NF-(kappa)B-inducing kinase controls lymphocyte and osteoclast activities in inflammatory arthritis

Abstract

NF-(kappa)B is an important component of both autoimmunity and bone destruction in RA. NF-(kappa)B-inducing kinase (NIK) is a key mediator of the alternative arm of the NF-(kappa)B pathway, which is characterized by the nuclear translocation of RelB/p52 complexes. Mice lacking functional NIK have no peripheral lymph nodes, defective B and T cells, and impaired receptor activator of NF-kappaB ligand-stimulated osteoclastogenesis. We investigated the role of NIK in murine models of inflammatory arthritis using Nik-/- mice. The serum transfer arthritis model is initiated by preformed antibodies and required only intact neutrophil and complement systems in recipients. While Nik-/- mice had inflammation equivalent to that of Nik+/+ controls, they showed significantly less periarticular osteoclastogenesis and less bone erosion. In contrast, Nik-/- mice were completely resistant to antigen-induced arthritis (AIA), which requires intact antigen presentation and lymphocyte function but not lymph nodes. Additionally, transfer of Nik+/+ splenocytes or T cells to Rag2-/- mice conferred susceptibility to AIA, while transfer of Nik-/- cells did not. Nik-/- mice were also resistant to a genetic, spontaneous form of arthritis, generated in mice expressing both the KRN T cell receptor and H-2. Thus, NIK is important in the immune and bone-destructive components of inflammatory arthritis and represents a possible therapeutic target for these diseases.

Figure 1

Equivalent inflammatory response to serum transfer arthritis. Response of Nik+/+ and Nik–/– mice to arthritogenic serum was evaluated from days 0 to 14 by clinical scoring (A) and measurement of hind paw thickness (B). Clinical scores were obtained by observation of all 4 paws, with a maximum score of 4 indicating whole-paw redness and swelling in all 4. Hind paw thickness was measured by digital gauge at the ankle, and the sum of the data for both paws was plotted. Graphs show mean ± SEM at each data point for 8 mice. There were no significant differences (P < 0.05) in either parameter between Nik+/+ and Nik–/– mice. (C) Semiquantitative RT-PCR analysis of RNA derived from hind paws at days 0, 3, and 4 shows that RANKL and TNF-α are both induced by injection of arthritogenic serum in Nik+/+ and Nik–/– animals.

Figure 2

Histological evaluation of STA. Hind paws from control mice (A, D, and G) and from Nik+/+ (B, E, and H) and Nik–/– (C, F, and I) mice with STA at day 14 were sectioned and stained with H&E (A–F) or TRAP (G–I). In control sections (A and D), bones were closely apposed, with smooth surfaces and no inflammatory infiltrate. The TRAP stain (G) shows scattered OCs (stained red) at bone/marrow interfaces (arrowheads) but none on bone surfaces. In contrast, Nik+/+ paws from serum-injected mice (B and E) had an extensive inflammatory infiltrate in the joint spaces. In the TRAP stain (H), OCs were prominent in marrow (arrowheads) and on bone surfaces (arrows), particularly where the inflammatory pannus was adjacent. In these areas, the bone surface was irregular, which indicates bone erosion. Nik–/– mice with arthritis (C and F) show an inflammatory infiltrate similar to that of Nik+/+ mice. However, bone contours remain smooth. The TRAP stain (I) shows OCs within the marrow (arrowhead) but few on bone surfaces, even where adjacent to pannus (arrow). Scale bar: 500 μm for A–C; 200 μm for D–F; and 100 μm for G–I. (J) Histological sections were scored for inflammation (left) based on H&E stains and for bone erosion (right) based on TRAP stains according to an arbitrary scale of 0–3. (K) TRAP-stained sections were evaluated by histomorphometry in the regions shown in H–I. The mean number of OCs per mm (± SD) present on the bone surface (left) and at the marrow interface (right) is shown. (*P < 0.01; **P < 0.00005, Nik–/– compared with Nik+/+; n = 7–8 per group). (L) Serum levels of TRAP5b, a form of the enzyme specific for active OCs, were determined 1 day prior to first serum injection (pre) and at day 14 (STA). *P < 0.01 compared with pre-injection Nik+/+ by paired Student’s t test; n = 4)

Figure 3

Histological evaluation of AIA. (A) PBS-injected control joint, with a relatively acellular joint space (between arrows). (B) mBSA-injected Nik+/– knee joint showing a severe inflammatory infiltrate (between arrows) that extends beyond the joint capsule, accompanied by bone erosion (cortical thinning; arrowhead). (C) mBSA-injected Nik–/– knee joint resembling control joint. (D) mBSA-injected Lt−α^–/– joint, showing severe inflammation despite a lack of lymph nodes. (E) mBSA-injected joint from Rag2–/– recipient of Nik+/+ unfractionated splenocytes showing severe arthritis. (F) mBSA-injected joint from Rag2–/– recipient of Nik–/– unfractionated splenocytes showing no arthritis. (G) mBSA-injected joint from Rag2–/– recipient of Nik–/– T cells and Nik+/+ B cells, showing no arthritis. (H) mBSA-injected joint from Rag2–/– recipient of Nik+/+ T cells and Nik–/– B cells, showing severe arthritis. Magnification in all panels, ×40.

Figure 4

Spontaneous K/BxN arthritis. Nik–/– mice and control littermates were bred to express both the KRN T cell receptor and H-2^g7 allele, a combination that leads to severe spontaneous arthritis in wild-type mice, apparent by 5 weeks. (A) Total paw thickness (sum of results for all 4 paws) in Nik+/? and Nik–/– mice at 5 and 11 weeks of age. *P < 0.0002 compared with Nik+/?. (B) Arthritic forepaw from Nik+/+ mouse. (C) Nonarthritic forepaw from Nik–/– mouse. (D) Arthritic hind paw from Nik+/+ mouse. (E) Nonarthritic hind paw from Nik–/– mouse. Magnification, ×40.