Adhesion and Migration of Monocytes and Dendritic Cells in Type 1 Diabetes

SAMENVATTING VOOR NIET-INGEWIJDEN Type 1 diabetes, vroeger ook wel jeugddiabetes genoemd, wordt gekenmerkt door een afweerreactie gericht tegen de insuline-producerende ß cellen. Bij deze afweerreactie valt het afweersysteem (immuunsysteem) de lichaamseigen ß cellen in de alvleesklier aan en vernietigt deze. Deze afweerreactie waarbij het lichaam zichzelf aanvalt wordt een auto-immuunreactie genoemd. Als gevolg van deze auto-immuunreactie vermindert de productie van insuline, die belangrijk is voor de glucose huishouding binnen het lichaam. Door de verminderde inType 1 diabetes is characterized by a T cell mediated destruction of the insulin-producing ß cells in the islets of Langerhans that are situated in the pancreas. Prior to the infiltration of lymphocytes into the pancreas, an accumulation of macrophages (mf) and dendritic cells (DC) is observed. It is generally thought that these mf and DC originate from blood monocytes that have entered the pancreas and have differentiated into mf or DC. On the basis of their normal physiological role, these cells presumably take up self-antigens and process these into peptides, which they present, after a so-called “steady-state” or “homeostatic” trafficking of the DC to the draining lymph nodes, to T lymphocytes in the para-cortical area. Normally this leads to tolerance induction and T regulatory cells are induced. However in the case of diabetes development, not regulatory T cells, but erroneously effector T lymphocytes become activated and islet autoimmunity is induced. These effector T cells that were primed in the lymph node, become re-activated after re-circulation upon recognition of the self-antigens in the pancreas and consequently – together with macrophages - initiate inflammation and mediate ß cell destruction, which is a hallmark of type 1 diabetes. In this thesis I have studied the processes involved in the early accumulation of mf and DC in the pancreas prior to the infiltration of lymphocytes. The extravasation of monocytes from the circulation into the pancreas is a complex process. Amongst other factors, adhesion molecules, chemokines and myeloid related proteins (MRPs) play an important role in the adhesive and migratory responses of the monocytes that enable effective extravasation. I studied the adhesive and migratory behaviour of human monocytes of type 1 diabetic patients and compared these functions with those of monocytes of type 2 diabetic patients and healthy control subjects. First of all, I was not able to detect any differences between patients and control subjects regarding the subdivision of circulating monocytes in mature and immature cells based on the expression of CD14 and CD16 (chapter 2.1). Secondly, monocytes of patients with type 1 diabetes displayed an intrinsically increased surface expression of the pro-inflammatory molecule MRP8/14 and an increased serum level of MRP8/14. When monocytes were allowed to adhere to the extra cellular matrix component fibronectin the cells showed an enhanced expression and production of MRP8/14. The monocytes of type 1 diabetes patients showed the strongest expression and production of MRP8/14 which was significantly increased over that of healthy control monocytes (chapter 2.2). Furthermore, such activated type 1 diabetic monocytes showed an even stronger adhesion to fibronectin, which was indeed found to be the effect of exposition of the monocytes to MRP8/14 (chapter 2.1). My findings suggest a positive feedback mechanism regarding the adhesive capacity of monocytes in type 1 diabetes: circulating monocytes express and secrete higher levels of MRP8/14 as compared to healthy control subjects, resulting in increased MRP8/14 in the serum. The increased serum MRP8/14 induces an increased adhesive capacity to fibronectin of the monocytes, which leads to an even larger secretion of MRP8/14 compared to healthy controls. In this thesis I also describe that the increased MRP8/14 in the serum induced an increased expression of CD11b/CD18 on the monocytes that is likely involved in the increased adhesion of the type 1 diabetic monocytes to endothelial cells that I observed (chapter 2.2). After the adhesion studies I investigated the migratory behaviour of monocytes of type 1 diabetes patients and observed a remarkably decreased response towards the pro-inflammatory chemokines CCL2 and CCL3. Both the transendothelial migration (measured in a Transwell system) and the chemotaxis (measured in the classical Boyden assay) towards these pro-inflammatory chem

Vignette studies of medical choice and judgement to study caregivers' medical decision behaviour: systematic review

BACKGROUND: Vignette studies of medical choice and judgement have gained popularity in the medical literature. Originally developed in mathematical psychology they can be used to evaluate physicians' behaviour in the setting of diagnostic testing or treatment decisions. We provide an overview of the use, objectives and methodology of these studies in the medical field. METHODS: Systematic review. We searched in electronic databases; reference lists of included studies. We included studies that examined medical decisions of physicians, nurses or medical students using cue weightings from answers to structured vignettes. Two reviewers scrutinized abstracts and examined full text copies of potentially eligible studies. The aim of the included studies, the type of clinical decision, the number of participants, some technical aspects, and the type of statistical analysis were extracted in duplicate and discrepancies were resolved by consensus. RESULTS: 30 reports published between 1983 and 2005 fulfilled the inclusion criteria. 22 studies (73%) reported on treatment decisions and 27 (90%) explored the variation of decisions among experts. Nine studies (30%) described differences in decisions between groups of caregivers and ten studies (33%) described the decision behaviour of only one group. Only six studies (20%) compared decision behaviour against an empirical reference of a correct decision. The median number of considered attributes was 6.5 (IQR 4-9), the median number of vignettes was 27 (IQR 16-40). In 17 studies, decision makers had to rate the relative importance of a given vignette; in six studies they had to assign a probability to each vignette. Only ten studies (33%) applied a statistical procedure to account for correlated data. CONCLUSION: Various studies of medical choice and judgement have been performed to depict weightings of the value of clinical information from answers to structured vignettes of care givers. We found that the design and analysis methods used in current applications vary considerably and could be improved in a large number of cases

NOD mice have a severly impaired ability to recruit leukocytes into sites of inflammation

The accumulation of macrophages (MPhi) and dendritic cells (DC) in the pancreas plays a crucial role in the pathogenesis of autoimmune diabetes. We studied the recruitment of monocytes, MPhi and DC to sites of inflammation, i.e. the peritoneal cavity and a subcutaneously elicited air pouch in the NOD mouse model of autoimmune diabetes. The leukocyte recruitment was studied from 1 to 7 days after injection of thioglycollate (peritoneum), C5a (peritoneum, air pouch), CCL2 and CCL3 (air pouch). C57BL/6 and BALB/c mice served as controls. Morphological and flow cytometric analysis of the recruited cells was performed, IL-1beta, TNF-alpha, IL-6, IL-12 and IL-10 in exudates measured, and in vitro CCL2-chemotaxis of exudate MPhi (Boyden chamber) determined. NOD mice were strongly impaired in the recruitment of MPhi, DC, monocytes, and granulocytes. Chemokine-injected air pouches of NOD mice showed an increased IL-10 and a decreased IL-1beta level, while the other cytokines were normally or very lowly expressed. In addition, NOD exudate MPhi displayed an impaired in vitro CCL2-induced migration. Our data show that NOD mice have an impaired ability to recruit leukocytes into sites of inflammation elicited in the peritoneum and the air pouch. A raised IL-10/ IL-1beta ratio at these sites and a deficient migratory capacity of NOD monocytes are important determinants in this impairment

Epigenetically quantified immune cells in salivary glands of Sjogren's syndrome patients: a novel tool that detects robust correlations of T follicular helper cells with immunopathology : a novel tool that detects robust correlations of T follicular helper cells with immunopathology

OBJECTIVE: To investigate whether epigenetic cell counting represents a novel method to quantify immune cells in salivary glands of patients with different forms of Sjögren's and sicca syndrome and to capture immunopathology and potentially aid in diagnosis. METHODS: DNA from frozen salivary gland tissue sections of sicca patients was used for bisulphite conversion of demethylated DNA cytosine residues, followed by cell-specific quantitative PCR to calculate cell percentages in relation to total tissue cell numbers as quantified by housekeeping gene demethylation. The percentages of epigenetically quantified cells were correlated to RNA expression of matched salivary gland tissue and histological and clinical parameters. RESULTS: The percentages of epigenetically quantified CD3, CD4, CD8, T follicular helper (Tfh) cells, FoxP3+ regulatory T cells and B cells were significantly increased in the salivary glands of patients with SS. Unsupervised clustering using these percentages identified patient subsets with an increased lymphocytic focus score and local B cell hyperactivity and classifies patients different from conventional classification criteria. In particular, Tfh cells were shown to strongly correlate with the expression of CXCL13, lymphocytic focus scores, local B cell hyperactivity and anti-SSA positivity. CONCLUSION: Epigenetic cell counting is a promising novel tool to objectively and easily quantify immune cells in the labial salivary gland of sicca patients, with a relatively small amount of tissue needed. In view of the potential of this technique to include a huge number of (cell-specific) biomarkers, this opens up new standardized ways of salivary gland analysis with high relevance for patient classification, understanding of immunopathology and monitoring of drug responses in clinical trials

Epigenetically quantified immune cells in salivary glands of Sjogren's syndrome patients: a novel tool that detects robust correlations of T follicular helper cells with immunopathology : a novel tool that detects robust correlations of T follicular helper cells with immunopathology

OBJECTIVE: To investigate whether epigenetic cell counting represents a novel method to quantify immune cells in salivary glands of patients with different forms of Sjögren's and sicca syndrome and to capture immunopathology and potentially aid in diagnosis. METHODS: DNA from frozen salivary gland tissue sections of sicca patients was used for bisulphite conversion of demethylated DNA cytosine residues, followed by cell-specific quantitative PCR to calculate cell percentages in relation to total tissue cell numbers as quantified by housekeeping gene demethylation. The percentages of epigenetically quantified cells were correlated to RNA expression of matched salivary gland tissue and histological and clinical parameters. RESULTS: The percentages of epigenetically quantified CD3, CD4, CD8, T follicular helper (Tfh) cells, FoxP3+ regulatory T cells and B cells were significantly increased in the salivary glands of patients with SS. Unsupervised clustering using these percentages identified patient subsets with an increased lymphocytic focus score and local B cell hyperactivity and classifies patients different from conventional classification criteria. In particular, Tfh cells were shown to strongly correlate with the expression of CXCL13, lymphocytic focus scores, local B cell hyperactivity and anti-SSA positivity. CONCLUSION: Epigenetic cell counting is a promising novel tool to objectively and easily quantify immune cells in the labial salivary gland of sicca patients, with a relatively small amount of tissue needed. In view of the potential of this technique to include a huge number of (cell-specific) biomarkers, this opens up new standardized ways of salivary gland analysis with high relevance for patient classification, understanding of immunopathology and monitoring of drug responses in clinical trials