Pharmacological blockade of CCR1 ameliorates murine arthritis and alters cytokine networks in vivo

Abstract

Background and purpose: The chemokine receptor CCR1 is a potential target for the treatment of rheumatoid arthritis. To explore the impact of CCR1 blockade in experimental arthritis and the underlying mechanisms, we used J-113863, a non-peptide antagonist of the mouse receptor.

Experimental approach: Compound J-113863 was tested in collagen-induced arthritis (CIA) and three models of acute inflammation; Staphylococcus enterotoxin B (SEB)-induced interleukin-2 (IL-2), delayed-type hypersensitivity (DTH) response, and lipopolysaccharide (LPS)-induced tumour necrosis factoralpha (TNFalpha) production. In the LPS model, CCR1 knockout, adrenalectomised, or IL-10-depleted mice were also used. Production of TNFalpha by mouse macrophages and human synovial membrane samples in vitro were also studied.

Key results: Treatment of arthritic mice with J-113863 improved paw inflammation and joint damage, and dramatically decreased cell infiltration into joints. The compound did not inhibit IL-2 or DTH, but reduced plasma TNFalpha levels in LPS-treated mice. Surprisingly, CCR1 knockout mice produced more TNFalpha than controls in response to LPS, and J-113863 decreased TNFalpha also in CCR1 null mice, indicating that its effect was unrelated to CCR1. Adrenalectomy or neutralisation of IL-10 did not prevent inhibition of TNFalpha production by J-113863. The compound did not inhibit mouse TNFalpha in vitro, but did induce a trend towards increased TNFalpha release in cells from synovial membranes of rheumatoid arthritis patients.

Conclusions and implications: CCR1 blockade improves the development of CIA, probably via inhibition of inflammatory cell recruitment. However, results from both CCR1-deficient mice and human synovial membranes suggest that, in some experimental settings, blocking CCR1 could enhance TNF production.

Figure 1

Pharmacokinetic profile of J-113863 after a single i.p. injection of 3 or 10 mg kg⁻¹ to mice. The values of the area under the curve for each dose during the period studied (AUC_0-24) are also shown. Three animals were used for each time point.

Figure 2

Effect of J-113863 on collagen-induced arthritis. DBA-1 mice with established disease received either vehicle (V) or the test drug for 11 days by i.p. route. (a) Effect of J-113863 at 3 mg kg⁻¹ (J-3) and 10 mg kg⁻¹ (J-10) on the clinical manifestations of the disease (arthritic index). (b) Effect of the compound on anti-collagen II antibodies of the IgG1 and IgG2A subtypes, measured at the end of the experiment. Results shown are representative of three independent experiments using seven mice per treatment group. ^*P<0.05; ^**P<0.01 vs vehicle, Student's t-test.

Figure 3

Effect of the CCR1 antagonist on paw histology. Microscopic images (× 10 for (a, c); 20 × for (b)) representative of the paw of arthritic mice treated with vehicle or 10 mg kg⁻¹ of J-113863 illustrating the joint space (asterisks), the invasive pannus (arrowheads), and the presence of osteoclasts (arrows). (d) Inhibition of the joint damage score by J-113863 at 3 and 10 mg kg⁻¹ vs vehicle. Results shown are representative of three independent experiments using seven mice per treatment group. ^*P<0.05 vs vehicle-treated mice, Student's t-test.

Figure 4

Effect of J-113863 on the plasma levels of TNFα (a) and IL-10 (b) at 1.5 h after LPS challenge to Swiss mice. Results are the mean and s.e.m. of three independent experiments, each using five animals per treatment group. ^*P<0.05, ^**P<0.01 vs vehicle-treated mice, Student's t-test.

Figure 5

Effect of CCR1 deficiency on the plasma levels of TNFα and IL-10. Control (c) and CCR1 knockout mice (ko) were challenged with saline or LPS; plasma levels of TNFα and IL-10 were analyzed. Results are the means and s.e.m. of three independent experiments, each using five animals/treatment group. ^*P<0.05 ko vs control mice, Student's t-test.

Figure 6

Effect of vehicle or J-113863 at 10 mg kg⁻¹ on the plasma levels of TNFα (a) and IL-10 (b) in CCR1 ko mice challenged with LPS. Results are the means and s.e.m. of three independent experiments, each using five animals per treatment group. ^*P<0.05, ^**P<0.01 vs corresponding vehicle-treated mice, Student's t-test.

Figure 7

Effect of anti-IL-10 treatment on the TNF inhibition by J-113863 in LPS-challenged mice. Mice were treated with an anti-IL-10 antibody or corresponding isotype before the administration or vehicle or compound J-113863 at 10 mg kg¹. LPS was injected and plasma contents of TNFα (a) and IL-10 (b) measured. Results shown are representative of two independent experiments using five animals per group. ^**P<0.01 vs corresponding vehicle-treated mice, Student's t-test.

Figure 8

Involvement of the HPA axis in the effect of J-113863 on TNFα and IL-10. Adrenalectomized or sham-operated mice received vehicle (V) or 10 mg kg⁻¹ of J-113863 (J) or rolipram (also 10 mg kg⁻¹; R) followed, half an hour later, by an LPS challenge. Results shown are representative of two independent experiments using five animals per group. ^*P<0.05, ^**P<0.01 vs corresponding vehicle-treated mice, Student's t-test.