Proteolytic control of immunological defense systems
Neutrophils (neutrophilic granulocytes) and killer cells form the first line of defense against invading bacteria and viruses. In response to the invasion of pathogens, these cells are quickly recruited to the site of infection and destroy bacteria and virally infected host cells by a number of mechanisms. Secretory lysosome-like granules of neutrophils and killer cells harbor a significant arsenal of lethal destructive weapons, of which serine proteases are most prominent. Eradication of tissue invading bacteria and viruses infecting host cells in peripheral tissues is an emergency measure with collateral tissue damage and potential progression to chronic disease. The same immune effector mechanisms that can save our life can also result in chronic pathological inflammation and autoimmune disease in the absence of infectious agents.
We are particularly interested in the functions and target substrates of neutrophil serine proteases, their roles during immune defense, cell migration and cellular activation. Besides the three known serine proteases of primary granules, we discovered a fourth member and have started to elucidate its function. Using genetically modified mice, that lack one or a combination of these protease genes, we are able to analyze the molecular defects and alterations in neutrophil-mediated responses, in cell migration and in clinical disease models after elimination of these proteases. As these four serine proteases appear to generate diverse proinflammatory effects in preclinical mouse models, their specific inhibition should ameliorate a number of chronic disease conditions. Moreover, autoantibodies to endogenous neutrophil proteins develop in chronic inflammatory diseases with aggravating consequences. In particular, autoantibodies to proteinase 3 and myeloperoxidase are specific and diagnostically useful for systemic vasculitides and trigger neutrophil activation and tissue injury during disease exacerbations. Why these autoantibodies are regularly formed against these two host proteins in systemic vasculitis, is still a challenging puzzle which we are trying to solve.
Background of research
Proteases play crucial roles as posttranslational protein scissors in the biosynthesis and functional control of immune mediators, antigen processing, cell migration and immune effector mechanisms. A distinct subgroup of closely related serine proteases ("granzymes") exist in secretory lysosome-like granules of terminally differentiated cytotoxic T lymphocytes and natural killer cells, neutrophils, macrophages and mast cells.
The neutrophil specific enzymes, proteinase 3 and leukocyte elastase, are key pathogenic factors in the development of lung emphysema and inflammatory tissue damage. Mast cell chymase is an important inflammatory mediator generating e.g. angiotensin II and endothelin-31 (residues 1-31). Moreover, proteinase 3 is the principal target antigen in systemic vasculitis.
Two members of the granzyme family, the granzymes A and B, are highly efficient exogenous activators of programmed cell death (apoptosis) during target cell attack. The co-released perforin binds to target cell membranes and helps granzymes into the cytosol after their endocytic uptake by the target cells
Research projects
One focus of our work is to explore different natural and artificial protein transduction routes and the therapeutic potential of apoptosis inducing serine proteases in the treatment of cancer and chronic inflammation. Another project tries to identify the primary substrates of naturally transduced granzymes which damage mitochondria after their cleavage and initiate caspase-independent pathways of apoptosis. Besides the granzymes A and B, additional granzymes exist in the human genome whose biological functions in health and disease need to be clarified.
The autoimmune response against human proteinase 3 is highly correlated with disease activity, severity, risk of relapse and progression in Wegener’s granulomatosis, a small vessel vasculitis that is lethal without aggressive immunosuppressive therapy. Antibody responses to different conformational epitopes on the surface of PR3 appear to display different pathogenic potential and clinical relevance. The structural and functional basis of these epitope differences is investigated and anti-PR3 autoantibody specificities will be discriminated in WG patients during active disease, remission and relapses.
The key role of granule-associated serine proteases in chronic inflammatory and autoimmune diseases makes them attractive targets for pharmaceutical intervention. Protease inhibitors are potent drugs that can be delivered intracellularly and can protect host cells from inadvertent tissue damage. Moreover, granzymes and related proteases are enticing means to induce apoptotic cell death in tumor cells and autoreactive lymphocytes after targeted, membrane traversing delivery. The apoptosis-controlling and therapeutic potential of both proteases and specific inhibitors will be evaluated at the structural level, with purified recombinant proteins, in cell culture systems and preclinical animal models.
Key technologies
These include recombinant engineering and large scale production of active serine proteases and their precursors for crystallization trials, biochemical characterization of protease-substrate interactions, protein transduction of cultured cells to trigger apoptosis and preclinical mouse models (mainly transgenic) to study granzyme-containing immunoconjugates and inhibitors.
