Tumour necrosis factor receptor gene therapy affects cellular immune responses in collagen induced arthritis in mice

Abstract

Background: Collagen induced arthritis (CIA) is an animal model of rheumatoid arthritis (RA) amenable to immunotherapy directed against tumour necrosis factor alpha (TNFalpha).

Objective: To evaluate whether local TNF receptor (TNF-R) gene therapy in DBA/1 mice exerts an influence beyond anti-inflammatory effects. Two measures of CIA pathogenesis were investigated-namely, immunity to collagen II (CII) 245-270 peptide (the major immunodominant epitope within bovine CII) and the preferential activation of T cell Vbeta8.2 variable region receptors in arthritic DBA/1 mice.

Methods: DBA/1 mice received single periarticular injections of media or retroviral vectors containing LacZ or human TNF-R into affected arthritic paws at disease onset. Disease severity was monitored, immune responses towards the immunodominant bovine CII 245-270 and subdominant CII 334-360 peptide epitopes were assessed by ELISA, and T cell Vbeta usage was analysed by real time polymerase chain reaction for the LacZ transduced, TNF-R, and viral-free media treated control animals. The therapeutic influence of TNF-R gene transduction was compared with other groups at different times after treatment.

Results: Reduced disease severity was seen 15-35 days after treatment, with a concomitant increase in immunity towards the subdominant CII 334-360 peptide epitope rather than the immunodominant CII 245-270 peptide in TNF-R treated animals. Early in the disease, TNF-R treated animals demonstrated a reduction of bias towards the otherwise predominant Vbeta8.2 T cell subset.

Conclusions: TNF-R gene therapy influences cellular immunity in CIA, leading to overall disease amelioration, thus suggesting that TNF inhibition may have therapeutic potential beyond the control of inflammation in RA.

Figure 1

X-gal stain to show the expression of LacZ in the mouse paws given MFG-LacZ. (A) A paw at 3 days; and (B) 21 days after viral infection.

Figure 2

Influence of a single periarticular administration of retrovirus mediated MOIN-sTNF-Rc-Ig on the clinical progression of CIA: disease activity represented by the mean paw score was visually scored up to 49 days after disease onset. Mean paw score is the overall average of the paw scores of all animals in each group. Data are expressed as mean (SEM) for each group for each time point. Significant differences among the groups are indicated (_*p<0. 05). The figure shows normalised mean data from three different individual experiments with similar results.

Figure 3

IFNγ responses to CII 245–270 and CII 330–360 peptides after MOIN-sTNF-Rc-Ig gene therapy: mononuclear cells from lymph nodes of (A) MOIN-sTNF-Rc-Ig or (B) media (control) treated mice were cultured with either 100 µg/µl of immunodominant CII 245–270 peptide or with subdominant CII 330–360 peptide for 72 hours. Cells cultured with media and concanavalin A were used as negative and positive controls respectively (not shown). IFNγ levels in resulting culture supernatants were measured by ELISA to evaluate CII peptide epitope usage. TNF-R treated animals showed an enhanced IFNγ response towards the subdominant epitopes rather than the immunodominant epitopes (A), while the normal response to the dominant epitope was observed in the controls (B). Data are expressed as mean for each group at each time point. n = 5 for each time point for both control and TNF-R treated groups. _*p<0.05.

Figure 4

Comparison of Vß expression levels in joints of arthritic animals: cDNA made from RNA preparations from (A) injected (B) ipsilateral (C) contralateral joints of media or MOIN-sTNF-Rc-Ig injected joints were amplified with different Vß primer pairs using real time PCR. The figure shows the relative mRNA expression levels of the six Vß genes quantitatively analysed normalised against the housekeeping gene 18S rRNA used as an internal control. n = 5 for both control and TNF-R treated groups.

Figure 5

Local TNF-R treatment affects the TCR Vß repertoire in CIA over time: Vß mRNA levels in the joints of control and MOIN-sTNF-Rc-Ig treated groups were compared by real time RT-PCR at (A) 3; (B) 7; (C) 21; and (D) 49 days after disease onset. cDNA was obtained from RNA preparations from joints of the two groups of mice. Relative Vß gene expression in both groups, normalised against the housekeeping gene 18S rRNA used as an internal control, is represented. n = 5 for both control and TNF-R treated groups.