Schematic illustration of PTMs of Arg and Lys. A, Deimination (citrullination) is an enzymatic modification characterized by cleavage of NH₃ groups from Arg residues mediated by peptidylarginine deiminases. B, Carbamylation (homocitrullination) is characterized by the addition of a CONH₂⁻ group to Lys residues.

Immunization with carbamylated peptide predisposes to the development of erosive arthritis. Mice were immunized with Hcit-peptide (Hcit) and Cit-peptide (Cit) (75 μg/mouse) on days 0 and 14. Intra-articular injections of Cit-peptide (A) and Hcit-peptide (B) (1 μg/knee) were given on day 21. Histological evaluation of injected knees was undertaken on day 28; data are presented as arthritis index. Values from three independent experiments using NMRI mice were pooled (n = 60). The arthritis index for Hcit-immunized mice was 2.21-fold higher compared with Cit-immunized and nonimmunized mice (p < 0.001). Values represent the mean ± SEM. Horizontal lines indicate the medians. C, Representative histological changes seen in the injected joints of mice immunized with Hcit (1) and Cit (2) followed by intra-articular injection of Cit-peptide, and nonimmunized controls (3, 4). Arthritis induction was performed in five independent experiments, giving a 92% incidence of arthritis in three wild-type strains (NMRI, BALB/c, and Asn) (n = 126). Immunological staining of Hcit-immunized and Cit-immunized mice was done using rat anti-mouse CD3 Ab. Large numbers of CD3⁺ cells (brown staining) were observed in the synovia of Hcit-immunized mice (5) compared with Cit-immunized mice (6). Original magnification ×500. Tissue sections were stained with H&E (C1–C4). Immunostaining for CD3 was developed with 3-amino-9-ethylcarbazole. Hematoxylin was used as counterstain (C5, C6).

Immunization with carbamylated peptide (Hcit) increases cell migration, T cell proliferation, and cytokine production upon CD3-mediated activation. A, Splenocytes from Hcit-immunized (n = 5), Cit-immunized (n = 5), and nonimmunized mice (n = 4) were allowed to migrate along a CXCL13 gradient (1 μg/ml) for 12 h. Flow cytometry analysis of the transmigrated cells showed more CD4⁺ T cells (1) in cultures from Hcit-immunized mice compared with Cit-immunized mice (p < 0.001) and nonimmunized controls. The transmigrated CD8⁺ (2) and CD19⁺ (3) populations were similar in Hcit- and Cit-immunized mice. Values represent mean ± SEM. Horizontal lines indicate the median. B, Thymidine [³H] proliferation assay following stimulation of spleen cells (1 × 10⁶ cells/ml) from Hcit-immunized mice (n = 8), Cit-immunized mice (n = 8), and nonimmunized controls (n = 6) with anti-CD3 Abs (1 μg/ml) shows a 2.2-fold increase in the proliferation rate of Hcit-immunized mice compared with Cit-immunized mice (p < 0.0001). Splenocytes from Cit-immunized mice and nonimmunized controls show no differences in their proliferation rates. Nonstimulated cells were used as a negative control. Results represent pooled values from three independent experiments. Values are presented as mean ± SEM. Horizontal lines indicate the median. C, Cytokine production was measured in the supernatants of anti-CD3–stimulated splenocyte cultures. Cells from Hcit-immunized mice produced higher levels of IL-10 (1), IFN-γ (2), and IL-17 (3) than Cit-immunized mice or nonimmunized controls (p < 0.01).

T and B lymphocytes of mice immunized with carbamylated peptide (Hcit) predispose to Cit-induced arthritis. CD3⁺ and CD19⁺ cells were isolated by negative selection from the spleens of Hcit-immunized BALB/c mice and injected i.v into naive BALB/c recipients (5 × 10⁶ cells/mouse, n = 10). Control BALB/c mice received the same number of CD3⁺ or CD19⁺ cells isolated from naive BALB/c mice (n = 10). On day 12 following lymphocyte transfer, all mice received intra-articular injections of Cit-peptide (1 μg/knee). A, Histological evaluation of injected knee joints 7 d later showed development of severe arthritis in 100% of the recipients of CD3⁺ cells from Hcit-immunized mice (arthritis index, 2.19 ± 0.54) (1, 3), whereas only two recipients of naive CD3⁺ cells showed evidence of arthritis (arthritis index, 0.4 ± 0.41; p = 0.003) (2, 3). Knees were embedded in paraffin, sectioned, and stained with H&E. Original magnification ×500. B, Recipients of Hcit-immunized CD3⁺ cells also showed a significant increase in the T cell proliferation rate (p < 0.0001) (1) and produced more IFN-γ compared with controls (2). C, Recipients of Hcit-immunized CD3⁺ or CD19⁺ cells showed significantly higher levels of Hcit-specific Abs, as assessed by ELISPOT. Values represent mean ± SEM. Horizontal lines indicate the median.

Quantitative evaluation of carbamylated (Hcit) and citrullinated (Cit) peptides in proteins recovered from blood and synovial fluid of patients with RA. A, Quantitative evaluation of Hcit and Cit proteins recovered from blood and synovial fluid was done by mass spectrophotometry and expressed as absolute concentrations (μmol). Hcit Ab levels are presented as OD at 450 nm with 1:100 serum dilution. Levels of Hcit-Abs in serum and synovial fluid were measured using ELISA. Patient material was stratified according to radiological findings into erosive, nonerosive, and control groups. The erosive group has higher levels of Hcit-Ab compared with controls (p = 0.019) and the nonerosive group (p = 0.024). B, Levels of Hcit-peptides measured by mass spectrometry in proteins recovered from blood are increased in erosive and aCCP⁺ RA patients compared with erosive and aCCP⁻ patients and the control group (p < 0.05). C, Cit-peptide levels in blood of RA patients with erosive changes are higher compared with the control group but not within the nonerosive group. D, Hcit-Abs in synovial fluid of erosive RA patients are higher that in patients with nonerosive RA (p < 0.05). E, Absolute levels of Hcit-peptides are identical among the groups. F, Cit-peptide levels in synovial fluid are higher than in control group, regardless of aCCP status of RA patients. Blood and synovial fluid samples were collected from RA patients (n = 72) and controls with knee trauma (n = 41) and osteoarthritis (n = 40). Values are presented as mean ± SEM. Horizontal lines indicate the median.