Differential effects of infliximab on absolute circulating blood leucocyte counts of innate immune cells in early and late rheumatoid arthritis patients

Abstract

Anti-tumour necrosis factor (TNF) biologics have revolutionized therapy of rheumatoid arthritis (RA). We compared the effects of infliximab on numbers of circulating leucocyte subsets in early RA (disease/symptom duration of ≤1 year) and late RA patients (>1 year). A control group consisted of early RA patients treated with a combination of methotrexate (MTX) and methylprednisolone. Blood samples were obtained at baseline (pre-therapy) from all RA patients, divided into three groups: (i) late RA receiving infliximab/MTX, (ii) early RA-infliximab/MTX, (iii) early RA-steroid/MTX, and also from follow-up patients at 2 and 14 weeks. Significant differences in absolute counts of monocytes and granulocytes were observed between healthy controls and RA patients. At baseline CD14(bright) monocytes and CD16(+) granulocytes were increased in both early RA and late RA patients. CD4(+) T cells, CD8(+) T cells and B cells were all increased at baseline in early RA, but not in late RA. At 2 weeks following infliximab treatment decreased granulocytes were observed in both early and late RA and decreased natural killer (NK) cells in late RA. CD16(+) granulocytes and NK cells were also decreased at 14 weeks post-infliximab in early RA. Biotinylated infliximab was used to detect membrane-associated TNF (mTNF)-expressing leucocytes in RA patients. CD16(+) granulocytes, NK cells and CD14(dim) monocytes all expressed higher levels of mTNF in RA patients. In summary infliximab is associated with decreased CD16(+) granulocyte and NK cell counts, possibly through binding of mTNF. Differential effects of infliximab between early and late RA suggest that pathogenic mechanisms change as disease progresses.

Fig. 1

Basic gating strategy for absolute cell count analysis. (a) Cells were gated on forward/side scatter (FSC/SSC). The thick black boxes show cell populations measured. Isotype control antibodies were used to define negative populations and single-stained samples were tested to ensure that fluorochromes were well compensated. (b) CD19⁺CD8⁻B cells, (c) CD3⁺CD8⁺ T cells, (d) CD3⁺CD4⁺ T cells and (e) CD3^-CD56⁺ natural killer (NK) cells were gated within the lymphocyte gate on the FSC/SSC. (f) CD14⁺ monocytes were gated within the monocyte gate on the FSC/SSC and subdivided into CD14^bright and CD14^dim. (g) Counting beads were displayed on a SSC/phycoerythrin (PE) plot to distinguish clearly the two populations of beads, labelled as X and Y. The proportion of the bead populations acted as an internal control. (h) Example calculation of absolute cell counts.

Fig. 2

Baseline leucocyte cell counts in early and late rheumatoid arthritis (RA) compared to healthy control. Absolute cell counts for (a) CD14^bright monocytes, (b) CD14^dim monocytes (c) CD14^brighCD16⁺ monocytes, (d) CD16⁺ granulocytes, (e) CD4⁺ T cells, (f) CD8⁺ T cells, (g) CD19⁺ B cells and (h) CD3^-CD56⁺natural killer (NK) cells were analysed in healthy controls (n = 22), early RA (n = 45) and late RA (n = 18) patients. **P < 0·01; ***P < 0·001 by Kruskal–Wallis test.

Fig. 3

Absolute cell counts at week 2 of infliximab or steroid treatment in early and late disease. The change in cell number between baseline and week 2 was calculated in the late rheumatoid arthritis (RA) group receiving infliximab (n = 11), the early RA–infliximab group (n = 13) and the early RA–steroid group (n = 16). Changes were analysed for (a) CD14^bright monocytes, (b) CD14^dim monocytes, (c) CD16⁺ granulocytes and (d) CD3^-CD56⁺ NK cells. Significant differences from baseline to week 2 of treatment were calculated using Wilcoxon's signed-rank test. P < 0·05 was considered significant and marked with an asterish (*).

Fig. 4

Absolute cell counts at week 14 after infliximab or steroid treatment in early and late disease. The change in cell number between baseline and week 14 was calculated in the late rheumatoid arthritis (RA) group receiving infliximab (n = 7), the early RA–infliximab group (n = 11) and the early RA–steroid group (n = 11). Changes were analysed for (a) CD14^bright monocytes, (b) CD14^dim monocytes, (c) CD16⁺ granulocytes and (d) CD3^-CD56⁺ natural killer (NK) cells. Significant differences from baseline to week 14 of treatment were calculated using Wilcoxon's signed rank test. P < 0·05 was considered significant and marked with a star (*).

Fig. 5

Biotinylated immunoglobulin (Ig)G isotype control binding on leukocyte populations. Human IgG, mouse IgG and infliximab were biotinylated. Binding of each IgG class to the cell surface of leucocyte populations was determined using healthy control (HC) blood. The lymphocyte, monocyte and granulocyte populations were gated on forward/side scatter (FSC/SSC), as depicted in Fig. 1. Each histogram represents streptavidin-phycoerythrin (PE) binding to IgG in each cell population. Data were analysed using BD FACSDiva version 5.0.2 (BD Biosciences, Oxford, UK).

Fig. 6

Expression of membrane-associated tumour necrosis factor (mTNF) on the surface of different leucocyte populations. The expression of mTNF was detected by biotinylated infliximab in three rheumatoid arthritis (RA) patients who were infliximab-naive. Histograms showing binding of infliximab on (a) CD14^dim and CD14^bright monocytes, (b) CD4⁺ and CD8⁺ T cells, (c) CD16⁺ granulocytes and (d) natural killer (NK) cells and B cells. Median fluorescence intensity (MFI) was analysed using FlowJo (Tree Star, Inc.). The respective isotype controls (IC) were shown as dotted lines.