In this study, we examined whether IL21 regulates the synthesis of MMPs by intestinal fibroblasts, given that fibroblasts are a major source of MMPs in the gut and that MMP production can be enhanced by T cell‐produced cytokines.3,4,5,6,7,8,9,10
Initially we showed that IL21R is constitutively expressed by intestinal fibroblasts, and that such expression can be enhanced by TNF α and IL1β. In this context, however, it is worth noting that IL21R protein expression was semiquantitatively assessed by western blotting, as our attempts to characterise IL21R by flow cytometry using commercially available antibodies were unsuccessful. We cannot therefore exclude the possibility that the arbitrary units we measured may not exactly reflect the biological quantities of IL21R protein in those cells. Intestinal fibroblasts also express the common γ chain receptor, suggesting that these cells may be potential targets of IL21 in vivo. Indeed, intestinal fibroblasts responded to IL21 by increasing the production of MMPs. The effects of IL21 on MMP synthesis were observed with concentrations of IL21 that are similar to those used by other authors to test the biological effects of this cytokine in vitro.15
We do not know whether such concentrations reflect the amount of IL21 produced in the gut of patients with IBD, as no commercial kit is yet available to quantify human IL21. However, our previous western blotting analysis of IL21 showed that the intensity of immunoreactive bands in patients with IBD was not different from that obtained with 50 ng/ml rhIL‐2117
—that is, the maximal dose we used to stimulate fibroblasts. We restricted our analysis of the effect of IL21 on the synthesis of MMP‐1, MMP‐2, MMP‐3 and MMP‐9, as these proteases are produced in excess in the gut of patients with IBD.3,4,6,11
MMP production or activity can be regulated at multiple levels including gene activation and transcription, mRNA stability, secretion, proenzyme activation and inactivation by endogenous inhibitors.1,2
We thus examined how IL21 regulates MMP synthesis in gut fibroblasts. Our data suggest that regulation of MMPs by IL21 does not occur at the transcriptional level. In fact, stimulation of fibroblasts with IL21 did not alter the expression of MMP RNA transcripts. Additionally, the intracellular level of MMP proteins was not increased by IL21, and the IL21‐induced MMP synthesis was not affected by inhibitors of gene transcription and de novo protein synthesis. It is thus plausible that IL21 preferentially increases the secretion of either preconstituted or newly synthesised MMPs, as treatment of fibroblasts with brefeldin A inhibited IL21‐induced MMP secretion, and increased secretion of MMP was seen after exposure to IL21 for a short time.
Although it was once considered that MMPs were transcriptionally regulated and rapidly secreted, recent evidence shows that MMPs are stored in secreted granules, ready for rapid release. For example, coculture of a monocytic cell line with metastatic colon cancer cells results in an increased production of MMP‐2 without any increase in mRNA levels.24
Subcellular compartmentalisation of MMPs within caveolar structures has been described in endothelial cells, and activation of these cells by various stimuli results in enhanced MMP secretion, with no change in MMP RNA or de novo protein synthesis.25,26,27,28
By contrast, TNF α augmented the RNA expression of MMP‐1, MMP‐3 and MMP‐9, but not MMP‐2, supporting data of previous reports showing that MMP‐2 can be regulated via a different pathway from that of other MMPs, including MMP‐9.1,2,29,30
These data suggest that inflammatory cytokines such as TNF α may transcriptionally increase MMP production, but this effect is amplified by IL21 at the post‐transcriptional level.
The proteolytic activity of MMPs is tightly controlled by TIMPs.1,2
One of the most striking features of this study is that TIMP‐1 and TIMP‐2 protein remained unchanged after IL21 stimulation. These findings are consistent with previous reports showing high MMP production without concomitant TIMP elevation in other systems. For example, in IBD tissue, increased expression of MMP‐3 occurs with no change in TIMP‐1 production.5,6
Stimulation of scleral fibroblasts from patients with necrotising scleritis by TNF α resulted in a twofold increase in TIMP‐1 mRNA compared with a sevenfold increase in stromelysin‐1 mRNA.31
There was no similar increase in TIMP‐1 mRNA in the aqueous humour of patients with uveitis, although MMPs were increased.32
In conclusion, we have shown that IL21 increases MMP production by fibroblasts, thus confirming and expanding on our previous data showing that cytokines are important mediators of the cross talk between immune and non‐immune cells in the gut.5,9,18
The in vivo relevance of our findings relates to the fact that supernatants of Crohn's disease LPMC rapidly increase MMP production by fibroblasts and this is partially inhibited by IL21R/Fc. The fact that the IL21R/FC reduces but does not abrogate the effect of Crohn's disease LPMC supernatants on fibroblast MMP production indicates that Crohn's disease LPMC make additional MMP‐inducing molecules other than IL21. Indeed, molecules such as IL1β, IL6, IL17, IL22 and TNF α, all of which could regulate MMPs, are produced in excess in Crohn's disease.2,5,10,18,33,34
Although IL21 was originally described as an important regulator of T, B and NK cells, recent studies have shown that IL21 modulates the activity of multiple cell types and triggers several inflammatory pathways.14,35,36
These observations, with the demonstration of upregulation of IL21 in IBD tissue and enhancement of MMPs in fibroblasts by IL21, suggest that blocking IL21 can help in limiting the tissue‐damaging inflammatory response in IBD.