The contemporary theory of the immune response states that the immune system is not programmed to respond to foreign particles, as it was conceived for many years during the self-nonself era of immunology. What turns the immune system on and off is a very important question in understanding disease and pathology. The condition of the tissue is very central to understanding homeostasis, tolerance and the immune response. The central dogma of immunology is that the immune system receives signals to respond from the ‘tissue’ and that the immune system is far less concerned with things that are foreign than with those that do damage.
Tissue Damage and Immune Response
We emphasise on tissue damage as it is the central driver of an immune response whether the damage is an outcome of a drug molecule or a pathogen or endogenous metabolites. The prediction of a drug molecule’s immunogenicity or cytotoxicity to non-target tissue will be best understood at this upstream event.
A case example of severe tissue damage is the incidence of Trovafloxacin, an antibiotic that was withdrawn from the market as it proved to be lethal to some patients, ending in financial loss and lawsuits. Trovafloxacin (sold as Trovan by Pfizer and Turvel by Laboratorios Almirall) was developed as a broad spectrum antibiotic to inhibit the uncoiling of supercoiled DNA in various bacteria. The lethality of the drug was found to be through causing necrosis of hepatocytes leading to liver failure.
Tissue damage that is non-lethal/non-toxic will generate an immune response that may lead to autoimmune pathogenesis. The immune system is programmed to respond to distressed tissue that is associated with an altered microenvironment (protein expression) and is typically followed by inflammation.
Any drug has the potential to cause unwanted tissue damage. The outcome of such damage is the release of intracellular molecules to the immune system. Conventional ‘immunogenicity’ testing considerations are limited to ‘antigenicity’ testing of the drug molecule only. In standard toxicology studies, immune response data such as infiltrations of lymphocytes in tissue sites are ignored as transient immunity and the possibility of disease occurring from chronic immunogenic events is overlooked. It is this broader immunogenicity of a drug that we consider serious and our assays are designed to answer your questions about drug-induced tissue damage and immune response.
Cell cultures (with or without tissue damage induction) are assessed for tissue damage in our lab. Methods we currently use are:
- Microscopy based histochemistry and immunohistochemistry, such as H&E staining or antibody-based staining
- Cell viability assays
- Trypan Blue staining (microscopy or cell counter reading)
- AnnexinV and PI staining (flow cytometry reading)
- Crystal violet staining (absorbence reading and/or microscopy imaging)
- MTT assay
- TUNEL assay
- Oxidative stress assay
- Cleaved caspase (Western or FC)
- Mass spectrometry imaging
In a study on autoimmunity we induced targeted cell death of beta cells in pancreatic islets with streptozotocin. The images show clear difference between healthy live and damaged beta cells in microscopy of tissues. Note the disintegration of the nuclei in B.