Figure 1.
The presence of Fc receptors has a negative effect on the inhibitory efficacy of infliximab.
Different cell types composing intestinal wall were examined for the presence of Fc gamma receptors. Villin and α-SMA were used to confirm cell type-specific phenotypes (A). Immunofluorescence analysis of CD64-expressing CCD-18Co fibroblasts. Cells expressing CD64 at the plasma membrane are indicated by arrows. Size bar: 50 µm (B). Intestinal fibroblasts CCD-18Co (C), intestinal epithelial cells Caco2BBE (D) and THP-1 cells (E) were pre-incubated with anti-TNF therapeutics, and subsequently treated with human recombinant TNF to induce pro-inflammatory response. Values on Y axis represent mRNA expression levels relative to ß-actin. Columns represent mean values of four independent experiments measured in triplicate. Error bars represent SD. *p<0.05; **p<0.01; ***p<0.001.
Figure 2.
Adalimumab and infliximab, but not certulizumab-pegol have limited efficacy in PBMCs.
Peripheral blood of healthy donors (n = 4) and IBD patients (n = 3) was treated with different anti-TNFs and subsequently with soluble TNF to induce pro-inflammatory responses in PBMCs and plasma. The mRNA expression levels of CD64 (A), CD32 (B) and CD16 (C) Fc gamma receptors in healthy individuals and IBD patients were assessed by RT-PCR as compared to THP-1 cells (set to 1). The extent of inhibition by each drug was determined by measuring mRNA levels of ICAM-1 (D), TNF (E), and IL-8 (F) after 2 h of TNF stimulation, as well as by the release of IL-8 after 24 h of combined TNF and anti-TNF stimulation (G). The inhibitory efficacies were determined after eliminating intra-individual differences by setting the TNF-induced responses to 100% for each donor and target (H–K). Error bars represent SEM. *p<0.05; **p<0.01; ***p<0.001.
Figure 3.
Infliximab is more potent in activating Fc receptor CD64 than adalimumab.
Both ADA and IFX immune complexes were added to THP-1 cells to activate CD64 receptor in the presence (+FCS) or absence (−FCS) of serum. ADA/TNF complexes were more potent in inducing phospho-tyrosine signaling (A), but IFX either alone or in complex with TNF induced phosphorylation of distinct tyrosine residues (enlarged insert, right panel). Activation of CD64 downstream signaling leads to elevated transcription of GM-CSF (B, C), MCP-1 (D, E) and IL-8 (F, G) genes. Human serum (HS) was used as a positive control for activation of Fc gamma receptor(s). Error bars represent SEM. *p<0.05; **p<0.01; ***p<0.001.
Figure 4.
Blocking of Fc receptors with IgG1 partially restores the efficacy of infliximab.
CCD-18Co fibroblasts (A), Ko77 fibroblasts (C) and THP-1 cells (E) were pre-incubated with human IgG1 isotype (10 µg/ml) prior to subsequent incubation with infliximab (1 µg/ml) and TNF (50 ng/ml). Columns show mean values from a single, representative experiment measured in triplicates. Error bars indicate SD of three measurements. The recovery of anti-TNF activity was also evaluated as percentage of inhibition of TNF-induced responses for each cell line (B, D, F).
Figure 5.
Fc and Fab fragments of infliximab contribute differently to its inhibitory efficacy.
Blocking of Fc fragments of adalimumab and infliximab changes their inhibitory efficacy in Ko77 fibroblasts (A). Graph shows mean values from four independent experiments measured in triplicates. Error bars represent SD. Fab fragments of anti-TNFs have different inhibitory efficacies as compared to the whole IgG1 molecules in THP-1 cells (B). Columns represent mean values of three independent experiments measured in triplicates and set to 100 percent for TNF-treated cells. Error bars indicate SEM. *p<0.05.
Figure 6.
Changes in expression levels of CD64 affect the efficacy of infliximab in THP-1 cells.
Treatment with IFN-γ elevates the mRNA expression levels of CD64 (A), which result in the loss of efficacy of IFX and ADA (B). Decreased expression levels of CD64 (C) correlate with increased inhibitory efficacy of IFX (D). PMA treatment decreases the expression levels of CD64 (E), which results in complete recovery of efficacy of IFX (F). Graphs show mean values of at least three independent experiments measured in triplicates and columns are set to 100 percent for TNF-treated cells. Error bars indicate SEM. *p<0.05.
Figure 7.
Loss of response to infliximab correlates with increased levels of CD64 in IBD intestines.
The level of intestinal inflammation was assessed independently by endoscopic score and mRNA expression levels of pro-inflammatory target genes. The levels of Fc receptors were assessed in colonic biopsies from IFX responders (n = 13) tissues and compared to IFX non-responders in both non-inflamed (n = 6) and inflamed (n = 3) tissues. IL-18 mRNA was used as a specific negative control, since it is not elevated upon stimulation with pro-inflammatory cytokines (not shown). The expression levels of each gene were normalized to those of a housekeeping gene β-actin. *p<0.05; **p<0.01; ***p<0.001.
Figure 8.
Schematic representation of the working model.
IFN-γ induces its receptor downstream signaling involving JAK and STAT proteins (A). STAT dimers elevate the transcription of the CD64 (FCGR1) gene (B). Translated CD64 protein is targeted into plasma membrane, where it resides in an inactive state until binding immune complexes IFX-TNF (C). Propagation of downstream signaling (D) occurs through immunoreceptor tyrosine-based activation motif (ITAM) leading to the transcription of relevant target genes (E) via parallel activation of p38, JNK and ERK kinases [58]. Production and release of IL-8 and other pro-inflammatory factors contribute to the limited outcome of IFX treatment.