Immunohistochemistry and Cellular Immunology Laboratory
The main interests of the Immunohistochemistry and Cellular Immunology Laboratory are the pathogenesis of T cell-mediated inflammation, in the context of autoimmune diseases. Other areas of current or past interest are also listed.
Pathogenesis of T cell-mediated inflammation
This work is undertaken in a rat model of T cell-mediated polyarthritis. In the DA strain, adjuvant-induced arthritis (AA) is a robust model of polyarthritis in which the onset follows immunization with Complete Freund's Adjuvant (CFA) with a highly reproducible prodrome. We have found that lymphocytes in thoracic duct (TD) lymph collected in the late prodrome are effective in transferring polyarthritis to naive recipients. The cells responsible for the adoptive are recently activated CD4 T cells, which have the ability to enter both normal and inflamed synovium. Because the adoptive transfer does not require additional antigens or the co-transfer of donor antigen presenting cells (APC), it appears that the arthritogenic effector cells encounter cognate antigens presented by resident APC. This adoptive transfer system provides, therefore, an opportunity to study the effector phase of T cell-mediated autoimmune inflammation in isolation from the afferent phase of the disease and from the the mycobacterial antigens in CFA.
Function and Fate of CD4 Effector T Cells
The replicative potential and fate of adoptively transferred arthritogenic CD4 T cells is studied in the hand paws of rats, using TD cells from DA CD45.2 congenic rats developed in this laboratory. Both donor- and host-derived T cells are studied using a collagenase-perfusion technique developed in the laboratory to recover cells from the synovium-rich tissues of the hind paws. Cell division, apoptosis, cell surface phenotype and intracellular cytokine production are analysed by flow cytometry.
Synovial Antigen Presenting Cells in Polyarthritis
The arrival of adoptively transferred arthritogenic T cells in normal synovium initiates polyarthritis, indicating that resident APC are competent to present cognate synovial antigens. Macrophage and dendritic cell (DC) populations have been studied and described in normal synovium of the hind paws and in the synovial lining of the knee joint. Current work in the laboratory is focussed on the effects of adoptively transferred arthritogenic T cells on the recruitment, differentiation and functional properties of DC and macrophages in synovial tissues. Studies are undertaken using the collagenase-perfusion techinique to obtain cells from the synovium-rich tissues of hind paws and a technique of synovial lavage that has been developed to observe changes in the knee joint. Flow cytometry, confocal microscopy, cell sorting and in vitro stimulation assays are used to assess DC morphology, phenotype, subsets, endocytic activity and antigen presentation.
Recruitment of Dendritic Cell Precursors
Studies include defining the heterogeneity of circulating monocytoid cells in normal blood and during the induction of adjuvant-induced arthritis. DA CD45.2 congenic donors are being used to observe recruitment of monocytes and monocyte subsets into normal and inflamed synovium and to observe their differentiation under the influence of arthritogenic T cells.
Regulatory T cells
Subpopulations of CD4 T effector and T regulatory (Treg) cells have been defined in TD lymph from normal rats and rats in the late prodrome of AA. Ongoing work aims to study the role of these subsets in the initiation and regulation of polyarthritis, using the DA CD45.2 congenic adoptive transfer system.
Mucosal Immunology
The laboratory has past interests in mucosal antibody production, IgE antibodies and mucosal mast cells in nematode infection of the small intestine, and in extra-thymic T cell differentiation in the small intestine. These interests continue to influence the nature of research undertaken in the laboratory.
Regulatory T cells
The laboratory has embarked on a new program of work aimed at understanding the origin and physiology of Treg cells, making use of access to TD, intestinal and hepatic lymph to define the origins of Tregs in normal TD lymph. DA CD45.2 congenic rats are being used to examine the physiology of these cells after adoptive transfer. It is planned to extend these studies to investigate the role of Tregs in the phenomenon of oral tolerance.
Mast cells
T cells play an important part in regulating the numbers and function of mast cells at mucosal surfaces. In contrast to mucosal mast cells (MMC), numbers of mast cells in connective tissues (CTMC) appear to be regulated mainly by T-independent sources of growth and differentaition factors. Nevertheless, studies in the polyarthritis model indicate that MC in synovium-rich tissues increase in number in response to adoptive transfer of arthritogenic TD lymphocytes. Using antibody against the IgE-FcRI, we have studied MC by flow cytometry in cells prepared from the hind paws of normal and arthritic rats by the collagenase-perfusion technique. A new marker of MC maturation has been identified that allows enumeration of both immature and mature MC subpopulations. Studies are being undertaken on the functional properties of immature and mature subpopulations of CTMC.
Scavenger Receptor CD36
The laboratory has produced and characterized a monoclonal antibody (UA009) against rat CD36. The antibody has proved useful as a marker for monocytes and immature DC in studies on the pathogenesis of polyarthritis (see above). CD36 is scavenger receptor that has a variety of ligands that includes anionic phospholipids exposed on the surfaces of apoptotic cells, oxidized LDL, HDL, collagen and thrombospondin. However, it also has a function in fatty acid transport and has, therefore, the alternative name fatty acid translocase (FAT). In a study of the distribution of FAT/CD36 in rats using mAb UA009, we discovered that the molecule is expressed by hepatocytes in a gender dependent fashion. While expressed at low levels in male liver, hepatocytes in female liver express large amounts of the molecule.
and the Arthritis Research Laboratory work together and have joint weekly laboratory meetings. Our main research focus is the immunopathogenesis of rheumatoid arthritis. We use a rat model, in which we can investigate the early stages of the disease – something that is very difficult to do in humans. This model allows us to study the generation of arthritogenic T cells, how these cells are distributed through the blood to the many joints that are affected (reference 1) and the interactions that occur with dendritic cells in the synovial lining tissues to cause inflammation. We are investigating also the recruitment and differentiation of dendritic cells in the synovium and their role in the maintenance of normal peripheral tolerance and during the induction of autoimmunity (reference 2). This work involves the collection of central lymph to harvest arthritis causing T cells and the enzymatic digestion of synovial tissues to analyse effector T cells and dendritic cells. We use an adoptive transfer system (reference 3) and genetically marked donor cells to identify disease causing T cells, to examine local proliferation and to monitor antigen induced apoptosis. Flow cytometry is used to identify subsets of T cells and dendritic cells, to measure cytokine production by individual cells and to identify donor- and host-derived cells. We also use immunohistochemistry and immunofluoresence laser confocal microscopy to identify cells in situ and to examine the subcellular distribution of MHC class II molecules. Lymphocyte and dendritic cell subsets are purified by using immunomagnetic beads and by fluorescence activated cell sorting FACS). Sorted cells are used to assay responses to antigens and cytokines in vitro.
