQuest software.

Activation of NKT and T cells

For in vitro NKT cell activation, spleen cells (1–2 × 10⁶/ml) were incubated with titrated doses of synthetic α-GalCer (KRN7000; Kirin Brewery, Gunma, Japan) (15). For T cell activation, splenocytes (2 × 10⁵/well) were stimulated with plate-bound anti-CD3 mAb (1–10 μg/ml) and supernatants were collected after 48 h of culture for the measurement of cytokines.

Detection of cytokines

A standard sandwich ELISA was used to measure cytokines (Figs. 3⇓ and 5⇓a), as previously described (4). TNF-α levels were measured using the BD PharMingen mouse cytokine cytometric bead array kit (Fig. 4⇓) according to the manufacturer’s instructions. To examine the cellular sources of cytokines in α-GalCer-stimulated cultures (Fig. 5⇓, b–e), a cytokine secretion assay was performed using the MACS cytokine secretion assay kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s protocol with some modifications. Briefly, stimulated or control spleen cells (1 × 10⁷) were incubated at 37°C for 45 min with the cytokine Catch Reagent, which attaches to all leukocytes via CD45 Ag and binds to the specific cytokine. After washing, cells were stained with PE-conjugated cytokine detection Ab, followed by incubation with anti-PE microbeads. Cytokine-secreting cells were then positively selected using AutoMACS (Miltenyi Biotec). Cytokine-enriched cells were counterstained with DX5, CD1d/α-GalCer tetramer, and TCRβ and analyzed by flow cytometry. Dead cells and B cells, which can nonspecifically bind to cytokine detection Ab via PE, were excluded by staining with propidium iodide and PerCP-conjugated B220 (BD PharMingen), respectively.

Statistical analysis

Levels of Abs and cytokines, lymphocyte percent and numbers, and renal scores were compared using Student’s t or Mann-Whitney U test. Frequencies of Abs, proteinuria, and BUN were compared using the two-sided Fisher’s exact test.

CD1d deficiency accelerates pristane-induced lupus nephritis

Inoculation of hydrocarbon oils such as pristane induces lupus-like autoantibody production and mild glomerulonephritis in otherwise normal mouse strains such as BALB/c (Refs. 23 and 24 , and see data in CD1d⁺ mice in Figs. 1⇓ and 2⇓). To determine whether CD1d is involved in the development of pristane-induced lupus, we backcrossed CD1d1° 129/B6 mice onto the BALB/c background for nine generations and inoculated the final CD1d° and control BALB/c mice with pristane or PBS. All mice were bled before and at 3 and 6 mo postinoculation and monitored for proteinuria.

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FIGURE 1.

CD1d deficiency accelerates nephritis in pristane-inoculated BALB/c mice. a, CD1d° (n = 24) and CD1d⁺ (n = 19) mice were inoculated with pristane and monitored for proteinuria. Results are shown as the percentage of mice with 0+ to 4+ proteinuria at 6 and 10 mo postinoculation. (0+, 0 mg/dl; 1+, 30 mg/dl; 2+, 100 mg/dl; 3+, 300 mg/dl; and 4+, >2000 mg/dl). b, BUN levels in 10-mo-old CD1d° (n = 11) and CD1d⁺ (n = 7) mice. Results are shown as the percentage of mice with normal (1+, 5–15 mg/dl) or elevated (2+, 15–26 and 3+, 30–40 mg/dl) BUN. c, Kidney biopsy scores: Individual components (GAS, TIAS, and CLS) and a composite KBS (see Materials and Methods) in 10-mo-old animals are shown as the mean ± SE scores (∗, p < 0.05, Mann-Whitney U test). d, Representative periodic acid-Schiff (upper panels) and H&E-stained (lower panels) kidney sections from 10-mo-old mice are shown. Left panels, PBS-injected CD1d° mice show normal-appearing glomeruli (G) and tubules (T). Although pristane-inoculated CD1d⁺ mice have mild focal and mesangial proliferative glomerulonephritis (FP, MP, middle panels), pristane-inoculated CD1d° mice (right panels) develop a more advanced glomerular and tubulointerstitial disease with diffuse glomerular proliferation and glomerulosclerosis (GS) and almost complete occlusion of capillary lumens, severe interstitial inflammation (INF), dilated atrophic tubules (AT) with flattened tubular nuclei, and severe fibrous crescents (CR). Magnification, ×400. Results represent two independent experiments. e, Kidney sections from pristane-inoculated CD1d° and CD1d⁺ mice stained for total T cells (Thy1.2), its CD4⁺ and CD8⁺ subsets, and macrophages (CD11b). Note increased staining in CD1d° animals. Few cells stained for B cells in both groups (data not shown).

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FIGURE 2.

CD1d deficiency increases serum autoantibody production in the pristane-induced lupus model. Sera collected from BALB/c mice inoculated with pristane or PBS were tested for Abs against certain cellular Ags by immunoprecipitation (a and b) or ELISA (c). a, ³⁵S-Labeled K562 cell extract was immunoprecipitated and analyzed in 12.5% gel with sera from CD1d° (left panel) or CD1d⁺ (right panel) BALB/c mice 6 mo after injection with PBS (lanes 1–3 in both panels) or pristane (lanes 4–11 in the left and lanes 4–10 in the right panel). Left panel, Sera in lanes 4–9 and 11 are anti-nRNP Ab positive (indicated by appearance of A, B′/B, C, D, E/F, and G bands); sera in lanes 5 and 6 show three (P₀, P₁, and P₂) bands of anti-ribosomal P Ab. Right panel, In CD1d⁺ mice, sera in lanes 4–7 are anti-nRNP Ab positive. None of the CD1d⁺ sera show anti-ribosomal P Ab bands. None of the PBS-injected control mice are positive for any of these autoantibodies (lanes 1–3 in both panels). On the left are shown molecular mass and the bands are indicated on the right. b, Immunoprecipitation using 8% gel with sera from CD1d° mice shown in lanes 5–8 of the left panel of a. Sera in lanes 5–7 are anti-Su Ab positive (100-kDa band; long arrow); sera in lanes 5 and 6 are anti-OJ Ab positive (short arrows), and sera in lanes 5–7 are anti-Sm Ab positive (U5–200 kDa doublet; bold arrow). c, Results of ELISA for IgG anti-ribosomal P (∗, p < 0.01) and anti-dsDNA (∗, p < 0.05) Abs are expressed as the mean ± SE. Pristane-inoculated, n = 23 CD1d°, 16 CD1d⁺; PBS-injected, n = 5 CD1d°, 6 CD1d⁺ mice. Similar results were obtained in two independent experiments.

Proteinuria developed early and was more severe in the pristane-inoculated CD1d° mice than in the CD1d⁺ littermates (Fig. 1⇑a). At 6 mo postinoculation, 54% of the mice in the CD1d°, but none in the CD1d⁺ group, had ≥100 mg/dl (moderate to severe) proteinuria (p < 0.0001). The frequency of moderate to severe proteinuria was still high in CD1d° mice at 10 mo (75% in CD1d° vs 16% in CD1d⁺; p = 0.005). None of CD1d⁺, but 63% of CD1d°, mice developed severe (≥300 mg/dl) proteinuria at 10 mo postinoculation (p = 0.0001).

BUN was also elevated in pristane-inoculated CD1d° mice (Fig. 1⇑b), suggesting an advanced renal disease in these mice. Nine (82%) of 11 CD1d°, but 0 of 7 CD1d⁺, mice had elevated (>15 mg/dl) BUN (p = 0.002). Two (18%) of 11 CD1d° mice had a severely elevated BUN (≥30 mg/dl).

Mice were sacrificed at 10 mo of age to harvest kidneys and their renal histology was analyzed (Fig. 1⇑, c and d). Mild and focal mesangioproliferative glomerulonephritis was found in 60% of pristane-inoculated CD1d⁺ mice (Fig. 1⇑d, middle panels), the remaining 40% of mice had no evidence of nephritis by light microscopy, and none of the CD1d⁺ mice had diffuse proliferative or chronic lesions. CD1d° mice, however, developed diffuse proliferative glomerulonephritis with fibrous crescents, glomerulosclerosis, tubular atrophy, and interstitial fibrosis in 50% of mice (Fig. 1⇑d, right panels); another 25% of pristane-inoculated CD1d° mice had mild to moderate mesangioproliferative lesions; and the remaining 25% of mice had mild focal or no lesions. A composite KBS (see Materials and Methods) was increased in pristane-inoculated CD1d° mice (p < 0.05). Further analysis of the KBS revealed an increase in active (GAS) as well as chronic kidney lesions (CLS) in CD1d° mice (Fig. 1⇑c). None of the PBS-injected mice (six CD1d⁺, five CD1d°) had proteinuria or renal histological changes (Fig. 1⇑d, left panels).

As shown in Fig. 1⇑d, inflammatory cell infiltration was increased in CD1d° mice. To determine the phenotype of kidney-infiltrating cells, kidney sections were stained with conjugated anti-Thy1.2, anti-CD4, anti-CD8, anti-CD11b, and anti-B220 Abs. Infiltrating cells that were mostly T cells and macrophages were increased in CD1d° mice as compared with CD1d⁺ mice (Fig. 1⇑e). B220⁺ cells were rarely detected in kidney sections from both groups of animals (data not shown).

CD1d deficiency enhances pristane-induced autoantibody production

Pristane-inoculated BALB/c mice develop autoantibodies to several cellular Ags (23, 24). We detected these Abs with an immunoprecipitation assay using cell extract from an erythroleukemia cell line, K562, as a source of autoantigens. Overall, reactivity to cellular Ags was higher in sera from pristane-inoculated CD1d° mice than in sera from pristane-inoculated CD1d⁺ or PBS-injected CD1d° or CD1d⁺ mice (Fig. 2⇑a). Anti-ribosomal P (Fig. 2⇑a) and anti-OJ (isoleucyl tRNA synthetase complex) Abs (Fig. 2⇑b), which are generally not induced in pristane-inoculated wild-type BALB/c mice (23, 24, 26), were detected in 3 (13%) of 23 CD1d°, but in none of 16 CD1d⁺, mice. Levels of some of these autoantibodies were quantitated by ELISA. As shown in Fig. 2⇑c (left panel), anti-ribosomal P peptide Abs were significantly increased in the CD1d° mice as compared with wild-type littermates (p < 0.01). Serum IgG anti-dsDNA Ab levels, as measured by ELISA, were also higher in CD1d° than in CD1d⁺ mice at 6 mo postinoculation (p < 0.05; Fig. 2⇑c).

T cell cytokine responses in pristane-injected CD1d-deficient BALB/c mice

Abnormalities in cytokine production contribute to the development of lupus (24). To determine whether exacerbation of lupus in CD1d° animals is related to abnormalities in cytokine production, we measured cytokine responses in the spleens of pristane-inoculated CD1d° and CD1d⁺ BALB/c mice. After 12–24 h, 10–12 days, 6 wk, and 6 mo of pristane inoculation, spleen cells were stimulated in vitro with α-GalCer, anti-CD3 Ab, or Con A (Figs. 3⇓ and 4⇓ and data not shown). As expected, CD1d° mice exhibited no cytokine response to α-GalCer stimulation. Upon stimulation with anti-CD3 (Fig. 3⇓) or Con A (data not shown), IL-4 levels were significantly decreased, whereas IFN-γ and IL-2 levels were unchanged in CD1d° mice as compared with CD1d⁺ mice; there was no difference in the levels of these cytokines between PBS vs pristane-inoculated mice at 12 h and 11 days after pristane inoculation. This change in cytokine profile in CD1d° mice was not associated with significant alterations in various spleen cell populations, including CD4, CD8, DC, B220, macrophages, and neutrophils (Ref. 25 and data not shown). At 6 mo, IL-4 and IL-13 production was lower in pristane-injected CD1d° and CD1d⁺ mice than in PBS-injected CD1d⁺ mice, while there was no effect of CD1d deficiency or pristane injection on T cell production of IFN-γ or IL-2. Such a cytokine profile, i.e., decreased type 2 with unchanged type 1 cytokines, may contribute to the development and exacerbation of autoimmune disease in wild-type and CD1d° mice, respectively.

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FIGURE 3.

Effect of CD1d deficiency on T cell cytokine production in the pristane-induced lupus model. PBS- or pristane-injected CD1d⁺ or CD1d° mice were sacrificed at 12–24 h, 11 days, or 6 mo after pristane inoculation. Their spleen cells were stimulated with anti-CD3 for 48 h and culture supernatants were tested for cytokines. ∗, p < 0.05 to < 0.0001, n = 3–7 mice/group.

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FIGURE 4.

T cell production of TNF-α is selectively decreased during the initial phases of development of pristane-induced lupus. Ten- to 14-wk-old CD1d° and CD1d⁺ BALB/c mice were injected with PBS or pristane (n = 3–5 mice/group). Their spleen cells were cultured with medium alone, α-GalCer, Con A, or anti-CD3 Ab. TNF-α levels were measured in culture supernatants. Note that PBS-injected CD1d° mice had no TNF-α response to α-GalCer, but had normal TNF-α response when their spleen cells were stimulated with anti-CD3 or Con A. Pristane-injected CD1d⁺ mice, however, had decreased TNF-α response to α-GalCer stimulation at all times tested (∗∗, p < 0.01, pristane- vs PBS-injected CD1d⁺ mice). TNF-α response to Con A or anti-CD3 stimulation was decreased on days 10 and 11 (∗, p < 0.05, pristane- vs PBS-injected CD1d⁺ or CD1d° mice), but not at 12–20 h or 6 mo time points. Results shown are from one of two similar experiments.

Results of TNF-α production by T cells revealed a striking pattern (Fig. 4⇑). TNF-α levels, upon Con A or anti-CD3 stimulation, were similar between PBS-injected CD1d° and CD1d⁺ mice. In pristane-inoculated mice, however, TNF-α levels in Con A or anti-CD3-stimulated cultures significantly decreased on days 10–12 postinoculation (p < 0.05), regardless of the CD1d status of the mice. At 6 mo postinoculation, TNF-α levels were slightly increased in pristane-inoculated mice (p = NS). Such selective deficiency in T cell production of TNF-α during the initial phases of disease development may contribute to the development and exacerbation of disease in wild-type and CD1d° mice, respectively.

NKT cell functions and numbers are reduced in pristane-injected animals

Although the lack of cytokine production upon α-GalCer stimulation was expected in CD1d° mice, it was surprising that in wild-type mice α-GalCer-induced TNF-α production was significantly lower in pristane-inoculated than in PBS-injected animals (Fig. 4⇑). This suggested that pristane inoculation itself alters NKT cell functions. To further evaluate the effect of pristane on NKT cell functions, spleen cells were harvested from BALB/c mice at various time points (12–24 h, 10–12 days, 6 wk, and 6 mo) after pristane or PBS injection and stimulated with α-GalCer for 40–48 h. Culture supernatants were assayed for various cytokines, which were significantly decreased in the pristane group at 10–12 days (p < 0.05–< 0.01; Fig. 5⇓a, left panel), 6 wk, and 6 mo postinoculation (data not shown); TNF-α (Fig. 4⇑) and IL-2 levels (data not shown) decreased as early as 12–24 h postinoculation in the pristane group as compared with the PBS group.

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FIGURE 5.

α-GalCer-induced cytokine responses are markedly reduced in pristane-inoculated mice. a, Effect of α-GalCer stimulation on cytokine production: 10- to 14-wk-old BALB/c mice were inoculated with pristane or PBS (n = 24/group). Left panels, Ten- to 11-day after inoculation, spleens were harvested and single spleen cells were cultured with α-GalCer for 40–48 h. Culture supernatants were assayed for cytokines (IFN-γ, IL-2, IL-4, IL-5, and IL-10). Values of p < 0.05–0.01 at all α-GalCer concentrations, n = 4–5 mice/group. Cytokine levels in control cultures containing synthetic β-GalCer or vehicle in which galactosylceramides were dissolved were similar to or <2-fold of the baseline value (cells with medium alone). Right panels, Twelve hours, 10 days, or 6 mo after PBS or pristane inoculation, wild-type and CD1d° mice were injected i.v. with 4 μg of α-GalCer, bled 2 h later, and serum samples were assayed for IFN-γ, IL-2, IL-4, IL-10, and IL-13. ∗, p < 0.05; ∗∗, p < 0.001, n = 3–5 mice/group, ±SE. In pristane-inoculated CD1d° mice at 6 mo postinoculation, the mean IFN-γ, IL-2, IL-4, IL-10, and IL-13 levels were 0.3, 0.1, 0.03, 0.3, and 0.02 ng/ml, respectively (data not shown). The background serum cytokine levels in wild-type mice injected with vehicle alone ranged from undetectable (<0.02) to 0.15 ng/ml. Data are from one of three similar experiments. b–e, Cytokine-secreting cells that are mostly NKT cells in α-GalCer-exposed animals are reduced after pristane injection. PBS- or pristane-inoculated mice were injected i.v. with α-GalCer (b–d). Two hours after injection, mice were sacrificed; their spleen cells were stained for IFN-γ (upper two rows), IL-2 (3rd and 4th rows), and IL-4 (5th and 6th rows) using a cytokine secretion assay (see Materials and Methods). b, CD1d/α-GalCer tetramer and cytokine staining cells are shown as the percentage of gated live B220⁻ lymphocytes. Tetramer/cytokine double-positive cells are decreased in pristane-injected mice (highlighted in bold). c, Spleen cells (1 × 10⁷) were enriched for cytokine-secreting cells and stained for the tetramer. Numbers of cells (×10²) in each quadrant are shown. Most enriched cytokine⁺ cells are tetramer⁺. The cytokine⁺ cells (right upper and lower quadrants) were then gated to determine the percentage of TCRβ⁺tetramer⁺ cells (d, left panels) or of TCRβ⁺DX5⁺ cells (d, right panels). e, Unstimulated spleen cells from control PBS-injected BALB/c mice were stained with tetramer and cytokines. Stained cells are indicated as the percentage of gated live B220⁻ lymphocytes. f, Cytokine expression on CD1d/α-GalCer tetramer⁺ cells. α-GalCer-stimulated spleen cells were enriched for cytokine⁺ cells as shown in c. The enriched cells were then analyzed for cytokine expression on gated CD1d/α-GalCer tetramer⁺ cells. Note decreased IL-4 and increased IFN-γ and IL-2 expression on tetramer⁺ cells.

We then assessed the effect of pristane on in vivo NKT cell functions. Mice were injected with pristane or PBS and 12–24 h, 10–11 days, or 6 mo later injected i.v. with 4 μg α-GalCer and bled 2 h later for detection of cytokines in serum samples (Fig. 5⇑a, right panel). All cytokines tested were markedly reduced at 6 mo postinoculation (p = 0.002–0.00003, pristane vs PBS-injected mice). Serum IL-2 and IL-13 levels decreased as early as 12–24 h after pristane inoculation, while serum levels of other cytokines were variable at 12 h and 10 days postinoculation. Thus, NKT cell cytokine responses, as assessed by brief in vivo or in vitro exposure to α-GalCer, markedly decline after pristane inoculation.

α-GalCer-induced responses shown in Fig. 5⇑a may reflect its direct effect on NKT cells as well as the secondary effects of NKT cell activation on other immune cells (27). To investigate this further, PBS- or pristane-inoculated mice were injected i.v. with 4 μg α-GalCer. Two hours later, mice were sacrificed and their spleen cells were enumerated for cytokine-secreting CD1/α-GalCer tetramer⁺ cells using a cytokine secretion assay. Less than 2% of live B220⁻ lymphocytes in α-GalCer-primed (Fig. 5⇑b) and few cells in unprimed (Fig. 5⇑e) animals were positive for IFN-γ, IL-2, or IL-4. Most cytokine⁺ cells were tetramer positive (Fig. 5⇑b). To further confirm this, spleen cells (1 × 10⁷) were enriched for cytokine⁺ cells (Fig. 5⇑c), which was highly efficient (>98.5%) for all three cytokines tested. Among all IFN-γ⁺ cells in PBS-injected mice, 88% were NKT cells (TCRβ⁺tetramer⁺) and ∼6% each were conventional T cells (TCRβ⁺tetramer⁻) or NK cells (TCRβ⁻DX5⁺) (Fig. 5⇑d). Only a few cytokine-secreting tetramer⁺ cells expressed DX5 (Fig. 5⇑d). Intriguingly, IL-4-secreting cells expressed more DX-5 than IL-2 or IFN-γ-secreting NKT cells (Fig. 5⇑d). Gadue and Stein (28) made a similar observation that more DX5⁺ thymic NKT (tetramer⁺) cells secrete IL-4 than DX5⁻ thymic NKT cells. Thus, most IFN-γ-, IL-2-, or IL-4 secreting cells after brief in vivo α-GalCer exposure are NKT cells. The percent (Fig. 5⇑b) and numbers (Fig. 5⇑c) of these cells were lower in pristane-inoculated mice than in PBS-injected mice. Interestingly, the remaining NKT cells in pristane-injected mice had a Th1 phenotype, i.e., decreased IL-4 and increased IFN-γ expression as compared with NKT cells from PBS-injected mice (Fig. 5⇑f): The mean fluorescence intensities on tetramer⁺ cells were 157 vs 202 for IL-4 and 220 vs 194 for IFN-γ in pristane- vs PBS-injected mice, respectively.

α-GalCer stimulation induces the expression of activation markers on spleen cells (27). To assess whether this function of NKT cells is also compromised in pristane-induced lupus, α-GalCer-stimulated spleen cells from PBS- or pristane-inoculated wild-type and CD1d° mice were analyzed for activation and memory markers, CD25, CD44, CD62 ligand, CD69, CD80, and CD86, by flow cytometry (Fig. 6⇓ and data not shown). Significant decreases in the induction of CD25 and CD69 on B and T cells were observed at 6 mo postinoculation (p = 0.03–0.0005; Fig. 6⇓), but not significantly at earlier time points, i.e., 12 h and 10–12 days postinoculation (data not shown). On CD1d/α-GalCer tetramer⁺ cells, however, decreases in CD25 and CD69 expression were observed as early as 10 days postinoculation. For example, the percent CD69⁺tetramer⁺ cells (6.6 ± 0.6 vs 12 ± 1% of gated TCRβ⁺ cells, p < 0.05) as well as the mean fluorescent intensity of CD69 on tetramer⁺ cells (51 ± 2.3 vs 63 ± 5.7, p < 0.05) were significantly reduced in pristane-inoculated as compared with PBS-injected mice. Pristane inoculation also affected the induction of other activation and memory markers. For example, the induction of CD44 and CD86 expression was significantly decreased on TCR⁺ and B220⁺ cells, respectively (p < 0.01–0.001; Fig. 6⇓). As expected, induction of all activation markers was markedly lower in CD1d° mice (Fig. 6⇓).

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FIGURE 6.

α-GalCer-induced expression of activation markers on spleen cells is reduced in pristane-inoculated mice. Six months after pristane inoculation, mice were injected with 4 μg of α-GalCer i.v., their spleens were harvested 2 h later, and the spleen cells were cultured with α-GalCer for 40 h. Expression of activation markers, CD25, CD44, CD69, CD86, and CD62 ligand, on B and/or T cells is shown. The (mean ± SE) percentage of cells expressing these markers is shown in the table. ∗, p < 0.05 and ∗∗, p < 0.01 compared with PBS-injected wild-type mice (WT/PBS/α-GC group); n = 3–5 mice/group. Similar levels of the expression of activation markers were observed in CD1d° mice injected with α-GalCer and wild-type mice injected and cultured with vehicle alone (WT/PBS/Vehicle group). Results are from one representative of three independent experiments.

Results shown in Figs. 4–6⇑⇑⇑ describe the effect of pristane on α-GalCer-induced NKT cell responses. To examine whether NKT cell numbers decline spontaneously (i.e., without any α-GalCer priming) in pristane-injected mice, we enumerated CD1d/α-GalCer tetramer⁺TCRβ⁺ cells in the thymus, spleen, and liver of BALB/c mice at 12–24 h, 10–12 days, 6–8 wk, or 12 mo after pristane injection. We found that the most severe deficiency in NKT cells found was in thymus 6–8 wk after (Fig. 7⇓a), but not at earlier time points (12–24 h and 10–12 days) after pristane injection (data not shown). The percent and total thymic tetramer⁺ cells were significantly reduced at 6–8 wk in pristane-inoculated mice as compared with PBS-injected control animals (p <0.05-<0.01); the percent and total splenic tetramer⁺ cells, however, were not significantly different between the two groups (p = 0.06–0.08; Fig. 7⇓a). In the liver, results on tetramer⁺ cells at 12 mo postinoculation were difficult to interpret (data not shown) because of pristane-induced changes in the liver architecture and cellularity.

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FIGURE 7.

Decreased NKT cell numbers in pristane-inoculated mice. Eight weeks after inoculation with pristane or PBS, 16-wk-old BALB/c mice were sacrificed and their thymus and spleen cells were analyzed for CD1d/α-GalCer tetramer⁺ cells. a, TCRβ⁺tetramer⁺ cells are expressed as the mean ± SE (percent) of gated lymphocytes or as the absolute numbers ×10⁴. b, NKT cell subsets (CD4⁺tetramer⁺, CD4⁺CD8⁺tetramer⁺, and CD4⁻CD8⁻tetramer⁺ cells) are expressed as the mean percentage of gated lymphocytes. Note that TCRβ⁺tetramer⁺ thymocytes (a) and both CD4⁺ and CD4⁻ subsets of tetramer⁺ thymocytes or splenocytes (b) were significantly lower in pristane-injected than in control animals. A minor population of double-positive (CD4⁺CD8⁺) tetramer⁺ thymocytes was also decreased in pristane-injected mice as compared with control mice (b). ∗, p <0.05; ∗∗, p <0.01, n = 3–6 mice/group, ±SE. Results are representative of three independent experiments.

NKT cells comprise diverse phenotypes of T cells (19); some subsets that may contribute to immune regulation and protection against autoimmunity might be particularly deficient in lupus mice. To examine such a possibility, we analyzed CD4 and CD8 expression on tetramer⁺ cells at 6–8 wk after pristane injection (Fig. 7⇑b). In the thymus, the reduction in percent and total tetramer⁺ cells in pristane-injected animals was seen in all three subsets, CD4⁺, CD4⁻CD8⁻, and CD4⁺CD8⁺ double-positive cells. In the spleen, the percent, but not total, CD4⁺ and CD4⁻CD8⁻tetramer⁺ cells were reduced in pristane-inoculated mice (p < 0.05). Thus, all subsets of invariant NKT cells decline in pristane-injected animals.

Thus, primary NKT cell functions as well as secondary effects of NKT cells on other immune cells are markedly compromised in pristane-inoculated BALB/c mice. Although a significant decrease in NKT cell numbers and most NKT cell functions are detectable 6 wk after pristane inoculation, some impairment of NKT cell functions, such as TNF-α and IL-2 production upon α-GalCer stimulation, begins as early as 12–24 h after pristane exposure.

To explore the mechanisms of decline in NKT cell functions and numbers after pristane inoculation, we examined the effect of pristane inoculation on CD1d-expressing APC (Fig. 8⇓). Overall, the expression of CD1d was decreased in spleen cells of pristane-inoculated mice as compared with untreated control animals (mean fluorescence intensity, 79 ± 0.8 vs 98 ± 3.6, p < 0.05). Specifically, the number of CD1d-expressing CD11c⁺ DCs was significantly decreased in pristane-injected mice as compared with control animals (6.7 ± 0.2 vs 9.4 ± 0.3, p < 0.01). The CD1d⁺CD11c⁺ cells could be subdivided into three subsets, namely, CD1d⁺CD11c^high, CD1d^highCD11c^low, and CD1d^lowCD11c^low cells. Significant differences between the pristane-inoculated and control mice were seen in the numbers of CD1d⁺CD11c^high (1.7 ± 0.2 vs 2.5 ± 0.3, p < 0.05) and CD1d^highCD11c^low subsets (3.3 ± 0.3 vs 5.2 ± 0.1, p = 0.02) of CD11c⁺ cells. The CD1d-expressing B220⁺ cells were also slightly decreased in pristane-inoculated mice (p = 0.04–0.06). There was no significant effect of pristane injection on the numbers of CD1d-expressing macrophages (CD11b⁺) and T cells. Thus, pristane inoculation results in decreased numbers of CD1d-expressing DCs and B cells, which, in turn, may be responsible for the decreased NKT cell functions in pristane-induced lupus.

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FIGURE 8.

Reduced numbers of CD1d-expressing DCs in pristane-inoculated mice. Twelve days after inoculating with pristane or PBS, BALB/c mice were injected i.v. with 4 μg of α-GalCer and sacrificed 2 h later. Total spleen cell numbers were similar between the two groups of animals. CD1d⁺CD11c⁺, CD1d⁺B220⁺, CD1d⁺CD11b⁺, and CD1d⁺TCRβ⁺ cells are expressed as the mean percentage of spleen cells. ∗, p < 0.01, n = 5 mice/group. Results are representative of three independent experiments.

Effect of CD1d deficiency on marginal zone B cells in pristane-inoculated BALB/c mice

Results in Fig. 8⇑ show that, while overall CD1d expression was decreased on B cells, the CD1d^highB220⁺ cells that correspond to marginal zone B cells (29) were increased in pristane-injected mice in two separate experiments (total number of cells (×10⁶) were 3.7 ± 0.7 and 8.1 ± 0.9 in PBS- vs pristane-injected mice, p < 0.05). To further examine the effect of pristane inoculation and CD1d deficiency on marginal zone B cells, we enumerated CD21^highCD23^low B cells in the spleens of CD1d° and CD1d⁺ mice on day 11 or 30 (Fig. 9⇓, a and b) or at 6 or 12 mo (data not shown) after pristane or PBS injection. We found that marginal zone B cells were increased by ∼1.5- to 2-fold in the pristane group at 1, 6, and 12 mo after injection, regardless of the CD1d status of mice. Interestingly, this expansion of marginal zone B cells was restored to normal in pristane-injected BALB/c mice treated with a CD1d ligand, α-GalCer (A. K. Stanic, J.-Q. Yang, L. V. Kaer, and R. R. Singh, manuscript in preparation). Additionally, although serum IgG1 and IgG2a levels were similar between the two groups of animals (data not shown), serum levels of IgG3 isotype that is known to be preferentially secreted by marginal zone B cells (29) were significantly higher in pristane-injected CD1d° mice than in pristane-injected CD1d⁺ mice (Fig. 9⇓c). Previous studies have reported reduced CD23 expression on B cells in lupus-prone NZB mice (30). We found that the mean fluorescent intensity of CD23 expression on B cells was also reduced in pristane-injected mice as compared with PBS-injected mice, regardless of the CD1d status of mice, although the differences were not statistically significant (data not shown).

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FIGURE 9.

Effect of pristane inoculation and CD1d deficiency on marginal zone B cells. CD1d° or CD1d⁺ mice were injected with PBS or pristane and sacrificed on day 11 or 30. Their spleen cells were stained with CD21, CD23, and B220 and analyzed by flow cytometry. Marginal zone B cells (CD21^highCD23^low) are shown as the mean ± SE (percent) of B220⁺ cells on day 30 (a) or on days 11 and 30 (b) after injection with PBS or pristane. Note 1.5- to 2-fold expansion of marginal zone B cells on day 30, but not on day 11, in pristane-injected mice. Results represent three independent experiments (n = 2–4 mice/group). c, Serum total IgG3 levels (mean ± SE, μg/ml) in CD1d° and CD1d⁺ BALB/c mice 9–12 mo after pristane injection (n = 13–16 mice/group, p < 0.02).

Effect of α-GalCer-activated NKT cells on autoantibody production in vitro

Our findings suggest that the development of lupus-like disease in pristane-inoculated wild-type mice and its exacerbation in CD1d° animals is related, at least in part, to the induced or genetic deficiency of NKT cells, respectively. To provide further support for this possibility, we tested whether NKT cell activation can suppress autoantibody production by B cells from pristane-injected mice. For this purpose, B cells isolated from the spleens of pristane-inoculated BALB/c mice were cocultured with T cells isolated from the spleens of PBS-injected control animals, with or without α-GalCer. Supernatants collected on day 6 were tested for IgG anti-DNA Ab and rheumatoid factor. IgG anti-DNA Ab, and rheumatoid factor levels in these cultures were generally low and were not affected by the addition of α-GalCer (data not shown). However, in LPS-stimulated cultures, which had high levels of rheumatoid factor, addition of α-GalCer significantly decreased rheumatoid factor levels (Fig. 10⇓).

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FIGURE 10.

α-GalCer-stimulated T cells can decrease autoantibody production by LPS-activated B cells from pristane-inoculated mice in vitro. Splenic B cells from BALB/c mice 6 mo after pristane inoculation were cocultured with splenic T cells from PBS-injected control animals, with or without α-GalCer. Supernatants collected on day 6 were tested for rheumatoid factor, which are expressed as the mean ± SD triplicate OD.