Humoral immune responses in DBA mice immunized with type II collagen (CII) and complete Freund's adjuvant (CFA) and treated with 1-methyl-tryptophan (1-MT) or vehicle

<p><b>Copyright information:</b></p><p>Taken from "Inhibition of indoleamine 2,3-dioxygenase-mediated tryptophan catabolism accelerates collagen-induced arthritis in mice"</p><p>http://arthritis-research.com/content/9/3/R50</p><p>Arthritis Research & Therapy 2007;9(3):R50-R50.</p><p>Published online 18 May 2007</p><p>PMCID:PMC2206348.</p><p></p> Concentrations of antibodies (total concentrations of IgGAM [immunoglobulins G, A, and M]) to heterologous (human) CII were determined in the serum of DBA/1 mice immunized with CII and CFA and treated with 1-MT or vehicle. Concentrations of antibodies (total concentrations of IgGAM) to autologous (mouse) CII were determined in the serum of DBA/1 mice immunized with CII and CFA and treated with 1-MT or vehicle. Sera were obtained on days 5, 22, 33, and 54. Values are presented as the mean and standard error of the mean of 25 1-MT-treated and 20 vehicle-treated DBA/1 mice per group and represent two independent experiments. *< 0.05 between vehicle and 1-MT-treated mice on the corresponding days

Incidence and severity of collagen-induced arthritis (CIA) in mice treated with 1-methyl-tryptophan (1-MT) or vehicle

<p><b>Copyright information:</b></p><p>Taken from "Inhibition of indoleamine 2,3-dioxygenase-mediated tryptophan catabolism accelerates collagen-induced arthritis in mice"</p><p>http://arthritis-research.com/content/9/3/R50</p><p>Arthritis Research & Therapy 2007;9(3):R50-R50.</p><p>Published online 18 May 2007</p><p>PMCID:PMC2206348.</p><p></p> Arthritis was first detected on day 26 after immunization with type II collagen on days 0, 21, and 42 (solid arrows) in mice treated with 1-MT or vehicle (open arrows). Incidence of CIA expressed as the percentage of arthritic animals. Disease severity expressed as the cumulative arthritis score in affected animals. Statistically significant differences in arthritis scores were found between days 37 and 47 (< 0.05). Values are presented as the mean and standard error of the mean of 25 1-MT-treated and 20 vehicle-treated DBA/1 mice per group and represent two independent experiments

Concentrations of interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the supernatants of spleen cells harvested from mice with collagen-induced arthritis and treated with 1-methyl-tryptophan (1-MT) or vehicle

<p><b>Copyright information:</b></p><p>Taken from "Inhibition of indoleamine 2,3-dioxygenase-mediated tryptophan catabolism accelerates collagen-induced arthritis in mice"</p><p>http://arthritis-research.com/content/9/3/R50</p><p>Arthritis Research & Therapy 2007;9(3):R50-R50.</p><p>Published online 18 May 2007</p><p>PMCID:PMC2206348.</p><p></p> The release of IL-4 and IFN-γ into the supernatants of spleen cells in response to human or mouse type II collagen (CII) was determined at the end of the experiment. Values are presented as the mean and standard error of the mean of 16 animals per group (*< 0.01)

Proliferation of spleen cells from mice with PG-induced arthritis (PGIA) in response to stimulation with peptide pools and individual peptides.

<p>Spleen cells from mice with PGIA were cultured with (A) Cit and R pairs of PG or OVA peptide pools or (B) Cit and R pairs of selected individual peptides (P) in triplicate wells. The Cit:R ratio of SI was calculated as described in the Methods. Data are expressed as the mean of Cit:R ratio of SI±SEM (n = 12 mice). Equal response to the Cit and R version of a peptide pool or peptide (theoretical Cit:R ratio of 1.0, at which no preference for the Cit or R version is assumed) is indicated with a dotted line. Statistical analysis was performed using Wilcoxon signed rank test (*p<0.05: Cit:R ratio vs 1.0) and Kruskal-Wallis test followed by Dunn’s multiple comparison test (#p<0.05: Cit:R ratio of PG13 vs Cit:R ratio of any other peptide pool).</p

Immune Recognition of Citrullinated Proteoglycan Aggrecan Epitopes in Mice with Proteoglycan-Induced Arthritis and in Patients with Rheumatoid Arthritis

<div><p>Background</p><p>Rheumatoid arthritis (RA) is an autoimmune inflammatory disease affecting the joints. Anti-citrullinated protein antibodies (ACPA) are frequently found in RA. Previous studies identified a citrullinated epitope in cartilage proteoglycan (PG) aggrecan that elicited pro-inflammatory cytokine production by RA T cells. We recently reported the presence of ACPA-reactive (citrullinated) PG in RA cartilage. Herein, we sought to identify additional citrullinated epitopes in human PG that are recognized by T cells or antibodies from RA patients.</p><p>Methods</p><p>We used mice with PG-induced arthritis (PGIA) as a screening tool to select citrulline (Cit)-containing PG peptides that were more immunogenic than the arginine (R)-containing counterparts. The selected peptide pairs were tested for induction of pro-inflammatory T-cell cytokine production in RA and healthy control peripheral blood mononuclear cell (PBMC) cultures using ELISA and flow cytometry. Anti-Cit and anti-R peptide antibodies were detected by ELISA.</p><p>Results</p><p>Splenocytes from mice with PGIA exhibited greater T-cell cytokine secretion in response to the Cit than the R version of PG peptide 49 (P49) and anti-P49 antibodies were found in PGIA serum. PBMC from ACPA+ and ACPA- RA patients, but not from healthy controls, responded to Cit49 with robust cytokine production. High levels of anti-Cit49 antibodies were found in the plasma of a subset of ACPA+ RA patients. Another PG peptide (Cit13) similar to the previously described T-cell epitope induced greater cytokine responses than R13 by control (but not RA) PBMC, however, anti-Cit13 antibodies were rarely detected in human plasma.</p><p>Conclusions</p><p>We identified a novel citrullinated PG epitope (Cit49) that is highly immunogenic in mice with PGIA and in RA patients. We also describe T-cell and antibody reactivity with Cit49 in ACPA+ RA. As citrullinated PG might be present in RA articular cartilage, Cit PG epitope-induced T-cell activation or antibody deposition may occur in the joints of RA patients.</p></div

Cit:R ratios of IL-17, IFNγ, IL-6, and IL-10 concentrations in peptide-stimulated culture supernatants of spleen cells from mice with PGIA or G1 domain-induced arthritis (GIA) or from naïve mice.

<p>Data are shown as the mean of Cit:R ratios±SEM for the concentrations of (A) IL-17, (B), IFNγ, (C) IL-6, and (D) IL-10 in peptide-treated spleen cell cultures from mice with PGIA (red bars) or GIA (blue bars) or from non-arthritic naïve mice (gray bars) (PGIA n = 10 mice; GIA n = 5 mice; Naïve n = 3 mice). Cit:R ratio of 1.0 is indicated by a dotted line in each graph. Wilcoxon signed rank test was used to identify Cit:R ratios significantly higher than 1.0 (*p = <0.05: Cit:R ratio vs 1.0). Cit:R ratios of cytokines induced by the P13 and P49 pairs of peptides in cells from the 3 groups of mice were compared using Kruskal-Wallis test followed by Dunn’s multiple comparison test (#p<0.05: PGIA or GIA vs naïve mice). Average net concentrations of IL-17 in the cell cultures stimulated with the Cit version of P13 (Cit13) were as follows: 86.4 pg/ml in the PGIA group, 70.5 pg/ml in the GIA group, and 76.8 pg/ml in the naïve group (graphs not shown). In the Cit49-stimulated cultures, the average net amounts of IL-17 were: 136.9 pg/ml in the PGIA group, 60.8 pg/ml in the GIA group, and 29.8 pg/ml in the naïve group (graphs not shown).</p

Cit:R ratios of IL-17, IFNγ, and IL-6, induced in human PBMC cultures by Cit and R pairs of P13 and P49.

<p>(A) Cit:R ratios of IL-17 induced by the P13 and P49 peptide pairs in PBMC cultures of anti-citrullinated protein antibody positive (ACPA+) and ACPA negative (ACPA-) RA patients and HC subjects. Data are expressed as the mean±SEM of Cit:R ratios of peptide-induced IL-17 in PBMC of RA all (purple bars), RA ACPA+ (red bars), RA ACPA- (blue bars) and HC (green bars) groups. (B) Net concentrations of IL-17 in the same PBMC cultures produced in the absence of a peptide (no peptide) or in the presence of peptide Cit13 or Cit49. Data shown are the mean±SEM of IL-17 (pg/ml). Cit:R ratios of (C) IFNγ and (D) IL-6 in the P13 and P49 peptide pair-stimulated PBMC cultures were determined as described for IL-17 in panel A above. Sample numbers for IL-17 (RA all n = 42; RA ACPA+ n = 32; RA ACPA- n = 10; HC n = 8), for IFNγ (RA all n = 28; RA ACPA+ n = 22; RA ACPA- n = 6; HC n = 8), and for IL-6 (RA all n = 41; RA ACPA+ n = 31; RA ACPA- n = 10; HC n = 7). Cit:R ratio of 1.0 (in panels A, C and D) is indicated by a dotted line. Cit:R ratios of cytokines (in A, C and D) were analyzed using Wilcoxon signed rank test (*p<0.05: Cit:R ratio vs 1.0) and Kruskal-Wallis test followed by Dunn’s multiple comparison test (#p<0.05: RA groups vs HC group). For the data in panel B, inter-group and inter-peptide comparisons were made using two-way ANOVA followed by Tukey’s test (#p<0.05: RA groups vs HC group) and Shidak’s test (^Xp<0.05: Cit49 vs Cit13 or no peptide).</p

Anti-PG peptide antibodies in serum samples from mice with PGIA or GIA, or from naïve mice.

<p>IgG antibodies reacting with Cit or R versions of 6 PG peptides were measured by ELISA using biotinylated Cit-R peptide pairs. Delta optical density at 450 nm (ΔOD 450 nm) was determined as described in the Methods. (A) Serum antibodies against the Cit versions of six PG peptides in mice with PGIA (red bars) or GIA (blue bars) or naïve mice (gray bars). Data are shown as the mean ΔOD 450 nm±SEM (PGIA n = 10 mice/peptide; GIA n = 3–5 mice/peptide; Naïve n = 3 mice/peptide). Inter-group comparisons were made using Kruskal-Wallis test followed by Dunn’s multiple comparison test (^#p<0.05: PGIA vs naïve mice). (B) Cit:R ratios of peptide-specific serum antibodies from mice with PGIA or GIA and from naïve mice. Data are expressed as Cit:R ratios of ΔOD 450 nm (mean±SEM) of serum antibodies specific for the peptide pairs in the groups of mice described above. Inter-group comparisons were made as described for graph A above (#p<0.05: GIA vs naïve or PGIA vs GIA mice). A Cit:R ratio of 1.0 is indicated by a dotted line. None of the Cit:R ratios were significantly different from 1.0 as determined by Wilcoxon signed rank test.</p

Cit:R ratios of cytokine concentrations in culture supernatants of peptide-stimulated spleen cells from mice with PGIA.

<p>Concentrations of cytokines IL-17, IFNγ, IL-6, and IL-10 in 4-day culture supernatants were measured by ELISA. Data are shown as the mean of Cit:R ratios±SEM for the concentrations of (A) IL-17, (B) IFNγ, (C) IL-6, and (D) IL-10. Cit:R ratio of 1.0 is indicated with a dotted line. Wilcoxon signed rank test was used to identify Cit:R ratios significantly higher than 1.0 (preference for a Cit pool or peptide) (*p<0.05; n = 10 mice). Cit:R ratio of the P13 pair-induced vs Cit:R ratio of any other peptide pair-induced production of cytokines in the same cultures was compared using Kruskal-Wallis test followed by Dunn’s multiple comparison test (#p<0.05).</p

Intracellular cytokines in CD4 cells from non-stimulated or peptide-stimulated PBMC cultures of RA patients and HC subjects.

<p>Representative flow cytometry panels of intracellular cytokines in CD4+ cells from non-stimulated or peptide-stimulated PBMC cultures of (A) an ACPA+ RA patient and (B) a HC subject. PBMC were cultured in the absence of peptide (no peptide) or in the presence of Cit or R versions of P13 (Cit13, R13) or P49 (Cit49, R49). Cells were labeled for cell-surface CD4 and intracellular IL-17 (IL-17A) and IFNγ with fluorochrome-tagged antibodies and subjected to flow cytometry as described in the Methods. In each case, the gate was set at CD4+ cells (gate not shown) and fluorescence signals were collected in 2-dimensional dot plots. Dots in the upper left quadrants of flow panels represent CD4 cells containing IL-17, dots in the lower right quadrants are cells containing IFNγ, the upper right quadrants show cells containing both IL-17 and IFNγ, and the cells in the lower left quadrants do not contain either of these cytokines. Cit:R ratios of (C) IL-17+ cells (D) IFNγ+ cells, and (E) double IL-17/IFNγ+ cells in P13 or P49-stimulated PBMC cultures of RA and HC groups. Data are expressed as mean±SEM of Cit:R ratios of cytokine-containing cells. Sample numbers for intracellular IL-17A, IFNγ, and IL-17/IFNγ (RA all n = 34; RA ACPA+ n = 23; RA ACPA- n = 9; HC n = 8). Cit:R ratio of 1.0 is indicated by a dotted line. Cit:R ratios significantly higher or lower than 1.0 were identified by Wilcoxon signed rank test (*p<0.05: Cit:R ratio vs 1.0) and Cit:R ratios of the groups were compared using Kruskal-Wallis test followed by Dunn’s multiple comparison test (#p<0.05: RA groups vs HC group).</p