CCR5 is involved in resolution of inflammation in proteoglycan-induced arthritis

Abstract

Objective: CCR5 and its ligands (CCL3, CCL4, and CCL5) may play a role in inflammatory cell recruitment into the joint. However, it was recently reported that CCR5 on T cells and neutrophils acts as a decoy receptor for CCL3 and CCL5 to assist in the resolution of inflammation. The aim of this study was to determine whether CCR5 functions as a proinflammatory or antiinflammatory mediator in arthritis, by examining the role of CCR5 in proteoglycan (PG)-induced arthritis (PGIA).

Methods: Arthritis was induced by immunizing wild-type (WT) and CCR5-deficient (CCR5(-/-)) BALB/c mice with human PG in adjuvant. The onset and severity of PGIA were monitored over time. Met-RANTES was used to block CCR5 in vivo. Arthritis was transferred to SCID mice, using spleen cells from arthritic WT and CCR5(-/-) mice. The expression of cytokines and chemokines was measured by enzyme-linked immunosorbent assay.

Results: In CCR5(-/-) mice and WT mice treated with the CCR5 inhibitor Met-RANTES, exacerbated arthritis developed late in the disease course. The increase in arthritis severity in CCR5(-/-) mice correlated with elevated serum levels of CCL5. However, exacerbated arthritis was not intrinsic to the CCR5(-/-) lymphoid cells, because the arthritis that developed in SCID mouse recipients was similar to that in WT and CCR5(-/-) mice. CCR5 expression in the SCID mouse was sufficient to clear CCL5, because serum levels of CCL5 were the same in SCID mouse recipients receiving cells from either WT or CCR5(-/-) mice.

Conclusion: These data demonstrate that CCR5 is a key player in controlling the resolution of inflammation in experimental arthritis.

Figure 1

Expression of CCR5 ligands in synovial fluid of arthritic mice. (A) Synovial fluid was obtained from inflamed joints of arthritic WT mice during peak of inflammation and examined by ELISA for CCL3, 4, 5 (MIP1α, MIP1β and RANTES respectively). Results shown are average ± SEM. (B) Synovial tissue cells were obtained from joints of arthritic mice and CD4⁺ T cells and GR-1⁺ neutrophils were stained for CCR5 expression by flow cytometry. Results are average ± SD.

Figure 2

Suppression of early arthritis and exacerbation of late arthritis in mice treated with CCR5/CCR1 antagonist Met-RANTES. (A–D) Spleen cells (2.5×10⁷) from arthritic WT mice were transferred to SCID mice along with 100 μg hPG i.p. on day 0. (A and B) Mice were administered 100 μg Met-RANTES in PBS (n=5) or PBS (n=5) as control on day 6 after cell transfer and every third day for nine days. (C and D) Mice were administered 100 μg Met-RANTES in PBS (n=5) or PBS (n=5) as control on day 12 after cell transfer and every third day for nine days. Arthritis incidence (A and C) and arthritis score (B and D) were monitored daily by a blinded observer. Data represents daily averages ±SEM. Asterisks (*) denote significant differences (p≤0.05). Data are representative of three experiments.

Figure 3. Arthritis is exacerbated late in CCR5^−/− mice

WT (n=10) and CCR5^−/− (n=13) age matched female BALB/c mice were immunized i.p. with human PG in adjuvant three times at three week intervals and monitored for arthritis onset and severity by a blinded observer. (A) Arthritis incidence is expressed as the percentage of mice that developed arthritis. (B) Arthritis score is the sum of paw inflammation scores for each mouse divided by the number of arthritic mice. Results are shown as weekly mean scores ±SEM. Asterisks (*) denote significant differences (p≤0.05) compared to WT. (C) WT and (D) CCR5^−/− ankle joint histology. Sections were stained with hematoxylin and eosin.

Figure 4

Inflammatory cytokine expression, autoantibody production, and T cell proliferation expression similar in WT and CCR5^−/− mice. (A, B, and C) Purified T cells were obtained from immunized WT and CCR5^−/− mice and stimulated in the presence of PG and irradiated naïve spleen cells. Culture supernatants were harvested at 4 days and assayed for IFN-γ, IL-17, and IL-4 by ELISA. (D) Purified T cells were stimulated as above in (A, B, and C). PG-specific T cell proliferation was measured by the incorporation of [³H]-thymidine. (E) Serum was obtained and anti-murine PG (mPG) antibody isotypes (IgG1 and IgG2a) were measured by ELISA. Results are the average ± SEM. (WT n=7–10, CCR5 n=10–11).

Figure 5

Elevated expression of CCL5 in CCR5^−/− mice. Serum was taken from WT and CCR5^−/− mice before immunization and 10 days after each immunization. Individual serum samples were tested for CCL3, CCL4, and CCL5 by ELISA. Results are the average ± SEM. (WT n=13, CCR5 n=13.). Asterisks (*) denote significant differences (p≤0.05) compared to WT.

Figure 6

Disease transferred to SCID mice effectively. Spleen cells (4×10⁷) from WT or CCR5^−/− mice were injected i.p. into SCID recipients with PG and monitored for arthritis incidence (A) and severity (B) by a blinded observer. Results shown are average ± SEM. (WT n=8, CCR5 n=9). (C) SCID recipients of CCR5^−/− spleen cells controlled serum CCL5 concentrations similar to WT cells. Individual serum samples from SCID mice repopulated with WT or CCR5^−/− spleen cells at week 3 were examined by ELISA for CCL5 expression. Results shown are average ± SEM. (WT n=6, CCR5 n=6).