TRPA1 contributes to the acute inflammatory response and mediates carrageenan-induced paw edema in the mouse

Abstract

Transient receptor potential ankyrin 1 (TRPA1) is an ion channel involved in thermosensation and nociception. TRPA1 is activated by exogenous irritants and also by oxidants formed in inflammatory reactions. However, our understanding of its role in inflammation is limited. Here, we tested the hypothesis that TRPA1 is involved in acute inflammatory edema. The TRPA1 agonist allyl isothiocyanate (AITC) induced inflammatory edema when injected intraplantarly to mice, mimicking the classical response to carrageenan. Interestingly, the TRPA1 antagonist HC-030031 and the cyclo-oxygenase (COX) inhibitor ibuprofen inhibited not only AITC but also carrageenan-induced edema. TRPA1-deficient mice displayed attenuated responses to carrageenan and AITC. Furthermore, AITC enhanced COX-2 expression in HEK293 cells transfected with human TRPA1, a response that was reversed by HC-030031. This study demonstrates a hitherto unknown role of TRPA1 in carrageenan-induced inflammatory edema. The results also strongly suggest that TRPA1 contributes, in a COX-dependent manner, to the development of acute inflammation.

Figure 1. TRPA1 agonist allyl isothiocyanate (AITC) and carrageenen (Car) induced an inflammatory paw edema, which could be prevented by pre-treatment with HC-030031 (HC) or ibuprofen (Ibu.).

HC-030031 (300 mg/kg) or ibuprofen (100 mg/kg) was injected intraperitoneally 2 h prior to intraplantar injection of carrageenan or AITC into the hind paw. The edema was measured 3 h and 6 h after intraplantar injection and compared to the basal level. The contralateral control paw injected with saline developed no measurable edema. Mean + SEM, n = 6, ***p<0.001.

Figure 2. In TRPA1 knock out (KO) mice, the carrageenan-induced paw edema formation was blunted when compared to the corresponding wild type (WT) mice (A).

TRPA1-deficient mice showed almost no response to the TRPA1 agonist AITC (allyl isothiocyanate) in contrast to the WT mice (B). Carrageenan or AITC were injected intraplantarly. The edema was measured after 3 h and 6 h and compared to the basal level. The contralateral control paw injected with saline developed no measurable edema. Mean + SEM, n = 5, **p<0.01, ***p<0.001.

Figure 3. HEK 293 cells transfected with human TRPA1 showed an upregulation of cyclo-oxygenase-2 (COX-2) mRNA in response to the TRPA1 agonist allyl isothiocyanate (AITC).

The expression was suppressed when the TRPA1 antagonist HC-030031 (10 μM) was given before AITC. Both TRPA1 transfected and non-transfected (wild type, WT) cells were incubated with HC-030031 or vehicle for 30 min and thereafter AITC was added. After 6 h the incubations were terminated and COX-2 mRNA was assayed by real-time RT-PCR. COX-2 mRNA was normalized against GAPDH mRNA. Mean + SEM, n = 4, *p<0.05, **p<0.01, n.s. = non-significant.

Figure 4. In calcium imaging experiments, HEK 293 cells transfected with human TRPA1 were exposed to either carrageenan or its vehicle for 60 s and subsequently to AITC (allyl isothiocyanate) for 36 s.

Whereas carrageenan or vehicle was without effect, AITC always evoked robust calcium responses as assessed by ratiometric Fura 2 imaging. Traces show the average calcium responses (A) and the bar graph (B) shows the maximum calcium responses in cells exposed to the various treatments. Mean + SEM, n = 4 (each experiment was performed in duplicate or triplicate).