Anti-interleukin-6 monoclonal antibody inhibits autoimmune responses in a murine model of systemic lupus erythematosus

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease resulting from dysregulation of the immune system. Interleukin-6 (IL-6) is a multifunctional cytokine produced by macrophages, monocytes and T and B cells. It stimulates B-cell differentiation/maturation, immunoglobulin secretion, and T-cell functions. Elevated levels of IL-6 in serum, urine and renal glomeruli were detected in patients with active SLE and in murine models of SLE. Our study investigated the role of IL-6 in an SLE-like disease in New Zealand Black/White (NZB/W) F1 mice by administration of an anti-murine IL-6 monoclonal antibody (mAb). Intraperitoneal administration of the anti-IL-6 mAb suppressed the production of anti-dsDNA autoantibody. B-cell proliferation induced by anti-IgM and anti-CD40 was lower in the anti-IL-6 mAb-treated mice, ex vivo studies demonstrated that anti-IL-6 mAb treatment inhibited anti-dsDNA production. Anti-CD3-induced T-cell proliferation and mixed lymphocyte reactions were inhibited by anti-IL-6 mAb treatment, indicating a partial down-regulation of T cells. Histological analysis showed that treatment with anti-IL-6 mAb prevented the development of severe kidney disease. These results suggest that treatment with anti-IL-6 mAb has a beneficial effect on autoimmunity in murine SLE and that autoreactive B cells may be the primary target for anti-IL-6 mAb treatment; its effect on autoreactive T cells is also indicated.

Figures

Figure 1

Anti-IL-6 mAb inhibited SAA production in NZB/W F1 mice. SAA levels measured by ELISA at 15 and 34 weeks of age are shown. *p < 0·05 versus saline and isotype control mAb-treated groups.

Figure 2

Anti-IL-6 mAb suppressed serum IL6 level. Serum samples were analysed for IL-6 levels by Luminex at 15 and 34 weeks of age. *p < 0·05 versus saline and control mAb-treated groups.

Figure 3

Anti-IL-6 mAb suppressed autoantibody production. Serum samples were analysed for anti-dsDNA autoantibody levels by ELISA at 15 and 34 weeks of age. OD index values represent individual data points normalized throughout the studies to a single positive control serum with anti-dsDNA. *p < 0·05 versus saline and control mAb-treated groups.

Figure 4

Histological analysis of kidney pathology. (a) Haematoxylin & esosin (H&E) section of a saline control mouse. Note prominent perivascular, predominantly lymphocytic infiltrates at the hilus (original magnification 10×). (b) PAS-stained section of a saline control mouse. Note thickened basement membranes and mesangial cell hyperplasia (original magnification 40×). (c) H&E section of an isotype control mouse. Note prominent perivascular, predominantly lymphocytic infiltrates at the hilus and extensive interstitial inflammatory infiltrates in the cortex (original magnification 10×). (d) PAS-stained section of an isotype control mouse. The extent of glomerular disease is greater than that seen in (b) (original magnification 40×). (e) H&E section of an anti-IL-6 mAb-treated mouse. Note lack of perivascular lymphocytic infiltrates at the hilus and interstitial inflammatory infiltrates in the cortex (original magnification 10×). (f) PAS-stained section of an anti-IL-6 mAb-treated mouse. The extent of glomerular disease is much less than that seen in (b) or (d) (original magnification 40×).

Figure 5

Anti-IL-6 mAb reduced the number of activated B and T cells. (a) Total splenocytes were analysed with anti-CD19, anti-CD21 and anti-CD23 antibodies by flow cytometry to determine the population of CD21+ CD23– marginal zone B cells at 34 weeks of age. The cells were gated on CD19+ B cells. One representative out of five dot plots from each treatment group is shown. (b) Total splenocytes were analysed with anti-CD4, anti-CD62L and anti-CD44 antibodies by flow cytometry to determine the population of naive, activated and memory T cells at 34 weeks of age. The cells were gated on CD4+ T cells. One representative out of five dot plots from each treatment group was shown.

Figure 6

Effect of anti-IL-6 mAb treatment on T-cell and B-cell responses at 34 weeks of age. (a) Anti-CD3-stimulated T-cell proliferation was examined in splenocytes harvested when mice were 34 weeks of age. ATPlite assay was performed to determine T-cell proliferation and the results are expressed as cells per count (cps). (b) B-cell proliferation induced by anti-CD40/anti-IgM antibodies was assessed in splenocytes harvested when mice were 34 weeks of age. ATPlite assay was performed and the results are expressed as cps. (c) T-cell proliferation in MLR was examined in splenocytes harvested when mice were 34 weeks of age. ATPlite assay was performed and the results are expressed as cps. *p < 0·05 versus saline and isotype control mAb-treated groups.

Figure 7

Anti-dsDNA autoantibody produced by autoreactive B cells from 16-week-old NZB/W F1 mice. Splenocytes were cultured with media, anti-CD40/anti-IgM mAbs, anti-CD40/anti-IgM plus recombinant mouse IL-6, and anti-CD40/anti-IgM plus anti-IL-6 mAb in triplicates for 7 days before the supernatants were harvested and the anti-dsDNA autoantibody level was measured by ELISA. OD index values represent individual supernatants normalized to a single positive control serum with anti-dsDNA.

Figure 8

Anti-IL-6 mAb inhibited autoantibody production from autoreactive B cells. Anti-dsDNA autoantibody produced by autoreactive B cells from 34-week-old NZB/W F1 mice treated with saline, isotype control mAb and anti-IL-6 mAb were examined in splenocytes cultured with media, anti-CD40/anti-IgM mAbs, anti-CD40/anti-IgM plus recombinant mouse IL-6, and anti-CD40/anti-IgM plus anti-IL-6 mAb and recombinant IL-6 in triplicates for 7 days before the supernatants were harvested and the anti-dsDNA autoantibody level was measured by ELISA. OD index values represent individual supernatants normalized to a single positive control serum with anti-dsDNA autoantibody. *p < 0·05 versus saline-treated group.

Figure 9

Spontaneous stat3 phosphorylation in T and B cells from 22-week-old C57BL/6 and NZB/W F1 mice. Splenocytes were cultured with complete culture media for 6 min at 37°. Surface markers CD4, CD19 and intracellular phosphorylated stat3 were determined by flow cytometry. Representatives of two experiments were shown.

Figure 10

Anti-IL-6 mAb inhibited stat3 phosphorylation in T and B cells. Stat3 phosphorylation in T and B cells from saline, isotype control antibody, and anti-IL-6 mAb-treated NZB/W F1 mice was examined in splenocytes cultured with 40 ng/ml of recombinant mouse IL-6 for 6 min at 37°. Surface markers, CD4, CD19 and intracellular phosphorylated stat3 were determined by flow cytometry. Representatives of CD4+ T cells from 24-week-old mice and CD19+ B cells from 34-week-old mice are shown.