Citrulline-specific antibodies mediate arthritis. (a) The frequency of arthritis on different days is shown. Groups (ACC1, n = 14; ACC4, n = 10; M2139 + PBS, n = 33; M2139 + ACC3, n = 10; M2139 + ACC4, n = 28; M2139 + ACC5, n = 10; M2139 + CIIC1, n = 20; and M2139 + GB8, n = 10) of 4-mo-old naive male B10.RIII mice were injected i.v. with 9 mg of a single mAb or an equal combination of two mAbs. M2139 with CIIC1 and 4.5 mg M2139 with PBS constituted positive and negative controls, respectively. 25 µg LPS per mouse was injected i.p. on day 5 to enhance the incidence and severity of arthritis. Results shown are pooled values from three similar experiments with balanced groups. M2139 + ACC4 versus M2139 + PBS, P ≤ 0.0113 (days 3–4) and P ≤ 0.001 (days 5–27); ACC1 versus ACC4, P ≤ 0.0034 (days 10–14 and days 21–27) and P ≤ 0.0013 (days 17–19). *, P < 0.05; **, P < 0.005; and ***, P < 0.001. (b) 2-mo-old QB = (BALB/c x B10.Q) F1 mice were injected with CII + CFA on day 0 and boosted with CII + IFA on day 35. These mice developed chronic arthritis that persisted for a minimum of 210 d. Mice that had no arthritis after 210 d were injected with 9 mg of a single mAb or an equal combination of two mAbs constituted different groups (ACC4, n = 9; PBS, n = 20; M2139 + PBS, n = 7; UL1 + PBS, n = 8; M2139 + ACC4, n = 10; and UL1 + ACC4, n = 9). M2139 with CIIC1 or UL1 and 4.5 mg M2139 or UL1 with PBS constituted positive and negative controls, respectively. LPS was not injected in these mice. The PBS-injected group denotes spontaneous relapse. All of the mice were used for calculations. Results shown are pooled values from two similar experiments with balanced groups. M2139 + ACC4 versus M2139 + PBS, P ≤ 0.026 (day 13) and P ≤ 0.026 (day 15); UL1 + ACC4 versus UL1 + PBS, P ≤ 0.0311 (day 5). *, P < 0.05 indicates a significant increase in arthritis frequency induced by ACC4 antibodies.

Molecular structural analysis of the ACC4 interacting with citrullinated collagen. (a) The structure of ACC4 Fab complexed with pCII-Cit1 peptide. The light chain of the Fab is shown in cyan, the heavy chain is shown in green, and the bound pCII-Cit1 peptide is shown in red. The overall shape of ACC4 Fab is same as the other Fab structures in the Protein Data Bank. The peptide has bound to the interface between CDR loops on top of the variable domain, as anticipated. The images were created with PyMOL (reference 60). (b) The structure of the peptide pCII-Cit1. The difference (2Fo-Fc) electron density map is shown in blue. The density is contoured at 1 σ. The water molecule is shown as a red ball. The electron density is strong enough to build nine residues of peptides, shown as sticks. The PCII-Cit1 peptide adopts a β turn conformation. Cit6P represents citrulline residue 6 and Hyp12P represents hydroxyproline residue 12 of the peptide. (c) Surface representations of ACC4 Fab–peptide complex from the top (left) and the side (right). The pCII-Cit1 peptide is shown as sticks. The water molecule on the peptide is shown as a red ball. The peptide fills a cavity on top of the variable domain. Although the C-terminal side of the peptide is buried inside the cavity, the N-terminal side is solvent exposed. (d) Fab–peptide interactions. (A) The residues that contact with citrulline (Cit6P). (B) The residues that contact with arginine (Arg11P). The black dashed lines represent hydrogen bonds. The green residues are from the heavy chain and the cyan residues are from the light chain.

ACC mAb antibody specificity in vitro and in vivo. (a) ACC mAb specificity in ELISA. Several arginine-containing peptides and their citrulline analogues have been tested to characterize the citrulline specificity of our generated antibodies. Some of the epitopes were accessible in the triple helical form of the citrulline-containing C1^III epitope (THPCII-Cit) for some of the antibodies (ACC1–3 and ACC5; A and B). Nevertheless, this epitope seems not to be accessible for ACC4. Some of the antibodies cross react to selected citrullinated peptides even with a noncollageneous backbone. ACC2, ACC3, and ACC5 bind not only to CII but also to the citrullinated derivative of fibrinogen (C and D). Furthermore, ACC4 and ACC5 bind stronger to the citrullinated form of cyclic filaggrin (cyc-Cit) than to its arginine-containing form (cyc-Arg; E and F). Enzymatic deimination of CII with PAD4 generates neoepitopes, which are recognized by ACC2–4 (G–I). C1^III peptides containing an additional biotinylated lysine have been synthesized, which in turn bind to NeutrAvidin-precoated ELISA plates (J–M). This setup allows free accessibility of the different antibodies specific for the citrulline-modified immunodominant CII epitopes. All of the assays were done in duplicate. (b) Staining of arthritic joints. Citrulline-specific antibodies bind to arthritic cartilage. Joint sections (10 mm) from arthritic BALB/c (A, C, and D), naive (BALB/c × B10.Q) F1 (B), or BALB/c (E) mice are shown. Results shown are representative histological pictures of arthritic (n = 4) and control (n = 3) mice used in the staining of joints with anticitrulline antibodies. Arthritis was induced in 4–6-mo-old naive male BALB/c mice (n = 36) by injecting 9 mg of an arthritogenic anti-CII mAb cocktail containing antibodies M2139 (binding to the J1 epitope) and CIIC1 (binding to the C1^I epitope). The arthritis induction experiment was performed four times independently with 100% incidence and a mean maximum arthritis score of 32.9 ± 3. The four mice that were used for histology had arthritis scores of 59, 60, 47, and 50. Paw samples were taken on day 11 after antibody transfer (5 d after LPS injection). Sections were treated with no antibodies (A), ACC1 (B), or ACC4 (C–E) mAbs. Magnification, ×10.

Detection of citrullinated CII in human synovial fluid. (a) Immunoprecipitation of citrullinated CII from synovial fluid. Western blot staining with the antimodified citrulline antibody (AMC) upon appropriate pretreatment of the nitrocellulose membrane and immunostaining with the anticitrullinated CII mAb ACC2 are shown (+, ELISA-positive synovial fluid; −, ELISA-negative synovial fluid [compare with b]). Shown is a representative result of three independently performed experiments. (b) Capture ELISA for detection of citrullinated CII in synovial fluid specimens obtained from patients with OA, Reac A, and RA. Synovial fluid specimens were collected from patients with OA (n = 47), RA (n = 10), and Reac A (n = 15).