M Y Alexander

The role of γδ T cells in the regulation of neutrophil-mediated tissue damage after thermal injury

Thermal injury induces an inflammatory response that contributes to the development of secondary tissuedamage. Neutrophil recruitment and activation are in part responsible for this tissue damage. Although γδ T cells have been shown to regulate the inflammatory responses in tissues that are prone to neutrophil‐mediated injury post‐burn, their role in the induction of secondary tissue injury post‐burn remains unknown. To study this, γδ T cell‐deficient (γδ TCR−/−) and wild‐type (WT) mice were subjected to thermal injury or sham procedure, and tissue samples were isolated 1–24 h thereafter. Burn injury induced neutrophil accumulation in the lung and small intestines of WT mice at 1–3 h post‐injury. No such increase in neutrophil tissue content was observed in γδ TCR−/− mice. An increase in tissue wet/dry weight ratios was also observed in these organs at 3 h post‐burn in WT but not in γδ TCR−/− mice. A parallel increase in plasma and small intestine levels of the chemokines macrophage‐inflammatory protein‐1β (chemokine ligand 4) and keratinocyte‐derived chemokine (CXC chemokine ligand 1) were observed in injured WT mice but not in injured γδ TCR−/− mice. Increased activation (CD120b expression) of the circulating γδ T cell population was also observed at 3 h post‐burn in WT mice. These results indicate the γδ T cells, through the production of chemokines, play a central role in the initiation of neutrophil‐mediated tissue damage post‐burn.

Endothelial progenitor cells enter the aging arena

Age is a significant risk factor for the development of vascular diseases, such as atherosclerosis. Although pharmacological treatments, including statins and anti-hypertensive drugs, have improved the prognosis for patients with cardiovascular disease, it remains a leading cause of mortality in those aged 65 years and over. Furthermore, given the increased life expectancy of the population in developed countries, there is a clear need for alternative treatment strategies. Consequently, the relationship between aging and progenitor cell-mediated repair is of great interest. Endothelial progenitor cells (EPCs) play an integral role in the cellular repair mechanisms for endothelial regeneration and maintenance. However, EPCs are subject to age-associated changes that diminish their number in circulation and function, thereby enhancing vascular disease risk. A great deal of research is aimed at developing strategies to harness the regenerative capacity of these cells. In this review, we discuss the current understanding of the cells termed “EPCs,” examine the impact of age on EPC-mediated repair and identify therapeutic targets with potential for attenuating the age-related decline in vascular health via beneficial actions on EPCs.

Relationships between burn size, immunosuppression, and macrophage hyperactivity in a murine model of thermal injury

Burn injury induces immune dysfunction and alters numerous physiological parameters. While clinical studies indicate that burn injury size profoundly impacts patient immune status, only limited experimental studies have systematically addressed its impact on immune functional parameters. In the present study, mice were subjected to burn injuries of varying sizes and splenic immune cells (splenocytes and macrophages) were isolated 7 days thereafter. Burn injury suppressed splenic T-cell proliferation in an injury size-dependent manner that correlated with the release of the immunosuppressive mediators PGE(2) and nitric oxide. In addition, a shift towards an immunosuppressive Th-2 cytokine profile and a hyperactive macrophage phenotype (increased release of inflammatory mediators) was observed post-injury, however, this effect was in part independent of burn size. Thus, unlike patient survival data, burn injury-induced changes in immune function do not necessarily correlate with the size of the injury.

Endothelial microparticles as conveyors of information in atherosclerotic disease

Endothelial microparticles (EMPs) are complex submicron membrane-shed vesicles released into the circulation following endothelium cell activation or apoptosis. They are classified as either physiological or pathological, with anticoagulant or pro-inflammatory effects respectively. Endothelial dysfunction caused by inflammation is a key initiating event in atherosclerotic plaque formation. Athero-emboli, resulting from ruptured carotid plaques are a major cause of stroke. Current clinical techniques for arterial assessment, angiography and carotid ultrasound, give accurate information about stenosis but limited evidence on plaque composition, inflammation or vulnerability; as a result, patients with asymptomatic, or fragile carotid lesions, may not be identified and treated effectively. There is a need to discover novel biomarkers and develop more efficient diagnostic approaches in order to stratify patients at most risk of stroke, who would benefit from interventional surgery. Increasing evidence suggests that EMPs play an important role in the pathogenesis of cardiovascular disease, acting as a marker of damage, either exacerbating disease progression or triggering a repair response. In this regard, it has been suggested that EMPs have the potential to act as biomarkers of disease status. In this review, we will present the evidence to support this hypothesis and propose a novel concept for the development of a diagnostic device that could be implemented in the clinic.

Hepatocyte growth factor and c‐Met expression in pericytes: implications for atherosclerotic plaque development

Intraplaque neovascularization contributes to the progression of atherosclerosis. Our aim is to understand the mobilization of cells and factors involved in this process. We investigated the localization of hepatocyte growth factor (HGF) and its receptor, c‐Met, in human atherosclerotic plaques, together with the effects of HGF on pericyte migration in vitro. Atherosclerotic femoral arterial segments were collected and analysed from 13 subjects who were undergoing lower limb amputation. Pericytes were identified in human lesions using a 3G5 antibody. Immunohistochemical analysis localized HGF mainly around microvessels, in association with some, but not all, CD31‐positive endothelial cells. c‐Met expression was mainly associated with smooth muscle cells and pericytes, around some, but not all, microvessels within the atherosclerotic lesions; no detection was apparent in normal internal mammary arteries. Using RT–PCR, we demonstrated expression of HGF and c‐Met in a rat pericyte cell‐line, TR–PCT1, and in primary pericytes. HGF treatment of TR‐PCT1 cells induced their migration, but not their proliferation, in a dose‐dependent manner (10–100 ng/ml, p < 0.01), an effect mediated by activation of the serine/threonine kinase Akt, shown by western blot analysis. Treating the cells with the PI3K inhibitors Wortmannin (0.1 µM) or LY294002 (10 µM) abolished these effects. This work demonstrates the expression of c‐Met and HGF in human atherosclerotic arteries, in association with SM‐actin‐positive cells and CD‐31‐positive cells, respectively. HGF induces pericyte migration via PI3‐kinase and Akt activation in vitro. HGF and c‐Met may be involved in neovascularization during plaque development, and may recruit pericytes to neovessels. Since pericytes are thought to mechanically stabilize new blood vessels, these factors may function to protect against haemorrhage. Copyright

Regulation of macrophage IL-10 production postinjury via beta2 integrin signaling and the P38 MAP kinase pathway.

Although LPS receptor (CD14) signaling is mediated in part by &bgr;2 integrins, the role of &bgr;2 integrins in macrophage LPS signaling postinjury remains unknown. To study this, splenic macrophages were isolated from mice 7 days postburn, and inflammatory mediator production was determined. Macrophages isolated from injured mice produced higher levels of PGE2, TNF-&agr;, IL-6, and IL-10 and lower levels of IL-12 in response to LPS stimulation than did cells from sham-treated mice. Blockade of &bgr;2 integrin signaling by addition of antibodies against the CD11b (&agr;CD11b) to the cultures increased IL-10 production by macrophages from injured mice without affecting other mediators. In contrast, sham macrophage responses to LPS were unaffected by &agr;CD11b. Inhibition of p38 MAP kinase activity attenuated IL-10 production and abrogated the enhanced IL-10 response induced by &agr;CD11b, whereas ERK 1/2 inhibition had no effect. Burn injury was associated with increased levels of total and phosphorylated p38 MAP kinase. These findings indicate that LPS signaling via &bgr;2 integrins acts to attenuate the exaggerated induction of IL-10 by macrophages postinjury. Moreover, this effect of &bgr;2 integrin signaling postinjury appears to be downstream of the p38 MAP kinase pathway and is independent of other markers of macrophage hyperactivity.

Nitric oxide contributes to the development of a post‐injury Th2 T‐cell phenotype and immune dysfunction

Severe injury induces immune dysfunction resulting in increased susceptibility to opportunistic infections. Previous studies from our laboratory have demonstrated that post‐burn immunosuppression is mediated by nitric oxide (NO) due to the increased expression of macrophage inducible nitric oxide synthase (iNOS). In contrast, others suggest that injury causes a phenotypic imbalance in the regulation of Th1‐ and Th2 immune responses. It is unclear whether or not these apparently divergent mediators of immunosuppression are interrelated. To study this, C57BL/6 mice were subjected to major burn injury and splenocytes were isolated 7 days later and stimulated with antiCD3. Burn injury induced NO‐mediated suppression of proliferative responses that was reversed in the presence of the NOS inhibitor L‐monomethyl‐L‐arginine and subsequently mimicked by the addition of the NO donor, S‐nitroso‐N‐acetyl‐penicillamine (SNAP). SNAP also dose‐dependently suppressed IFN‐γ and IL‐2 (Th1), but not IL‐4 and IL‐10 (Th2) production. Delaying the addition of SNAP to the cultures by 24 h prevented the suppression of IFN‐γ production. The Th2 shift in immune phenotype was independent of cGMP and apoptosis. The addition of SNAP to cell cultures also induced apoptosis, attenuated mitochondrial oxidative metabolism and induced mitochondrial membrane depolarization. However, these detrimental cellular effects of NO were observed only at supra‐physiologic concentrations (>250 µM). In conclusion, these findings support the concept that NO induces suppression of cell‐mediated immune responses by selective action on Th1 T cells, thereby promoting a Th2 response. J. Cell. Physiol. 208: 418–427, 2006.

Contribution of VCAF-positive cells to neovascularization and calcification in atherosclerotic plaque development.

Calcification of the vessel wall is a regulated process with many similarities to osteogenesis. Progenitor cells may play a role in this process. Previously, we identified a novel gene, Vascular Calcification Associated Factor (VCAF), which was shown to be important in pericyte osteogenic differentiation. The aim of this study was to determine the localization and expression pattern of VCAF in human cells and tissues. Immunohistochemical analysis of seven atherosclerotic arteries confirmed VCAF protein expression within calcified lesions. In addition, individual VCAF‐positive cells were detected within the intima and adventitia in areas where sporadic 3G5‐positive pericytes were localized. Furthermore, VCAF‐positive cells were identified in newly formed microvessels in association with CD34‐positive/CD146‐positive/c‐kit‐positive cells as well as in intact CD31‐positive endothelium in internal mammary arteries. Western blot analysis confirmed the presence of VCAF (18 kD) in protein lysates extracted from human smooth muscle cells, endothelial cells, macrophages, and osteoblasts. In fracture callus samples from three patients, VCAF was detected in osteoblasts and microvessels. This study demonstrates the presence of VCAF in neovessels and raises the possibility that VCAF could be a new marker for vascular progenitor cells involved in a number of differentiation pathways. These data may have implications for the prevention or treatment of vascular disease. Copyright

T cells of the γδ T-cell receptor lineage play an important role in the postburn wound healing process

Although &ggr;&dgr; T cells have been implicated in various aspects of the dermal wound healing process, their role in postburn wound healing processes has not been investigated. To study this, we subjected mice deficient in &ggr;&dgr; T cells (ie, T-cell receptor &dgr; gene [&dgr; TCR–/–]) and wild-type (WT; C57BL6J) mice to burn injury (25% TBSA) or sham treatment; skin samples were isolated 3 days later. Marked inflammation of the injury site was observed in WT mice but was markedly reduced in &dgr; TCR–/– mice. Postinjury fibroblast growth factor, platelet-derived growth factor granulocyte-colony stimulating factor levels, and nitrite/nitrate were elevated in skin samples from injured WT mice, whereas skin tissue levels of these growth factors and inflammatory mediators was significantly atteunuated in &dgr; TCR–/–mice. In conclusion, these findings support the concept that &ggr;&dgr; T cells are important to postburn wound healing via the production of growth factors and, potentially, regulation of inducible nitric oxide synthase activation.

MAP kinases differentially regulate the expression of macrophage hyperactivity after thermal injury.

Thermal injury increases the capacity of macrophages (Mϕ) to produce various inflammatory mediators, (i.e., Mϕ hyperactivity), which is believed to be involved in the development of subsequent immunosuppression, sepsis, and multiple organ failure. The signal transduction pathways involved in the expression of Mϕ hyperactivity post‐burn, however, remain to be clearly elucidated. To study this C57BL/6 female mice were subjected to a 25% TBSA burn and splenic Mϕs were isolated 7 days later. LPS‐stimulated inflammatory mediator production and MAPK expression (P38 ERK 1/2 and JNK) were determined. Burn injury increased LPS‐induced P38 MAPK, suppressed JNK activation and ERK 1/2 activation was unaltered. These changes in MAPK activation were paralleled by the increased production of PGE2, TNF‐α, IL‐1β, IL‐6, and IL‐10. Differential sensitivity to the inhibition of the MAPK pathways was observed with regard to the mediator evaluated and the presence or absence of burn injury. In general cytokine production in the burn group was in part resistant to the inhibition of a single MAPK pathway as compared with shams. Thus, burn injury increases cross‐talk between the MAPKs pathways, suggesting that alterations MAPK activation and signal transduction contribute to the development Mϕ hyperactivity post‐injury. J. Cell. Physiol. 201: 35–44, 2004.