Severe gunshot injuries in a porcine model: impact on central markers of innate immunity.

BACKGROUND: the mechanisms behind lipopolysaccharide (LPS) tolerance remain obscure. LPS signals through Toll-like receptor 4 (TLR4) and severe trauma/haemorrhage may influence binding and signalling through this receptor, e.g. by changing membrane expression or by releasing endogenous ligands like High Mobility Group Box 1 (HMGB1). The aim of this study was to examine these relations further in a porcine model with standardized trauma. METHODS: nine anaesthetized pigs sustained one gunshot through the femur and one pistol shot through the upper abdomen. Blood was sampled before and 90 min after shooting. The samples were stimulated for 4 h with LPS 10 ng/ml or an equivalent amount of normal saline. The leucocyte response was evaluated by measuring the tumour necrosis factor-α (TNF-α) and CXC ligand 8 (CXCL8) in the supernatant. Flow cytometry was used to measure the surface expression of TLR4 on CD14+ monocytes. HMGB1 concentrations were measured in the plasma. RESULTS: trauma and treatment caused a significant decline in the LPS-stimulated concentrations of TNF-α [4.53 ± 0.24 pg/ml (ln) at 0 min, 3.54 ± 0.35 pg/ml (ln) at 90 min, P=0.026], but did not modify the release of CXCL8. Monocyte TLR4 expression was unchanged. Plasma HMGB1 increased significantly [<0.92 vs. 3.02 ± 0.19 ng/ml (ln), P<0.001]. The concentrations of TNF-α and CXCL8 did not correlate with TLR4 expression or HMGB1 concentrations. CONCLUSION: the results suggest that trauma-induced LPS tolerance is not primarily regulated by TLR4 expression on circulating CD14+ monocytes or by the release of HMGB1 from damaged tissues.

Low thymic output in the 22q11.2 deletion syndrome measured by CCR9+CD45RA+ T cell counts and T cell receptor rearrangement excision circles.

Thymic hypoplasia is a frequent feature of the 22q11.2 deletion syndrome, but we know little about patients' age-related thymic output and long-term consequences for their immune system. We measured the expression of T cell receptor rearrangement excision circles (TREC) and used flow cytometry for direct subtyping of recent thymic emigrant (RTE)-related T cells in 43 patients (aged 1-54 years; median 9 years) from all over Norway and in age-matched healthy controls. Thymic volumes were estimated by ultrasound in patients. TREC levels correlated well with RTE-related T cells defined by co-expression of CD3, CD45RA and CCR9 (r=0.84) as well as with the CD4+ and CD8+ T cell subtypes. RTE-related T cell counts also paralleled age-related TREC reductions. CD45RA+ T cells correlated well with absolute counts of CD4+ (r=0.87) and CD8+ (r=0.75) RTE-related T cells. Apart from CD45RA- T cells, all T cell subsets were lower in patients than in controls. Thymic volumes correlated better with RTE-related cells (r=0.46) than with TREC levels (r=0.38). RTE-related T cells and TREC levels also correlated well (r=0.88) in patients without an identifiable thymus. Production of RTEs is impaired in patients with a 22q11.2 deletion, and CCR9 appears to be a good marker for RTE-related T cells.

Age-related changes in CCR9+ circulating lymphocytes: are CCR9+ naive T cells recent thymic emigrants?

The chemokine receptor CCR9 is reported to be predominantly expressed by thymocytes as well as by circulating gut-homing and resident T cells in the small intestinal mucosa. Its ligand thymus-expressed chemokine (TECK) is produced by thymic and small intestinal epithelium. Here we report that the proportion of circulating CCR9+ naive T cells (mostly CD4+) declines with age, from approximately 15% of all T cells at birth to around 1% in adults. The proportion of CCR9+ T cells lacking the classical gut-homing receptor alpha4beta7, was much higher in children than in adults. Therefore, circulating CD3+CCR9+CD45RA+ cells have most likely left the thymus quite recently. This notion was supported by the small number of CCR9+ naive T cells which was present shortly after thymectomy. Establishing a phenotypic marker for recent thymic emigrants might provide a powerful tool in the clinical assessment and follow-up after cancer chemotherapy, hematopoietic stem cell transplantation, and during antiretroviral treatment of human immunodeficiency virus (HIV)-infected patients.

Interferon-gamma-secreting T cells localize to the epithelium in coeliac disease.

Increased levels of interferon-gamma (IFN-gamma) transcripts have previously been found in duodenal biopsy specimens from patients with untreated coeliac disease (CD). Such samples and duodenal control mucosa were therefore studied to locate and phenotype cells spontaneously secreting IFN-gamma. Specimens were collected from consecutively recruited patients with untreated (seven), treated (four) or refractory (three) CD and from five histologically normal controls. Morphological and immunohistochemical examinations were performed, and epithelial and lamina propria cell suspensions were prepared from parallel samples. Unstimulated viable cells secreting IFN-gamma were identified and phenotyped with a new fluorescence-activated cell sorter-based assay, and IFN-gamma messenger RNA (mRNA) was analysed in snap-frozen aliquots of the same suspensions. Untreated CD cases had the highest fraction of IFN-gamma+ cells in the epithelial compartment (median 2.6%, range 1.6-6.2%) and, less strikingly, in the lamina propria compartment (1.6%, range 0.3-3.6%), followed by refractory (1.4%, 1.0-1.9%; and 0.3%, 0.0-1.2%) and treated (0.8%, 0.5-0.9%; and 0.7%, 0.2-1.1%) disease and finally the controls (0.5%, 0.3-0.9%; and 0.2%, 0.1-0.7%). IFN-gamma mRNA data supported these findings. IFN-gamma+ intraepithelial lymphocytes were mostly CD3+ and CD8+, whereas many positive lamina propria cells were CD8-. We conclude that isolated T cells spontaneously secreting IFN-gamma localize preferentially in the epithelium of patients with classical and refractory CD.