Type I interferon production by tertiary lymphoid tissue developing in response to 2,6,10,14-tetramethyl-pentadecane (pristane)

Abstract

Lymphoid neogenesis is associated with antibody-mediated autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. Although systemic lupus erythematosus is the prototypical B-cell-mediated autoimmune disease, the role of lymphoid neogenesis in its pathogenesis is unknown. Intraperitoneal injection of 2,6,10,14-tetramethyl-pentadecane (TMPD, pristane) or mineral oil causes lipogranuloma formation in mice, but only TMPD-treated mice develop lupus. We report that lipogranulomas are a form of lymphoid neogenesis. Immunoperoxidase staining of lipogranulomas revealed B cells, CD4(+) T cells, and dendritic cells and in some cases organization into T- and B-cell zones. Lipogranulomas also expressed the lymphoid chemokines CCL21, CCL19, CXCL13, CXCL12, and CCL22. Expression of the type I interferon (IFN-I)-inducible genes Mx1, IRF7, IP-10, and ISG-15 was greatly increased in TMPD- versus mineral oil-induced lipogranulomas. Dendritic cells from TMPD lipogranulomas underwent activation/maturation with high CD86 and interleukin-12 expression. Magnetic bead depletion of dendritic cells markedly diminished IFN-inducible gene (Mx1) expression. We conclude that TMPD-induced lupus is associated with the formation of ectopic lymphoid tissue containing activated dendritic cells producing IFN-I and interleukin-12. In view of the increased IFN-I production in systemic lupus erythematosus, these studies suggest that IFN-I from ectopic lymphoid tissue could play a role in the pathogenesis of experimental lupus in mice.

Figure 1

Flow cytometry of peritoneal exudate cells. BALB/c mice were treated intraperitoneally with TMPD or mineral oil (MO). Peritoneal exudate cells were harvested by lavage 12 or 20 weeks later (TMPD-12, MO-12, TMPD-20, and MO-20, respectively). Resident peritoneal cells (RPCs) from three untreated BALB/c mice were analyzed for comparison. Pooled lipogranulomas from individual mice were harvested at 20 weeks after TMPD or mineral oil injection (TMPD-Lip and MO-Lip, respectively) and treated with collagenase and dispase. A: Total peritoneal cell counts. Total cell counts were determined using a hemocytometer (mean and SE are shown). B and C: Cellular composition of peritoneal exudates by flow cytometry. Peritoneal cells were analyzed by flow cytometry using anti-B220 and CD19 (B cells), anti-CD3 (T cells), anti-CD11b (monocytes and some B cells), and CD11c (dendritic cells). The percent (B) and absolute numbers (C) of B and lymphocytes, CD11b⁺ cells, and CD11b⁺, CD11c⁺ monocyte-derived dendritic cells are shown. D: Representative examples of the staining for mineral oil- versus pristane-treated mice. Peritoneal cells were stained with anti-B220 and anti-CD19 mAbs (left), anti-CD11b and anti-I-A^d mAbs (middle), or anti-CD11c and anti-CD86 mAbs (right).

Figure 2

Pathology of lipogranulomas. A: Lipogranulomas adherent to the mesothelial surface of a mouse treated 12 weeks earlier with TMPD (arrow). B: Lipogranulomas adherent to the mesothelial surface of the diaphragm of a mouse treated 12 weeks earlier with mineral oil (arrows). C: H&E staining of lipogranulomas from a TMPD-treated mouse. Note numerous oil droplets and lymphocytic infiltration. D and E: Neovascularization of TMPD-induced lipogranulomas. D: H&E staining of a peritoneal lipogranuloma from a mouse treated 12 weeks earlier with TMPD showing a blood vessel filled with erythrocytes (arrow). E: Immunohistochemistry of frozen tissue (5-μm sections, immunoperoxidase technique) from a peritoneal lipogranuloma from a mouse treated 12 weeks earlier with TMPD and stained with MECA-79 antibody (anti-peripheral lymph node addressin, a marker for high endothelial venules). F–I: Immunohistochemistry of serial sections through a TMPD lipogranuloma. A snap-frozen lipogranuloma was sectioned at 5 μm and stained by the immunoperoxidase technique as follows: biotinylated anti-mouse CD45R/B220 (F), biotinylated anti-mouse CD11c (G), biotinylated anti-mouse CD4 (H), and biotinylated peanut agglutinin (I). Arrow indicates the location of a collection of B cells in the serial sections. J–L: Immunohistochemistry of mineral oil lipogranulomas. Snap-frozen lipogranulomas from mineral oil-treated mice were sectioned (5 μm) and stained by the immunoperoxidase technique using biotinylated MECA-70 (anti-PNAd) (J), B220 (B cells) (K), or CD4 (T cells) (L).

Figure 3

Expression of chemokines and chemokine receptors in lipogranulomas. RNA isolated from pooled lipogranulomas (12 weeks) from four TMPD-treated and four mineral oil-treated mice was used to synthesize cDNA. Expression of SLC (CCL21), ELC (CCL19), CCR7, BLC (CXCL13), and CXCR5 (top), as well as SDF-1 (CXCL12) and MDC (CCL22) (bottom) was analyzed by RT-PCR, normalized to β-actin (top) or 18S ribosomal RNA (bottom).

Figure 4

TMPD lipogranulomas contain activated dendritic cells. A: Accumulation of dendritic cells in the lipogranulomas. Peripheral blood mononuclear cells (PBMCs), spleen, peritoneal exudate cells, and pooled lipogranuloma cells from a representative BALB/c mouse 20 weeks after intraperitoneal TMPD injection were analyzed by flow cytometry. Top: Percentages of CD11c⁻/CD11b⁺ monocytes/macrophages, CD11c⁺/CD11b⁺ monocyte-derived (?) dendritic cells, and CD11c⁺/CD11b⁻ dendritic cells in each location (PBMCs, spleen, peritoneal exudate, and lipogranulomas) are indicated. Bottom: Percentages of PDCA-1⁺ dendritic cells in each location (peripheral blood, spleen, peritoneal exudate, and lipogranulomas) are shown. B: Lipogranuloma dendritic cells express high levels of CD86. Expression of CD86 by CD11c⁺ cells in the peripheral blood, spleen, peritoneal exudate, and lipogranulomas from representative TMPD-treated mice was determined by flow cytometry. Top: Mean fluorescence intensity of CD86 in CD11c-gated cells from spleen, peritoneum, and lipogranulomas. Bottom: CD11c and CD86 staining of PBMCs, peritoneal cells, and isolated lipogranuloma cells. C: Dendritic cells in lipogranulomas preferentially express CD86. Shown is the ratio of CD86⁺ to CD86⁻ dendritic cells (CD11c⁺) in peripheral blood mononuclear cells (PBMCs), peritoneal cells (PCs), and TMPD-treated mouse lipogranulomas. D: Increased expression of IL-12 p40 in TMPD lipogranulomas. RT-PCR was performed using cDNA from lipogranulomas from four mineral oil-treated mice and four TMPD-treated mice. Sequences of the IL-12 p40, IL-6, IL-4, IFN-γ, and β-actin primers used are shown in Table 1.

Figure 5

IFN-I-inducible gene expression in peritoneal exudate cells. A: Cytokine expression in peritoneal washings. Peritoneal lavage was performed in mice treated with TMPD or mineral oil and levels of IL-12, IL-6, IFN-β, and IFN-γ in the washings were measured by sandwich ELISAs. Levels were compared using the Mann-Whitney test. B: Stimulation of Mx1 gene expression by IFN-α. RAW 264.7 cells were treated for 24 hours with IFN-α (1000 U/ml) in the absence or presence of anti-IFN-α neutralizing antibodies (1 or 2 μg/ml). Mx1 expression (normalized to β-actin) was measured from cDNA by real-time PCR. C: Specificity of Mx1 expression for IFN-I. RAW 264.7 cells were treated with IFN-β (1000 or 500 U/ml), IL-6 (5 ng/ml), TNF-α (20 ng/ml), or IL-12 (10 or 20 ng/ml) or with medium (Med) alone. After 24 hours, cells were harvested and RNA extracted for cDNA synthesis. Mx1 expression was determined by real-time PCR, normalized to β-actin. D: Increased expression in TMPD- versus mineral oil-elicited peritoneal cells. RNA was extracted and cDNA was synthesized from freshly collected peritoneal exudate cells of mice treated with TMPD or mineral oil. Expression of mRNAs for the IFN-inducible genes Mx1, IRF-7, and IP-10 as well as Toll-like receptors (TLR3, TLR4) and adapter proteins MyD88, TRAM, and TRIF was measured by real-time PCR normalized to β-actin. E: Correlation of Mx1 and IRF-7 expression. Expression of Mx1 and IRF-7 was measured (real-time PCR) in RNA isolated from either unstimulated peritoneal cells or LPS-stimulated peritoneal cells from the same group of mice (seven measurements). Expression of the two IFN-inducible genes correlated highly (r² = 0.9788, P < 0.0001). F: Mx1 expression by individual cell types. Dendritic cells (CD11c⁺), B cells (CD19⁺), monocytes/macrophages (CD11b⁺), and T cells (CD3⁺) were isolated from peritoneal exudates of TMPD-treated mice using magnetic beads and RNA was extracted. Mx1 expression by each cell type was determined by real-time PCR normalized to β-actin.

Figure 6

IFN-I-inducible gene expression in lipogranulomas. BALB/c mice were treated with TMPD or mineral oil and RNA was isolated from lipogranulomas adherent to the peritoneal lining or diaphragm at 12 weeks and used to synthesize cDNA. A: Expression of IFN-inducible genes. RT-PCR was performed for β-actin, IRF-7, Mx1, IP-10, and ISG-15 using lipogranuloma cDNA from mineral oil (MO)- or TMPD-treated mice. B: Mx1 expression, individual lipogranulomas. Expression of Mx1 normalized to β-actin was measured in individual lipogranulomas (n = 6) from mice treated with either TMPD or mineral oil by real-time PCR. Mx1 expression was significantly higher in TMPD versus mineral oil lipogranulomas (P = 0.0087, Mann-Whitney test). C: Mx1 expression, pooled lipogranulomas. Expression of Mx1 normalized to β-actin was measured in pooled lipogranulomas from six mice treated with TMPD and six mice treated with mineral oil. Mx1 expression (real-time PCR) was significantly higher in TMPD versus mineral oil lipogranulomas (P = 0.0022, Mann-Whitney test). D: Expression of other IFN-I-inducible genes, individual lipogranulomas. Gene expression in individual lipogranulomas from representative TMPD or mineral oil mice (five lipogranulomas per mouse) was measured by real-time PCR, normalized to β-actin. Expression of IFN-inducible genes Mx1, IRF-7, ISG-15, and IP-10 as well as TLR3 and TLR4 expression is shown. E: IL-4 expression in pooled lipogranulomas and peritoneal exudate. Expression of IL-4 normalized to β-actin (real-time PCR) was measured in pooled lipogranulomas and peritoneal exudate cells from four mice treated with TMPD and four mice treated with mineral oil (MO). F: Mx1 expression in the spleen. Mx1 expression normalized to β-actin (real-time PCR) was measured in spleen cells from mice treated with mineral oil (MO, n = 7) or TMPD (n = 12).

Figure 7

IFN-I and IL-12 production by CD11c⁺ peritoneal cells. A: Reduced TLR ligand stimulated Mx1 expression after depleting CD11c⁺ cells. Unfractionated peritoneal exudate cells and peritoneal exudate cells depleted of CD11c⁺ cells (magnetic beads) were treated with LPS (10 μg/ml), CpG ODN no. 1826 (10 μg/ml), or poly (I:C) (50 μg/ml) for 4 hours before extracting RNA. Mx1 expression was quantified by real-time PCR (normalized to β-actin). B: Reduced TLR ligand stimulated IL-12 production after depleting CD11c⁺ cells. CD11c⁺ cells from peritoneal exudate of a mouse treated 5 months previously with TMPD were depleted with magnetic beads, and TNF-α (left) and IL-12 (right) production in culture supernatants in response to LPS (10 μg/ml) or CpG DNA (10 μg/ml ODN no. 1826) were determined 24 hours later by ELISA.