Proteomic characterisation of cell contact-dependent macrophage activation

The generation of tumour necrosis factor alpha (TNF-α) in the rheumatoid synovium is widely perceived as pivotal to disease pathogenesis in rheumatoid arthritis (RA). While TNF-α production in dispersed synovial cell cultures is T-cell-dependent [1] there is little evidence of antigen-dependent T-cell activation. One explanation for this apparent paradox is the well-documented ability of appropriately activated T cells to drive production of TNF-α from macrophages through a cell contact-dependent, antigen-independent mechanism [2]. Despite some promising leads (e.g. CD69 [3]), the identities of the relevant stimulating T-cell surface ligands, and the macrophage signalling pathways engaged following cell contact remain obscure. Our approach has been to develop new proteomic techniques to address these issues in a systematic and unbiased fashion.

We used cell surface biotinylation and solution-phase isoelectric focusing to extract and resolve plasma membrane proteins from murine T-cell hybridomas and primary splenocytes prior to identification using liquid chromatography tandem mass spectrometry (LC MS/MS). Using this method we identified 75 plasma membrane proteins, 13 of which appeared to be regulated following stimulation with phorbol 12-myristate 13-acetate and ionomycin. Six of these putative changes were verified independently by western blotting or by flow cytometry [4]. To develop protocols to examine macrophage signalling pathways we utilised anti-phosphotyrosine antibodies to recover tyrosine phosphoproteins from RAW 264.7 cells activated with the tyrosine phosphatase inhibitor, pervanadate. Recovered proteins were eluted using phenyl phosphate, then resolved and identified by solution-phase isoelectric focusing and LC MS/MS. We identified 113 proteins inducibly associated with anti-phosphotyrosine immunoprecipitates from distinct subcellular compartments (cytosol, nucleus and plasma membrane), many of which were known tyrosine phosphoproteins (e.g. Fyn, Syk, SHIP, SLP76) but also included several hypothetical proteins, hitherto not documented at the protein level.

We then used this methodology to identify macrophage signalling proteins phosphorylated following cell contact with T-cell receptor transgenic splenocytes stimulated for 48 hours with specific (influenza virus hemaglutinin), or non-specific (ovalbumin), peptide. Proteins recovered in RAW cell phosphotyrosine immunoprecipitates were analysed by western blotting using antibodies to phosphotyrosine or the signalling molecules phospholipase C gamma, Hck, p38 and ERK. Cell contact with either resting or activated splenocytes induced acute and transient tyrosine phosphorylation of the src kinase Hck and phospholipase C gamma. In contrast, sustained activation of the MAP kinases p38 and ERK was observed only after contact with activated splenocytes.

Application of these methods should shed new light on the molecular bases of the contact-dependent activation of macrophages by activated T cells and, thereby, suggest novel targets for therapeutic intervention in RA.