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. 2006 Nov;149(5):591-9.
doi: 10.1038/sj.bjp.0706905. Epub 2006 Sep 18.

Activation of mouse protease-activated receptor-2 induces lymphocyte adhesion and generation of reactive oxygen species

S Y Lim  1 , G M Tennant, S Kennedy, C L Wainwright, K A Kane
Affiliations

Activation of mouse protease-activated receptor-2 induces lymphocyte adhesion and generation of reactive oxygen species

S Y Lim et al. Br J Pharmacol. 2006 Nov.

Abstract

Background and purpose: Protease-activated receptor-2 (PAR-2) is expressed on lymphocytes and endothelial cells, and plays a significant role in inflammatory reactions. Since leukocyte-endothelial cell interaction and reactive oxygen species (ROS) generation are hallmarks of the development of inflammation, the effects of PAR-2 activation by trypsin on lymphocyte adhesion and ROS generation was examined utilising PAR-2 wild type and knockout (PAR-2-/-) mice.

Experimental approach: Lymphocyte adhesion to the luminal surface of mouse isolated aortae was measured using 51Cr-labelled leukocytes and ROS generation from isolated lymphocytes was quantified using chemiluminescence.

Key results: Trypsin induced adhesion of lymphocytes when added exogenously to the endothelial surface of the aorta for 30 min. Similarly, increased lymphocyte adhesion was also observed when mice were injected with trypsin intravenously 24 h prior to the adhesion assay, an effect which was partly ICAM-1 mediated. Trypsin also increased ROS generation from isolated mouse lymphocytes in a dose-dependent manner. The increase in lymphocyte adhesion and ROS production in response to trypsin were abolished in PAR-2-/- mice indicating a PAR-2 dependent mechanism. Superoxide dismutase had a greater inhibitory effect in PAR-2-/- mice compared to wild type mice when lymphocytes were stimulated with PMA but not trypsin.

Conclusions and implications: The present study indicates that activation of PAR-2 may be an important factor in modulating lymphocyte adhesion and ROS generation. The results have implications for developing anti-inflammatory strategies.

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Figures

Figure 1
Figure 1
Effect of drug treatments on lymphocyte adhesion in vitro. Effect of trypsin (1000 U ml−1, 30 min), TNFα (10 ng ml−1, 60 min) and PMA (50 ng ml−1, 30 min) on adhesion of lymphocytes to thoracic aorta isolated from PAR-2+/+ and PAR-2−/− mice. Numbers in parentheses indicate n numbers. *Indicates P<0.05 vs vehicle control.
Figure 2
Figure 2
Effect of drug treatments on lymphocyte adhesion ex vivo. Effect of intravenous trypsin injection (1 U g−1, 0.05 ml per 25 g) on adhesion of autologous lymphocytes to PAR-2+/+ and PAR-2−/− mice thoracic aorta. Adhesion was studied 4 or 24 h post-trypsin injection. Numbers in parentheses indicate n numbers. *Indicates P<0.01 vs vehicle control.
Figure 3
Figure 3
Adhesion molecule expression on endothelium. Quantification of ICAM-1 (A) or P-selectin (B) staining on thoracic aorta isolated from PAR-2+/+ and PAR-2−/− mice subjected to lymphocyte adhesion assay (ex vivo study). Arteries were isolated from mice injected with either saline (vehicle control) or trypsin (4 or 24 h). (C) photomicrographs showing the up-regulation of ICAM-1 staining in the thoracic aorta isolated from the PAR-2+/+ mice 24 h after trypsin injection compared with the vehicle control segments. Arrows indicate the endothelial nucleus. Numbers in parentheses indicate n numbers. *Indicates P<0.05 vs vehicle control.
Figure 4
Figure 4
Effect of PAR-2 receptor knockout on the chemiluminescent response to trypsin. The Figure shows the total chemiluminescence (CL) signal generated by PAR-2+/+ (n=6) and PAR-2−/− (n=6) mouse lymphocytes stimulated with varying concentrations of trypsin. The values shown are corrected for CL signals generated by saline alone. * and **Indicate P<0.05 and <0.001 vs PAR-2−/−, respectively.
Figure 5
Figure 5
Effect of ROS scavengers on PMA-induced chemiluminescence (CL). The effects of SOD, catalase and/or sodium azide on CL signal generated by lymphocytes from PAR-2+/+ (n=6) and PAR-2−/− (n=6) mice, stimulated with 10 ng ml−1 PMA . ‘All' indicates 50 U ml−1 of SOD+3000 U ml−1 catalase+10−5 M sodium azide. #indicates P<0.05 compared to 10 ng ml−1 PMA, * indicates P<0.01 compared to PAR-2+/+, and $ indicates P<0.05 compared to 50 U ml−1 of SOD, to 3000 U ml−1 catalase and to 10−5 M sodium azide.
Figure 6
Figure 6
Effect of SOD on trypsin-induced chemiluminescence. The effect of SOD on the chemiluminescence signal generated by lymphocytes from PAR2+/+ (n=8–14) and PAR2−/− (n=8–14) mice, stimulated with 1000 U ml−1 trypsin. Basal is the chemiluminescence response to saline (unstimulated) expressed as 100%. *Indicates P<0.05 compared to trypsin 1000 U ml−1 of respective PAR-2 mice and #indicates P<0.05.
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