Annexin A1: a novel molecular determinant for T cell differentiation and activation

Fulvio D’Acquisto [introduced by Mauro Perretti] William Harvey Research Institute, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ

It is now becoming clear that excessive activation of T cells through uncontrolled T Cell Receptor (TCR) signaling and T helper 1 (Th1)/Th2 imbalance are the main triggers pathogenesis of autoimmune diseases (Nicholson & Kuchroo, 1997). Current therapies for the treatment of autoimmune diseases include the use of glucocorticoids (GCs). Annexin A1 (AnxA1) is a GC-regulated protein with well-known anti-inflammatory activity; several studies have reported the effects of exogenous and endogenous AnxA1 on the innate immune system (Perretti & Gavins, 2003). In this study we performed a systematic analysis of the role of Anx-A1 in T cell activation.

Naïve lymph node T cells from C57/BL6 male mice (4-8 weeks of age) were pre-treated with human recombinant AnxA1 (hrAnxA1; 150-600 nM) and stimulated with plate bound anti-CD3 and anti-CD28 (anti-CD3/CD28; 1.25 m g/ml) for 24 h. Cell proliferation was measured by 3H-thymidine (Amersham Buckinghamshire, UK) incorporation, and interleukin (IL)-2 release quantified by ELISA (eBioscience, Middlesex, UK). In a second set of experiments, Jurkat cells were transfected with specific Nuclear Factor- κB (NF- κB), Nuclear Factor of Activated T cells (NFAT) and Activator Protein-1 (AP-1) reporter constructs and then stimulated with anti-CD3/CD28 in presence or absence hrAnxA1. To monitor Th1/Th2 profile, T cells were cultured in Th0 condition (anti-CD3/CD28, 5 μg/ml and IL-2, 20 U/ml) with or without hrAnx-A1 (300 nM) for four days and thereafter the profile of cytokines released in the supernatant during the subsequent 24 h determined. Data were analysed by ANOVA and Bonferroni test.

Addition of hrAnxA1 induced a concentration-dependent increase in cell proliferation (81 ± 5% and 221 ± 26% at 300 and 600 nM, respectively; n=5, P<0.05) as well as IL-2 production (61 ± 4% and 125 ± 10%, respectively; n=5, P<0.05, P<0.001).Pre-incubation of cells with hrAnx-A1 (300nM) before stimulation with anti-CD3/CD28 (1.25μg/ml) revealed a significant increase in NF- κB, NFAT and AP-1 luciferase activity (295.3 ± 15.7%, 281.6 ± 25.8% and 299.5 ± 19.2%, respectively; n=3, P<0.001) as compared to control cells. In experiments of Th1/Th2 differentiation, cells cultured in presence of hrAnx-A1 produced higher levels of IL-2 and IFN-γ, and lower levels of IL-4 (not shown, n=5, P<0.05).

Together these results show that Anx-A1 acts as molecular “tuner” of TCR signalling. Exogenous application of the protein strengthens TCR signalling favouring T cell skewing towards a Th1 phenotype. In conclusion, these results suggest that high level of Anx-A1 expression in T cells might contribute to the hyperactivation of T cells that characterizes several autoimmune diseases. Therefore, results obtained from this project represent a step forward for the identification of new molecular targets for the treatment of immune-mediated pathological disorders.

Nicholson & Kuchroo (1996) Curr Opin Immunol 8, 837-42.
Perretti & Gavins (2003) News Physiol Sci 18, 60-4.  

This study is funded by the Medical Research Council,