Adrenomedullin receptors on human T cells are glucocorticoid-sensitive.

Adrenomedullin (AM) is a novel vasodilatatory peptide which acts primarily through the calcitonin receptor-like receptor (CLR) in combination with either receptor-activity-modifying-protein (RAMP) 2 or 3 (forming receptors, AM(1) and AM(2) respectively). AM plays an important role during inflammation, with its expression increasing following cytokine treatment, promoting macrophage action in situ and high expression by T cells during hypoxic conditions. Examination of T cell AM receptor expression has previously been incomplete, hence we here consider the presentation of AM receptors and their responsiveness to AM and glucocorticoids (GC). AM receptor expression was examined by PCR and flow cytometry in primary human T cells, revealing that RAMP2, 3 and CLR are physiologically expressed in unstimulated T cells, both intracellularly and on the cell surface. PHA stimulation decreased receptor proteins, significantly so for CLR and RAMP3. Incubation with AM elicited limited receptor alterations however, GC treatment (10(-6) M; 24 h) markedly affected cell surface expression, significantly increasing receptor components in unstimulated cells and significantly decreasing the same in stimulated T cells. Our findings indicate that human T cells utilize both AM(1) and AM(2) receptors, which are GC-sensitive in an activation-state dependent manner.

Identification of PDL-1 as a novel biomarker of sensitizer exposure in dendritic-like cells.

The development of novel in vitro methods to assess risks of allergic sensitization are essential in reducing animal testing whilst maintaining consumer safety. The main research objectives of this study were to identify novel biomarkers to assess the sensitization predictability of chemicals. Phenotypic and cytokine responses of moDCs and MUTZ-3 cells were investigated following application of contact sensitizers; dinitrochlorobenzene (DNCB), cinnamaldehyde (Cin), eugenol (E), isoeugenol (IE), P-phenylenediamine (PPD) and non-sensitizers; salicyclic acid (SA) and sodium lauryl sulphate (SLS). CD86 was up-regulated on MUTZ-3 cells in response to DNCB, Cin and PPD, however, moDCs only modulated CD86 in response to DNCB and E. PDL-1 (Programmed death receptor ligand-1) proved a promising sensitization biomarker in MUTZ-3 cells where up-regulation occurred in response to DNCB, Cin, IE and PPD. Additionally, moDC-expressed PDL-1 was modulated in response to Cin, IE and E thus demonstrating improved sensitizer predictability when compared with CD86. MCP-1 and RANTES were identified as biomarkers of DNCB exposure but MCP-1 did not show any change in expression above controls for the other sensitizers investigated. However, RANTES was increased in MUTZ-3 cells by both DNCB and Cin. Our findings highlight novel biomarkers which, in MUTZ-3 cells, could be taken forward within a multiple biomarker in vitro assay ensuring strong and reliable predictability.

Nociceptin-induced modulation of human T cell function.

There is an accumulating evidence for the immunoregulatory role of the neuropeptide, nociceptin/orphanin FQ (N/OFQ) however its role on T cell function requires elucidation. This study has demonstrated an inhibitory role for N/OFQ on SEB-activated T cell function. N/OFQ decreases T cell proliferation, which is abrogated when the costimulatory receptors CD80 and CD86 are blocked. In addition, evidence suggests that the immunoregulatory cytokines TGF-beta, IFN-gamma and nitric oxide (NO) are involved in the N/OFQ effect. N/OFQ also, through involvement of IFN and NO, induces the expression of the immunosuppressive modulator indoleamine 2,3-dioxygenase (IDO), suggesting a central role for IDO in the N/OFQ effect on T cell proliferation. The data presented in this report indicate a multi-faceted mechanism of action used by N/OFQ to modulate T cell function.

Apoptotic cells induce dendritic cell-mediated suppression via interferon-gamma-induced IDO.

Dendritic cells (DC) are sensitive to their local environment and are affected by proximal cell death. This study investigated the modulatory effect of cell death on DC function. Monocyte-derived DC exposed to apoptotic Jurkat or primary T cells failed to induce phenotypic maturation of the DC and were unable to support CD4+ allogeneic T-cell proliferation compared with DC exposed to lipopolysaccharide (LPS) or necrotic cells. Apoptotic cells coincubated with LPS- or necrotic cell-induced mature DC significantly suppressed CD80, CD86 and CD83 and attenuated LPS-induced CD4+ T-cell proliferation. Reduced levels of interleukin-12 (IL-12), IL-10, IL-6, tumour necrosis factor-alpha and interferon-gamma (IFN-gamma) were found to be concomitant with the suppressive activity of apoptotic cells upon DC. Furthermore, intracellular staining confirmed IFN-gamma expression by DC in association with apoptotic environments. The specific generation of IFN-gamma by DC within apoptotic environments is suggestive of an anti-inflammatory role by the induction of indoleamine 2,3-dioxygenase (IDO). Both neutralization of IFN-gamma and IDO blockade demonstrated a role for IFN-gamma and IDO in the suppression of CD4+ T cells. Moreover, we demonstrate that IDO expression within the DC was found to be IFN-gamma-dependent. Blocking transforming growth factor-beta (TGF-beta) also produced a partial release in T-cell proliferation. Our study strongly suggests that apoptosis-induced DC suppression is not an immunological null event and two prime mediators underpinning these functional effects are IFN-gamma-induced IDO and TGF-beta.

A review of in vitro modelling approaches to the identification and modulation of squamous metaplasia in the human tracheobronchial epithelium.

Squamous metaplasia in the tracheobronchial epithelium (TBE) involves the replacement of the normal pseudostratified mucociliary epithelium with a stratified squamous epithelium. Squamous metaplasia is considered to be an adaptive response that protects the lumen from the effects of inhaled airborne pollutants, but which might also feature as a pre-neoplastic lesion preceding squamous cell carcinoma. With the exception of transglutaminase I, involucrin, and cytokeratins 5, 6 and 13, few markers that contribute to the squamous phenotype have been identified in human TBE that can be used in diagnosis or to monitor its development in laboratory investigations, and current models are inadequate to provide statistically meaningful data. Therefore, new predictive markers have been identified, and new techniques established, in epithelial in vitro models capable of expressing squamous characteristics, which will be used to identify hazardous exposures and elucidate the mechanisms by which they induce their effects. A protocol for the quantitative detection of transglutaminase activity has been standardised in keratinocytes, based on the enzymatic incorporation of fluorescein-cadaverine (FC) into bis(gamma-glutamyl) polyamine cross-links. The specificity of this compound as a transglutaminase substrate was demonstrated by using a range of competitive transglutaminase inhibitors, and by modulation of the squamous pathway. FC incorporation was localised to the cell membrane of terminally differentiating cells, and was not visible in basal, proliferating cells. High calcium-containing medium, nicotine and cigarette smoke condensates (CSC) induced an increase in FC incorporation, providing evidence of their role in enhancing the squamous pathway. Analysis by flow cytometry was used to provide a quantitative assessment of a range of optimised squamous differentiation markers, identified in normal human bronchial epithelia and in a bronchial cell line. Transglutaminase I was induced in a time-dependent manner, in post-confluent cells induced to differentiate down the squamous pathway, whereas involucrin was ubiquitously expressed and the levels of cytokeratins 5, 6 and 18 were reduced. The response of these and other differentiation markers to squamous-inducing conditions is being explored.

Activation marker expression and apoptotic susceptibility of T-cell clones derived from CD34(+), young and SENIEUR donors.

T-cell clones (TCC) derived from human peripheral blood lymphocytes of a young control, a healthy elderly (SENIEUR) donor, or from CD34(+) hematopoietic progenitor cells were utilised in this study to examine how in vivo and in vitro ageing affects T-cell apoptotic capability. The role of CD25, CD28 and the intracellular proteins, FLICE-inhibitory protein (FLIP), receptor-interacting protein (RIP) and caspase 3 were investigated. We observed an age-related decline in the expression of the IL-2 receptor alpha chain CD25, and absence of the co-stimulatory receptor CD28 on three of the four TCC studied. In young donor- and CD34 cell-derived TCC, but not in SENIEUR donor-derived TCC, we observed an age-related increase in susceptibility of the cells to mFas-L-induced apoptosis, which correlated with the age-related decrease of CD25 expression. Expression levels of full-length RIP and FLIP did not show any correlation to apoptotic susceptibility. However, expression levels of the cleaved form of RIP were greatly reduced in the SENIEUR donor-derived TCC, which together with a trend towards increased caspase 3 activity, could indicate an age-related alteration in utilisation of different apoptotic signalling pathways.

Nociceptin/orphanin FQ modulates human T cell function in vitro.

Although nociceptin/orphanin FQ (N/OFQ) and its receptor (ORL-1) are widely distributed throughout the immune system, its role has yet to be elucidated. This study shows that N/OFQ (10(-14)-10(-12) M) modulates T cell activation by up-regulating activation marker expression, e.g. CD28, leading to enhanced proliferation and modulation of TNFalpha secretion. However, on re-stimulated T cells N/OFQ causes inhibition of proliferation, which could be linked with N/OFQ up-regulating CTLA-4 expression. We have also shown that some of these effects are partly prostaglandin-dependent and that N/OFQ induces prostaglandin synthesis. This report suggests that N/OFQ could exert a key modulatory role in human T cell functions.

Identification of a secondary zinc-binding site in staphylococcal enterotoxin C2. Implications for superantigen recognition.

The previously determined crystal structure of the superantigen staphylococcal enterotoxin C2 (SEC2) showed binding of a single zinc ion located between the N- and C-terminal domains. Here we present the crystal structure of SEC2 determined to 2.0 A resolution in the presence of additional zinc. The structure revealed the presence of a secondary zinc-binding site close to the major histocompatibility complex (MHC)-binding site of the toxin and some 28 A away from the primary zinc-binding site of the toxin found in previous studies. T cell stimulation assays showed that varying the concentration of zinc ions present affected the activity of the toxin and we observed that high zinc concentrations considerably inhibited T cell responses. This indicates that SEC2 may have multiple modes of interaction with the immune system that are dependent on serum zinc levels. The potential role of the secondary zinc-binding site and that of the primary one in the formation of the TCR.SEC2.MHC complex are considered, and the possibility that zinc may regulate the activity of SEC2 as a toxin facilitating different T cell responses is discussed.

P-glycoprotein decreases with T cell maturation but is not responsible for resistance to CD95-induced apoptosis.

P-glycoprotein (Pgp) is a membrane transporter responsible for resistance to chemotherapy in cancer cells. Its presence in T cells is very well documented, but its function in the immune system is still poorly understood. Recent findings suggest that Pgp may be involved in regulating programmed cell death by inhibiting caspase 8 and caspase 3. Utilising antigenically-activated T cells and the physiologically relevant apoptotic ligand, membrane CD95-L, we have previously reported that while T cells are generally resistant to CD95-induced death at early stages of activation, their susceptibility to apoptosis increases with successive activation and clonal expansion. In this study we investigated whether changes in apoptotic susceptibility were related to T cell Pgp function. Results showed that Pgp expression and function in T cells decreases with maturation, with CD8 cells having the highest Pgp function. However, although Pgp function inversely correlated with caspase 3 activity, no difference was observed between apoptotic susceptible CD25- cells and resistant CD25+ cells. In addition sorting of cells with high and low Pgp function showed no correlation with apoptotic capability. Therefore, whilst Pgp modulates caspase activity, it is not responsible for resistance to apoptosis of early activated T cells nor the increased susceptibility observed at the later stages of maturation in antigenically activated cells.

Caspase involvement in RIP-associated CD95-induced T cell apoptosis.

CD95-induced apoptosis is an important regulatory mechanism in T cells and this complex signalling pathway is now thought to include the protein kinase RIP. Although, RIP is best known for its role in TNF signalling and NF-kappaB activation, it contains a death domain and it is capable of causing apoptosis upon cleavage. In the present study, the role of RIP in CD95-induced apoptosis and its inter-relationship with the caspase cascade was investigated. Studies were performed on both a RIP-/- T cell line and peripheral T lymphocytes, where RIP was degraded through the addition of geldanamycin. Apoptosis was induced by membrane CD95-L, thought to be the most physiological relevant form of CD95-L. Results showed that RIP-/- cells had a decreased susceptibility to death, thus confirming a role for RIP in CD95-induced apoptosis. Furthermore, it was confirmed that RIP is cleaved upon CD95-L stimulation, a process that can be inhibited by Z-VAD. However, only partial inhibition in peripheral T lymphocytes by Z-VAD was observed, suggesting a potential caspase-independent processing of RIP. Studies performed on the activity of effector caspase 3 and on the initiator caspases 2, 8, and 9 revealed that, in the absence of RIP, the activity of these caspases decreases, indicating that RIP-associated apoptosis is caspase-dependent. Hence, these studies support a caspase-related role for RIP in CD95-induced T apoptosis.

Activated T cells mediate direct blood-brain barrier endothelial cell death and dysfunction.

Neuro-inflammation is characterized by immune cell infiltration across the blood-brain barrier, a process instrumental in neuronal cell death. In neuro-inflammation the blood-brain barrier is also damaged and the consequences of activated lymphocytes on the integrity of the blood-brain barrier is not well characterized. Utilizing an blood-brain barrier model we demonstrate that endothelial cell viability and barrier integrity are directly altered following lymphocyte exposure. The effect of activated lymphocytes is cell number dependent, mostly mediated by direct contact, and is not associated with the pro-inflammatory cytokine TNF-alpha. For the successful treatment of neuro-inflammatory disease, intervention of this direct effect at the blood-brain barrier is warranted.

Evaluation of the role of P-glycoprotein in inflammation induced blood-brain barrier damage.

In this study we evaluated the role of the multi-drug transporter p-glycoprotein (Pgp) in the process of activated T lymphocyte-mediated blood-brain barrier dysfunction as described previously. Lymphocyte exposure induced significant endothelial cell death and there was an elevation of the expression of Pgp in the surviving cells. Inhibition of Pgp function using the antibody MRK16 and verapamil displayed a dose-dependent prevention of T cell mediated endothelial cell death and barrier breakdown. These data suggest that the activity of Pgp at the blood-brain barrier may play a role in lymphocyte induced barrier cell damage and a role as a possible survival mechanism to prevent further endothelial cell death in later stages of inflammation.

Age associated decline in CD25 and CD28 expression correlate with an increased susceptibility to CD95 mediated apoptosis in T cells.

Immunosenescence is believed to contribute to increase susceptibility to infectious diseases and cancer in the elderly, and is caused mainly by changes in the T cell compartment. Longitudinal studies were undertaken to examine T cell surface receptor expression and apoptotic susceptibility using Staphylococcal enterotoxin B (SEB) activated human T cells as an in vitro model of an ageing T cell culture. An intracellular stain Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to assess the number of population divisions (PD) occurring in the ageing T cell culture. One major biomarker of aged T cells is a decrease in expression of CD28 and since this is an essential co-stimulatory molecule, its decreasing expression with age could compromise their activation and apoptotic capacity. Activation of T cells resulted in initial up-regulation of CD25, CD95 and CD28, although expression of CD25 and CD28 subsequently decreased with increasing PD. CD4 and CD8 T cells expressed similar CD25 profiles although CD28 expression was unique in each subset. CD4+ cells expressed the highest CD28 levels, and showed a gradual decline in expression with increasing PD, whereas CD8+ cells were low CD28 expressers, but did not appear to lose their expression as they aged. To determine T cell susceptibility to apoptosis via CD95/CD95-L interactions with increasing age, cells were challenged with CD95-L transfected CHO cells at various PD. Increased death was observed as they aged, which correlated with the decreased expression of activation markers CD25 and CD28.