Blockade of collagen-induced arthritis post-onset by antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF): requirement for GM-CSF in the effector phase of disease

Abstract

There is mounting evidence for a role of the growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) in inflammatory disease, including arthritis. In the present study, we examined the effectiveness of treatment of collagen-induced arthritis (CIA) with a neutralizing mAb to GM-CSF. DBA/1 mice were immunized for the development of CIA and treated at different times, and with different doses, with neutralizing mAb to GM-CSF or isotype control mAb. Anti-GM-CSF mAb treatment prior to the onset of arthritis, at the time of antigen challenge, was effective at ameliorating the ensuing disease. Modulation of arthritis was seen predominantly as a reduction in overall disease severity, both in terms of the number of limbs affected per mouse and the clinical score of affected limbs. Importantly, anti-GM-CSF mAb treatment ameliorated existing disease, seen both as a reduction in the number of initially affected limbs progressing and lower numbers of additional limbs becoming affected. By histology, both inflammation and cartilage destruction were reduced in anti-GM-CSF-treated mice, and the levels of tumor necrosis factor-a and IL-1beta were also reduced in joint tissue washouts of these mice. Neither humoral nor cellular immunity to type II collagen, however, was affected by anti-GM-CSF mAb treatment. These results suggest that the major effect of GM-CSF in CIA is on mediating the effector phase of the inflammatory reaction to type II collagen. The results also highlight the essential role of GM-CSF in the ongoing development of inflammation and arthritis in CIA, with possible therapeutic implications for rheumatoid arthritis.

Figure 1

Effect of anti-GM-CSF treatment prior to the onset of arthritis. (a) Severity (mean clinical scores ? SEM). (b) Cumulative incidence. Mice were treated intraperitoneally every second day from days 21 to 31 with anti-GM-CSF mAb or control mAb. ^*P = 0.05 and ^**P = 0.01 compared with control mAb-treated. GM-CSF, Granulocyte macrophage-colony stimulating factor; mAb, monoclonal antibody.

Supplementary Figure 1

Effect of anti-GM-CSF treatment prior to the onset of arthritis on individual limb involvement. Data are from the experiment in Fig. 1 where mice were treated intraperitoneally with either 300 ?g anti-GM-CSF or 300 ?g isotype control every second day from days 21 to 31. (a) The number of mice that developed arthritis in a given number of limbs is presented. Anti-GM-CSF (n = 9) versus isotype control mice (n = 10) (P = 0.048; multiway chi-squared). There were no anti-GM-CSF-treated mice with all four limbs affected. (b) The number of individual limbs from arthritic mice only with a particular clinical score (severity) is presented. For the anti-GM-CSF-treated group, n = 20 limbs (5 mice); for the isotype control-treated group, n = 36 limbs (9 mice). There were no limbs from anti-GM-CSF-treated mice with a clinical score of 3. GM-CSF, Granulocyte macrophage-colony stimulating factor.

Supplementary Figure 2

Effect of timing of anti-GM-CSF treatment on arthritis development. (a) Mice treated on day 21 (n = 17, anti-GM-CSF) or days 21 and 23 (n = 20, anti-GM-CSF; n = 20, isotype control). ^* Days 21 and 23 treatment, anti-GM-CSF treated versus isotype control treated mice (day 40) (P = 0.044; Mann-Whitney test). (b) Mice treated at the time of primary immunization (day 0) only (n = 10, anti-GM-CSF; n = 9, isotype control). (c) Mice treated at the time of primary immunization (day 0) then weekly until day 21 (n = 10, anti-GM-CSF; n = 10, isotype control). For all treatments, 300 ?g anti-GM-CSF or isotype control was used. Results are expressed as the mean ? SEM. GM-CSF, Granulocyte macrophage-colony stimulating factor.

Figure 2

Effect of anti-GM-CSF treatment on established arthritis. Mice were treated daily (days 0-9) with anti-GM-CSF mAb or control mAb. Results are expressed as the mean ? SEM (P = 0.007). GM-CSF, Granulocyte macrophage-colony stimulating factor; mAb, monoclonal antibody.

Supplementary Figure 3

Effect of anti-GM-CSF treatment post-onset on individual limb involvement. Data are from the experiment in Fig. 2 where mice were treated intraperitoneally with 300 ?g anti-GM-CSF (n = 20 mice) or isotype control (n = 19 mice) daily from the day of disease onset (day 0) until day 9 (total, 10 times). (a) The progression of arthritis in limbs already affected prior to the commencement of treatment is presented: improvement, decrease in clinical score of individual limbs following treatment; no change, clinical score of individual limbs remained the same following treatment; mild progression, clinical score of individual limbs progressed from 1 initially to 2 at the end of treatment; progression, clinical score of individual limbs progressed from 1 or 2 initially to 3 at the end of treatment (n = 33 limbs [from 20 mice], anti-GM-CSF-treated group; and n = 31 limbs [from 19 mice], isotype control-treated group) (P = 0.01; multiway chi-squared). (b) The number of limbs, initially unaffected (clinical score 0, day 0), that developed a particular score at the end of the experiment is presented (n = 47 limbs [from 20 mice], anti-GM-CSF-treated group; and n = 45 limbs [from 19 mice], control-treated group) (P = 0.0007; multiway chi-squared). There were no limbs recruited in anti-GM-CSF-treated mice that developed a clinical score > 1. GM-CSF, Granulocyte macrophage-colony stimulating factor.

Figure 3

Effect of anti-GM-CSF treatment post-onset on joint histopathology of hind-limb distal interphalangeal joints with CIA. (a), (b) Normal joint; (c), (d) joint from a control mAb-treated mouse; and (e), (f) joint from an anti-GM-CSF mAb-treated mouse. For control and anti-GM-CSF mAb-treated mice, both limbs had a clinical score of 1 at onset. The control mAb-treated mouse limb progressed to a score of 3, whereas the anti-GM-CSF mAb-treated mouse limb showed no change over the course of treatment. These results are representative for each treatment group. (c) Severe inflammation and joint destruction is shown compared with (a). (e) Only mild infiltration and damage is evident with the architecture of the joint remaining intact. This is also reflected in (d), where there is loss of proteoglycan staining (arrowheads) compared with (b), while (f) shows intermediate staining. (a), (c) and (e) H & E staining; (b), (d) and (f) Safranin O, fast green staining. Magnification, x 125. B, Bone; C, cartilage; CIA, collagen-induced arthritis; GM-CSF, granulocyte macrophage-colony stimulating factor; J, joint space; M, bone marrow; mAb, monoclonal antibody.

Supplementary Figure 4

Effect of anti-GM-CSF treatment on CII-specific immune responses. (a) IgG Ab response to CII measured at the end of each experiment. Results are expressed as the mean ? SEM, where the mean level of anti-CII IgG for isotype control-treated mice was set at 100 U/ml for each experiment and compared with anti-GM-CSF-treated mice from the same experiment. (b) Proliferative response to CII measured in mice treated in vivo with 300 ?g of anti-GM-CSF or control mAb from every second day from days 21 to 31. (c) Proliferative response to CII measured in mice treated in vivo with control mAb, as in (b), except cells were cultured in the presence of 100 ?g/ml anti-GM-CSF or control mAb. (b), (c) Results are shown for cells cultured in the presence of 50 ?g/ml CII, and are expressed as a stimulation index (mean ? SEM) above background readings in the absence of CII (Stimulation Index = 1). CII, type II collagen; GM-CSF, Granulocyte macrophage-colony stimulating factor; mAb, monoclonal antibody.

Figure 4

Effect of anti-GM-CSF treatment on cytokine levels in the joints. GM-CSF, TNFa and IL-1? levels were measured by enzyme-linked immunosorbent assay in washouts from ankle joints of arthritic mice for mice treated with anti-GM-CSF or control mAb every second day from days 21 to 31 post immunization. Results are expressed as the mean level ? SEM (pg/ml). ^*P = 0.02 and ^**P = 0.007 compared with control mAb-treated mice. GM-CSF, Granulocyte macrophage-colony stimulating factor; IL-1?, interleukin-1?; mAb, monoclonal antibody; TNFa, tumor necrosis factor-a.