Arthritis suppressor genes TIA-1 and TTP dampen the expression of tumor necrosis factor alpha, cyclooxygenase 2, and inflammatory arthritis

Abstract

TIA-1 and TTP are AU-rich element-binding proteins that prevent the pathological overexpression of tumor necrosis factor alpha (TNF-alpha). TIA-1 inhibits the translation of TNF-alpha transcripts, whereas TTP promotes the degradation of TNF-alpha transcripts. Here we show that TIA-1 and TTP function as arthritis suppressor genes: TIA-1(-/-) mice develop mild arthritis, TTP(-/-) mice develop severe arthritis, and TIA-1(-/-)TTP(-/-) mice develop very severe arthritis. Peritoneal macrophages derived from all three genotypes overexpress cyclooxygenase 2 and TNF-alpha. Surprisingly, lipopolysaccharide-activated TIA-1(-/-)TTP(-/-) macrophages secrete less TNF-alpha protein than either TIA-1(-/-) or TTP(-/-) macrophages. In these mice, arthritogenic cytokine may be produced by neutrophils that accumulate in the bone marrow and peripheral blood. Our results suggest that TIA-1 and TTP are genetic modifiers of inflammatory arthritis that can alter the spectrum of cells that produce arthritogenic cytokines.

Fig. 1.

Growth curves for wild-type and knockout mice. TIA-1/TTP nullizygous mice are smaller than wild-type or TTP^+/– mice at birth and exhibit delayed growth.

Fig. 2.

Arthritis severity in wild-type and mutant mice. (A) Arthritis index. Wild-type, TIA-1^–/–, TTP^–/–, and TIA-1^–/–/TTP^–/– adult mice were blindly assessed for arthritis severity and were assigned a score based on the arthritis index described in Materials and Methods. Data show the means ± SE from 12 mice of each genotype. (B) Histologic assessment of arthritis. Shown is a hematoxylin/eosin stain of proximal interphalangeal joint from the front paw from the indicated genotype. (Magnification, ×20.) All histologic assessments were determined by using age-matched mice (6 months old). (Upper Left) Wild type (WT). (Upper Right) TIA-1^–/– (TIA1 KO). Arrows indicate bone erosion from the articular surface. (Lower Left) TTP^–/– (TTP KO). Arrows indicate hypercellular bone marrow with osteoclastogenic activation in the marrow space. (Lower Right) TIA-1^–/–/TTP^–/– (TIA1/TTP KO) mice. Arrows indicate thickened inflamed synovium with bone erosion from the articular surface.

Fig. 3.

Expression of COX-2 and TNF-α in LPS-activated peritoneal macrophages. (A) Peritoneal macrophages (derived from wild-type, TIA-1^–/–, TTP^–/–, and TIA-1/TTP^–/– mice) were cultured in the absence (–) or presence (+) of LPS (1 μg/ml for 4 h) before processing for Western blot analysis using antibodies reactive with COX-2. (B) LPS-activated macrophages of the indicated genotypes were processed for Western blot analysis by using antibodies reactive with TNF-α. The relative migration of molecular mass markers is shown at the left.

Fig. 4.

Expression of TNF-α mRNA and protein in LPS-activated peritoneal macrophages. (A) Quantitation of TNF-α mRNA using real-time PCR. Total cellular mRNA was extracted from LPS-activated peritoneal macrophages (derived from wild-type, TIA-1^–/–, TTP^–/–, and TIA-1^–/–/TTP^–/– mice) using the TRIzol method. TNF-α RNA and 18S rRNA were quantitated by using real-time PCR. The expression of TNF-α mRNA was normalized to 18S rRNA and is shown in arbitrary units. Pooled RNA from three mice was subjected to RT-PCR. Data show the average of two independent analyses. (B) Quantitation of TNF-α protein using ELISA. Supernatants from LPS-activated peritoneal macrophages derived from 12 wild-type, 10 TIA-1^–/–, 5 TTP^–/–, and 12 TIA-1/TTP^–/– mice were assayed for soluble TNF-α. Each bar shows the mean secretion ± SE. The open bar in the TIA-1^–/–/TTP^–/– sample depicts the expected production of TNF-α if the effects of TIA-1 and TTP were additive.

Fig. 5.

Quantitation of bone marrow and peripheral blood neutrophils. (A) Bone marrow histology. All histologic assessments were determined by using age-matched mice (4–6 months old). Bone marrow from wild-type, TIA-1^–/–, TTP^–/–, and TIA-1^–/–/TTP^–/– mice was analyzed by hematoxylin/eosin staining. (B) Peripheral blood counts from wild-type, TIA-1^–/–, TTP^–/–, and TIA-1^–/–/TTP^–/– mice shows total white blood count (WBC), hematocrit (HCT), absolute neutrophil count (ABS NEUTRO), percent of neutrophils (% NEUTRO), absolute lymphocyte count (ABS LYMPH), and percent of lymphocytes (% LYMPH). Data are the means and SE from three different mice. Statistical analysis (P < 0.05) compares wild-type and TIA-1^–/–/TTP^–/– mice.

Fig. 6.

Production of TNF-α by bone marrow cells and bone marrow-derived Gr-1⁺ neutrophils. (A) Supernatants harvested from LPS-activated bone marrow cells derived from wild-type, TIA-1^–/–, TTP^–/–, and TIA-1^–/–/TTP^–/– mice were assayed for TNF-α by using an ELISA. Data depict the means and SE from three independent experiments. (B) Percoll gradient-enriched, bone marrow-derived Gr-1⁺ neutrophils from wild-type, TIA-1^–/–, TTP^–/–, and TIA-1^–/–/TTP^–/– female mice were activated with LPS (1 μg/ml for 4 h) before harvesting supernatants for quantitation of TNF-α using an ELISA. Data depict the means and SE from three independent experiments.