Soluble GPVI is elevated in injured patients: shedding is mediated by fibrin activation of GPVI.

Soluble glycoprotein VI (sGPVI) is shed from the platelet surface and is a marker of platelet activation in thrombotic conditions. We assessed sGPVI levels together with patient and clinical parameters in acute and chronic inflammatory conditions, including patients with thermal injury and inflammatory bowel disease and patients admitted to the intensive care unit (ICU) for elective cardiac surgery, trauma, acute brain injury, or prolonged ventilation. Plasma sGPVI was measured by enzyme-linked immunosorbent assay and was elevated on day 14 after thermal injury, and was higher in patients who developed sepsis. sGPVI levels were associated with sepsis, and the value for predicting sepsis was increased in combination with platelet count and Abbreviated Burn Severity Index. sGPVI levels positively correlated with levels of D-dimer (a fibrin degradation product) in ICU patients and patients with thermal injury. sGPVI levels in ICU patients at admission were significantly associated with 28- and 90-day mortality independent of platelet count. sGPVI levels in patients with thermal injury were associated with 28-day mortality at days 1, 14, and 21 when adjusting for platelet count. In both cohorts, sGPVI associations with mortality were stronger than D-dimer levels. Mechanistically, release of GPVI was triggered by exposure of platelets to polymerized fibrin, but not by engagement of G protein-coupled receptors by thrombin, adenosine 5'-diphosphate, or thromboxane mimetics. Enhanced fibrin production in these patients may therefore contribute to the observed elevated sGPVI levels. sGPVI is an important platelet-specific marker for platelet activation that predicts sepsis progression and mortality in injured patients.

Changes in the pattern of plasma extracellular vesicles after severe trauma.

BACKGROUND: Extracellular vesicles (EV) released into the circulation after traumatic injury may influence complications. We thus evaluated the numbers of EV in plasma over 28 days after trauma and evaluated their pro-coagulant and inflammatory effects. METHODS AND FINDINGS: 37 patients suffering trauma with an injury severity score >15 were studied along with 24 healthy controls. Plasma samples were isolated by double centrifugation (2000g 20min; 13000g 2min) from blood collected from within an hour up to 28 days after injury. Plasma EV were counted and sized using nanoparticle tracking analysis (NTA); counts and cellular origins were also determined by flow cytometry (FC) using cell-specific markers. Functional effects were tested in a procoagulant phospholipid assay and in flow-based, leukocyte adhesion assay after endothelial cells (EC) were treated with EV. We found that EV concentrations measured by NTA were significantly increased in trauma patients compared to healthy controls, and remained elevated over days. In addition, or FC showed that patients with trauma had higher numbers of EV derived from platelets (CD41+), leukocytes (CD45+) and endothelial EC (CD144+). The increases were evident throughout the 28-day follow-up. However, the FC count represented <1% of the count detected by NTA, and only 1-2% of EV identified using NTA had a diameter >400nm. The procoagulant phospholipid activity assay showed that patient plasma accelerated coagulation on day 1 and day 3 after trauma, with coagulation times correlated with EV counts. Furthermore, treatment of EC for 24 hours with plasma containing EV tended to increase the recruitment of peripheral flowing blood mononuclear cells. CONCLUSIONS: EV counted by FC represent a small sub-population of the total load detected by NTA. Both methods however indicate a significant increase in plasma EV after severe traumatic injury that have pro-coagulant and pro-inflammatory effects that may influence outcomes.

Prehospital immune responses and development of multiple organ dysfunction syndrome following traumatic injury: A prospective cohort study.

BACKGROUND: Almost all studies that have investigated the immune response to trauma have analysed blood samples acquired post-hospital admission. Thus, we know little of the immune status of patients in the immediate postinjury phase and how this might influence patient outcomes. The objective of this study was therefore to comprehensively assess the ultra-early, within 1-hour, immune response to trauma and perform an exploratory analysis of its relationship with the development of multiple organ dysfunction syndrome (MODS). METHODS AND FINDINGS: The immune and inflammatory response to trauma was analysed in 89 adult trauma patients (mean age 41 years, range 18-90 years, 75 males) with a mean injury severity score (ISS) of 24 (range 9-66), from whom blood samples were acquired within 1 hour of injury (mean time to sample 42 minutes, range 17-60 minutes). Within minutes of trauma, a comprehensive leukocytosis, elevated serum pro- and anti-inflammatory cytokines, and evidence of innate cell activation that included neutrophil extracellular trap generation and elevated surface expression of toll-like receptor 2 and CD11b on monocytes and neutrophils, respectively, were observed. Features consistent with immune compromise were also detected, notably elevated numbers of immune suppressive CD16BRIGHT CD62LDIM neutrophils (82.07 x 106/l ± 18.94 control versus 1,092 x 106/l ± 165 trauma, p < 0.0005) and CD14+HLA-DRlow/- monocytes (34.96 x 106/l ± 4.48 control versus 95.72 x 106/l ± 8.0 trauma, p < 0.05) and reduced leukocyte cytokine secretion in response to lipopolysaccharide stimulation. Exploratory analysis via binary logistic regression found a potential association between absolute natural killer T (NKT) cell numbers and the subsequent development of MODS. Study limitations include the relatively small sample size and the absence of data relating to adaptive immune cell function. CONCLUSIONS: Our study highlighted the dynamic and complex nature of the immune response to trauma, with immune alterations consistent with both activation and suppression evident within 1 hour of injury. The relationship of these changes, especially in NKT cell numbers, to patient outcomes such as MODS warrants further investigation.

Primary adrenal insufficiency is associated with impaired natural killer cell function: a potential link to increased mortality.

OBJECTIVE: Mortality in patients with primary adrenal insufficiency (PAI) is significantly increased, with respiratory infections as a major cause of death. Moreover, patients with PAI report an increased rate of non-fatal infections. Neutrophils and natural killer (NK) cells are innate immune cells that provide frontline protection against invading pathogens. Thus, we compared the function and phenotype of NK cells and neutrophils isolated from PAI patients and healthy controls to ascertain whether altered innate immune responses could be a contributory factor for the increased susceptibility of PAI patients to infection. DESIGN AND METHODS: We undertook a cross-sectional study of 42 patients with PAI due to autoimmune adrenalitis (n = 37) or bilateral adrenalectomy (n = 5) and 58 sex- and age-matched controls. A comprehensive screen of innate immune function, consisting of measurements of neutrophil phagocytosis, reactive oxygen species production, NK cell cytotoxicity (NKCC) and NK cell surface receptor expression, was performed on all subjects. RESULTS: Neutrophil function did not differ between PAI and controls. However, NKCC was significantly reduced in PAI (12.0 ± 1.5% vs 21.1 ± 2.6%, P < 0.0001). Phenotypically, the percentage of NK cells expressing the activating receptors NKG2D and NKp46 was significantly lower in PAI, as was the surface density of NKG2D (all P < 0.0001). Intracellular granzyme B expression was significantly increased in NK cells from PAI patients (P < 0.01). CONCLUSIONS: Adrenal insufficiency is associated with significantly decreased NKCC, thereby potentially compromising early recognition and elimination of virally infected cells. This potential impairment in anti-viral immune defense may contribute to the increased rate of respiratory infections and ultimately mortality in PAI.

Neutrophil Dysfunction, Immature Granulocytes, and Cell-free DNA are Early Biomarkers of Sepsis in Burn-injured Patients: A Prospective Observational Cohort Study.

OBJECTIVE: The aim of this study was to measure neutrophil function longitudinally following burn injury and to examine the relationship between neutrophil dysfunction and sepsis. BACKGROUND: Sepsis prevalence and its associated mortality is high following burn injury, and sepsis diagnosis is complicated by the ongoing inflammatory response. Previous studies have suggested that neutrophil dysfunction may underlie high infection rates and sepsis postburn; however, neutrophil dysfunction has not been thoroughly characterized over time in burns patients. METHODS: Neutrophil phagocytosis, oxidative burst capacity, and neutrophil extracellular trap (NET) generation (NETosis) were measured from 1 day to up to 1 year postburn injury in 63 patients with major burns (≥15% total body surface area). In addition, immature granulocyte (IG) count, plasma cell-free DNA (cfDNA), and plasma citrullinated histone H3 (Cit H3) levels were measured. RESULTS: Neutrophil function was reduced for 28 days postburn injury and to a greater degree in patients who developed sepsis, which was also characterized by elevated IG counts. Plasma cfDNA and Cit-H3, a specific marker of NETosis, were elevated during septic episodes. The combination of neutrophil phagocytic capacity, plasma cfDNA levels, and IG count at day 1 postinjury gave good discriminatory power for the identification of septic patients. CONCLUSION: Neutrophil function, IG count, and plasma cfDNA levels show potential as biomarkers for the prediction/early diagnosis of sepsis postburn injury and neutrophil dysfunction may actively contribute to the development of sepsis. Targeting neutrophil dysfunction and IG release may be a viable therapeutic intervention to help reduce the incidence of nosocomial infections and sepsis postburn.

The diagnostic and prognostic value of systems biology research in major traumatic and thermal injury: a review.

As secondary complications remain a significant cause of morbidity and mortality amongst hospitalised trauma patients, the need to develop novel approaches by which to identify patients at risk of adverse outcome is becoming increasingly important. Centred on the idea that patients who experience "poor" outcome post trauma elicit a response to injury that is distinct from those who experience "good" outcome, tailored therapeutics is an emerging concept aimed at improving current treatment regimens by promoting patient-specific therapies. Making use of recent advancements in the fields of genomics, proteomics and metabolomics, numerous groups have undertaken a systems-based approach to analysing the acute immune and inflammatory response to major traumatic and thermal injury in an attempt to uncover a single or combination of biomarkers that can identify patients at risk of adverse outcome. Early results are encouraging, with all three approaches capable of discriminating patients with "good" outcome from those who develop nosocomial infections, sepsis and multiple organ failure, with differences apparent in blood samples acquired as early as 2 h post injury. In particular, genomic data is proving to be highly informative, identifying patients at risk of "poor" outcome with a higher degree of sensitivity and specificity than statistical models built upon data obtained from existing anatomical and physiological scoring systems. Here, focussing predominantly upon human-based research, we provide an overview of the findings of studies that have investigated the immune and inflammatory response to major traumatic and thermal injury at the genomic, protein and metabolite level, and consider both the diagnostic and prognostic potential of these approaches.

Inhibition of Histone Deacetylase 6 Reveals a Potent Immunosuppressant Effect in Models of Transplantation.

BACKGROUND: Current transplant immunosuppression regimens have numerous limitations. Recent evidence suggests histone deacetylase inhibitors (HDACis) may represent a class of drug with immunosuppressive properties. This study compares cyclosporin A (CyA) with the pan-HDACi suberoylanilide hydroxamic acid (SAHA) and a novel HDAC6-specific inhibitor (KA1010) in models of alloreactivity. METHODS: Proliferation and mixed lymphocyte reaction (MLR)-based assays were used to determine the immunosuppressive effect of compounds, and a murine model of allogeneic skin transplantation was adopted to assess the in vivo effects of HDAC6 inhibition. RESULTS: KA1010 displayed superior inhibitory effects on the activation of peripheral mononuclear cells using in vitro models of transplantation. In a 1-way MLR, KA1010 (5 µΜ) reduced parent cell proliferation from 92% to 64% (P = 0.001). A 2-way MLR, adopting IFN-γ production as a marker of alloresponse, resulting in up to 91% reduction. Dose-response curves revealed dose-dependent profiles with greater potency of HDACis over CyA (IC50 values of 82.0 nM and 13.4 nM for KA1010 and SAHA).Mice treated with KA1010 displayed no significant features of skin allograft rejection upon histological analysis at 70 days and graft survival of 80% in subjects treated with 160 mg/kg. Immunological assessment, revealed a significant increase in CD4CD25forkhead box P3 regulatory T cells (from 18% to 25%, P = 0.0002) and a corresponding reduction in CD4 T cells (from 58% to 42%, P = 0.0009). CONCLUSIONS: HDAC6 may represent an optimal target for future immunosuppressant therapeutics with a particular role in transplantation. In this article, we have demonstrated a superior immunosuppressive effect of KA1010 over both CyA and SAHA, in the models of allotransplantation adopted.

Immunesenescence and inflammaging: A contributory factor in the poor outcome of the geriatric trauma patient.

Compared to younger patients, traumatic injury in older patients is associated with increased mortality and a range of adverse outcomes such as higher rates of infectious episodes, longer length of hospital stay and poor functional outcome at follow up. Data emerging from human and murine-based studies suggest age-related changes in immune function, collectively termed immunesenescence, and the chronic sub-clinical systemic inflammatory state of older adults, termed inflammaging, may contribute to these poor outcomes. Here, we review the findings of these studies, whose results demonstrate that the geriatric trauma patient elicits an immune response to injury that is distinct to that of younger adults, being characterised by reduced immune cell activation, impaired function and abnormal haematopoiesis, defects that are accompanied by an altered inflammatory response that fails to return to a homeostatic baseline in the days following injury. Although considerable evidence is accumulating that demonstrates clear and significant age-related differences in the immune and inflammatory response to traumatic injury, our current understanding of the mechanism(s) that underlie these changes is limited. Future studies that provide a mechanistic explanation for the unique immune and inflammatory response of older adults to traumatic injury are therefore essential if we are to determine whether manipulation of the immune system has potential as a future therapeutic strategy by which to improve the outcome of the geriatric trauma patient.

N-Formyl peptides drive mitochondrial damage associated molecular pattern induced neutrophil activation through ERK1/2 and P38 MAP kinase signalling pathways.

Traumatic injury results in a systemic inflammatory response syndrome (SIRS), a phenomenon characterised by the release of pro-inflammatory cytokines into the circulation and immune cell activation. Released from necrotic cells as a result of tissue damage, damage associated molecular patterns (DAMPs) are thought to initiate the SIRS response by activating circulating immune cells through surface expressed pathogen recognition receptors. Neutrophils, the most abundant leucocyte in human circulation, are heavily implicated in the initial immune response to traumatic injury and have been shown to elicit a robust functional response to DAMP stimulation. Here, we confirm that mitochondrial DAMPs (mtDAMPs) are potent activators of human neutrophils and show for the first time that signalling through the mitogen-activated-protein-kinases p38 and extracellular-signal-related-kinase 1/2 (ERK1/2) is essential for this response. At 40 and/or 100 µg/ml, mtDAMPs activated human neutrophils, indicated by a significant reduction in the surface expression of L-selectin, and triggered a number of functional responses from both resting and tumour necrosis factor-α primed neutrophils, which included reactive oxygen species (ROS) generation, degranulation, secretion of interleukin-8 and activation of p38 and ERK1/2 MAPKs. Pre-treatment of neutrophils with Cyclosporin H, a selective inhibitor of formyl peptide receptor-1 (FPR-1), significantly inhibited mtDAMP-induced L-selectin shedding as well as p38 and ERK1/2 activation, suggesting that N-formyl peptides are the main constituents driving mtDAMP-induced neutrophil activation. Indeed, no evidence of L-selectin shedding or p38 and ERK1/2 activation was observed in neutrophils challenged with mitochondrial DNA alone. Interestingly, pharmacological inhibition of p38 or ERK1/2 either alone or in combination significantly inhibited L-selectin shedding and IL-8 secretion by mtDAMP-challenged neutrophils, revealing for the first time that MAPK activation is required for mtDAMP-induced neutrophil activation and function. Our findings demonstrate that signalling through FPR-1 and activation of p38 and ERK1/2 MAPKs are key events in mtDAMP-induced neutrophil activation. Gaining an understanding of the signalling pathways involved in mtDAMP-induced neutrophil activation may assist in the development of future therapeutic strategies aimed at targeting the SIRS response to improve the outcome of the hospitalised trauma patient. Reducing the severity of the inflammatory response may realise substantial benefits for the severely injured trauma patient.

NK cell immunesenescence is increased by psychological but not physical stress in older adults associated with raised cortisol and reduced perforin expression.

NK cell cytotoxicity (NKCC) reduces with age and this has been associated previously with increased mortality. The immune response is also modulated by stress, and here, we assessed the effect of the physical stress of hip fracture and the psychological stress of depression on NKCC in an aged immune system. NKCC was assessed in 101 hip fracture patients (81 female) 6 weeks and 6 months after injury and in 50 healthy age-matched controls (28 female). Thirty-eight patients were depressed at 6 weeks post-injury, and NKCC was reduced in patients who developed depression compared with non-depressed hip fracture patients (p = 0.004) or controls (p < 0.02). NKCC remained lower in the depressed patients compared to those without depression 6 months post-fracture (p = 0.017). We found reduced expression of perforin in NK cells of depressed hip fracture patients compared with controls at 6 weeks (p = 0.001) post-fracture. Serum cortisol levels were also elevated in patients with depression compared to non-depressed patients at 6 weeks (p = 0.01) and 6 months (p = 0.05). NK cells treated with dexamethasone showed a concentration-dependent reduction in NKCC and perforin expression. We propose that depression is the major factor affecting NK cell immunity after hip fracture.

The impact of trauma on neutrophil function.

A well described consequence of traumatic injury is immune dysregulation, where an initial increase in immune activity is followed by a period of immune depression, the latter leaving hospitalised trauma patients at an increased risk of nosocomial infections. Here, we discuss the emerging role of the neutrophil, the most abundant leucocyte in human circulation and the first line of defence against microbial challenge, in the initiation and propagation of the inflammatory response to trauma. We review the findings of the most recent studies to have investigated the impact of trauma on neutrophil function and discuss how alterations in neutrophil biology are being investigated as potential biomarkers by which to predict the outcome of hospitalised trauma patients. Furthermore, with trauma-induced changes in neutrophil biology linked to the development of such post-traumatic complications as multiple organ failure and acute respiratory distress syndrome, we highlight an area of research within the field of trauma immunology that is gaining considerable interest: the manipulation of neutrophil function as a means by which to potentially improve patient outcome.

Depressive symptoms in hip fracture patients are associated with reduced monocyte superoxide production.

Ageing is accompanied by reduced functioning of the immune system, termed immunesenescence which is associated with increased risk of infection and mortality. However the immune system does not operate in isolation and can be modified by many environmental factors, including stress. In this study we determined whether physical stress (hip fracture) and psychological distress (depressive symptoms) had additive effects upon the aged immune system, specifically on monocyte numbers and function. We assessed immune function in 101 hip fracture patients (81 female) 6weeks and 6months after injury and 43 healthy age matched controls (28 females). Thirty-eight of the hip fracture group were found to be depressed at the 6week sampling. No differences in peripheral monocyte count, distribution of monocyte subsets or TNFα secretion were observed between hip fracture patients and healthy controls. However we observed significantly reduced superoxide production in response to Escherichia coli in the monocytes of hip fracture patients who developed depressive symptoms compared with non-depressed hip fracture patients (p=0.002) or healthy controls (p=0.008) 6weeks after the fracture which remained decreased 6months following injury. In previous studies we have shown an effect of depression on neutrophil superoxide generation in hip fracture patients, suggesting a particular susceptibility of this aspect of immune cell function to psychological stress.

Up-regulation of anti-apoptotic genes confers resistance to the novel anti-leukaemic compound PEP005 in primary AML cells.

We showed previously that PEP005 induced apoptosis in leukaemic cell lines and blasts from patients with acute myeloid leukaemia (AML). Here we assess the anti-leukeamic effects of PEP005 in vivo and determine the mechanism of resistance of PEP005 non-responsive cells. We used 2 human xenograft mouse models of AML to assess the anti-leukaemic effects of PEP005 in vivo. Expression microarray analysis of primary AML blasts following treatment with PEP005 was used to determine patterns of gene expression that conferred resistance. PEP005 significantly reduced tumour burden in two human leukaemia mouse xenograft models. We also assessed responsiveness of 33 AML samples to PEP005, with 78% of the samples entering apoptosis at 100nM. Resistance to PEP005 was not restricted to a particular AML subtype. Expression microarray analysis of resistant samples following treatment with PEP005 revealed a significant up regulation of the anti-apoptotic genes Bcl-2A1, Mcl-1, and PHLDA1 which was verified using RT-PCR. We conclude that PEP005 shows broad efficacy against AML subtypes and that up regulation of anti-apoptotic genes underlies resistance to this agent and could be used to screen for patients unlikely to benefit from a therapeutic regime involving PEP005.

Depressive symptoms post hip fracture in older adults are associated with phenotypic and functional alterations in T cells.

BACKGROUND: Ageing is accompanied by reduced immunity, termed immunesenescence. The immune system does not act in isolation and is sensitive to both psychological and physical stress. Hip fracture is a common physical stressor in older adults with a high incidence of new onset depression, which relates to poorer prognosis. We therefore set out to examine the possible synergistic effects of physical stress (hip fracture) and psychological stress (depressive symptoms) on the aged immune system. RESULTS: T cell phenotype and function was assessed in 101 hip fracture patients (81 female) 6 weeks after hip fracture and 43 healthy age-matched controls (26 female). 38 fracture patients had depressive symptoms at 6 weeks. T cell frequency (p = .01) and numbers (p = .003) were both lower in depressed hip fracture patients compared to healthy controls. The frequency of senescent CD28(-ve) (p = .001), CD57(+ve) (p = .001), KLRG1(+ve) (p = .03) CD8 T cells, as well as senescent CD28(-ve) CD4(+ve) (p = .01) and CD57(+ve) CD4(+ve) (p = .003) T cells were higher in depressed hip fracture patients compared with healthy controls and the frequency of CD28(-ve) CD8 T cells was also higher when compared to patients with hip fracture alone (p = .01). Additionally, activated CD69(+ve) (p = .005) and HLADR(+ve) (p < .001) CD8 T cells, were also higher in depressed hip fracture patients compared to healthy controls. On examining cytokine production by activated T cells, a significant increase in TNFα (p = .03) and IL6 (p = .04) production was observed in CD4 T cells from hip fracture patients with depressive symptoms compared to healthy controls. CONCLUSIONS: As none of the patients in the study had a prior history of depression, our data suggest that the development of depressive symptoms in hip fracture patients is associated with altered T cell phenotype and increased pro-inflammatory function which is not seen in patients who do not develop depression after hip fracture. Treating depressive symptoms promptly in hip fracture patients may therefore improve immunity and outcomes in these patients.

Depressive symptoms are associated with reduced neutrophil function in hip fracture patients.

Hip fracture is a common trauma in older adults with a high incidence of depression, which relates to poorer prognosis including increased risk of infection. Ageing is accompanied by reduced immunity, termed immunesenescence, resulting in increased susceptibility to infection. We examined whether physical trauma (hip fracture) and psychological distress (depressive symptoms) had additive effects upon the aged immune system that might contribute to poor outcomes after injury. Neutrophil function was assessed in 101 hip fracture patients (81 female) 6 weeks and 6 months after injury and 43 healthy age-matched controls (28 female). Thirty eight fracture patients had depressive symptoms at 6 weeks. No difference in neutrophil phagocytosis of Escherichia coli was observed between controls and hip fracture patients, but superoxide production was significantly reduced in hip fracture patients with depressive symptoms compared with patients without symptoms (p=.001) or controls (p=.004) at 6 weeks. Superoxide production improved 6 months following fracture to the level seen in controls. We detected elevated serum cortisol, reduced dehydroepiandrosterone sulphate (DHEAS) and an increased cortisol:DHEAS ratio in fracture patients with depressive symptoms compared with patients without depressive symptoms or controls at 6 weeks and 6 months after injury. Serum IL6, TNFα and IL10 were higher among patients with depressive symptoms at 6 weeks. The cortisol:DHEAS ratio and IL6 levels related to depressive symptom scores but not to neutrophil function. In conclusion, depressive symptoms related to poorer neutrophil function after hip fracture, but this was not driven by changes in stress hormone or cytokine levels.

Neutrophil apoptosis and its induction as a potential treatment for chronic inflammatory disease.

PURPOSE OF REVIEW: Neutrophils are a key component of innate immunity. At sites of infection, they unleash cytotoxic molecules allowing them to kill invading pathogens. However, these molecules can also be deleterious to the host tissue. Therefore, neutrophils undergo apoptosis and are removed from the site of infection following elimination of the pathogen. When neutrophil apoptosis is significantly delayed, this can result in chronic inflammation. This review highlights recent advances in our understanding of neutrophil apoptosis, and discusses literature surrounding the induction of neutrophil apoptosis as a therapeutic strategy for the treatment of chronic inflammatory disease. RECENT FINDINGS: Recent studies have highlighted the role of cyclin-dependent kinases (CDKs) in the regulation of neutrophil lifespan. CDK activity appears to be involved in the maintenance of levels of Mcl-1, an antiapoptotic Bcl-2 family member. In addition, recent findings have demonstrated the induction of neutrophil apoptosis at sites of infection by bystander immune cells including T-regulatory cells (Tregs) and natural killer (NK) cells. SUMMARY: Recent advances in our understanding of the processes involved in neutrophil apoptosis, coupled with new insights into the mechanisms by which bystander immune cells induce neutrophil death, may provide novel and exciting possibilities with regards to the treatment of chronic inflammatory diseases.

Reduced release and binding of perforin at the immunological synapse underlies the age-related decline in natural killer cell cytotoxicity.

Physiological aging is accompanied by a marked reduction in natural killer (NK) cell cytotoxicity (NKCC) at the single cell level, but the underlying mechanisms are unknown. To address this issue, we isolated NK cells from healthy young (≤ 35 years) and old (≤ 60 years) subjects and examined the effect of age on events fundamental to the process of NKCC. Simultaneous assessment of NKCC and NK cell-target cell conjugate formation revealed a marked age-associated decline in NK cell killing but comparable conjugate formation, indicating a post-target cell binding defect was responsible for impaired NKCC. Despite a reduction in the proportion of NK cells expressing the activatory receptor NKp46, NK cells from old donors were not hyporesponsive to stimulation, as no age-associated difference was observed in the expression of the early activation marker CD69 following target cell coculture. Furthermore, intracellular levels of the key cytotoxic effector molecules perforin and granzyme B, and the fusion of secretory lysosomes with the NK cell membrane were also similar between the two groups. However, when we examined the binding of the pore-forming protein perforin to the surface of its target cell, an event that correlated strongly with target cell lysis, we found the percentage of perforin positive target cells was lower following coculture with NK cells from old subjects. Underlying this reduction in binding was an age-associated impairment in perforin secretion, which was associated with defective polarization of lytic granules towards the immunological synapse. We propose that reduced perforin secretion underlies the reduction in NKCC that accompanies physiological aging.

Total synthesis of bistramide†A and its 36(Z)†isomers: differential effect on cell division, differentiation, and apoptosis.

The total synthesis of bistramide A and its 36(Z),39(S) and 36(Z),39(R) isomers shows that these compounds have different effects on cell division and apoptosis. The synthesis relies on a novel enol ether-forming reaction for the spiroketal fragment, a kinetic oxa-Michael cyclization reaction for the tetrahydropyran fragment, and an asymmetric crotonylation reaction for the amino acid fragment. Preliminary biological studies show a distinct pattern of influence of each of the three compounds on cell division, differentiation, and apoptosis in HL-60 cells, thus suggesting that these effects are independent activities of the natural product.

Cyclin-dependent kinase 9 activity regulates neutrophil spontaneous apoptosis.

Neutrophils are the most abundant leukocyte and play a central role in the immune defense against rapidly dividing bacteria. However, they are also the shortest lived cell in the blood with a lifespan in the circulation of 5.4 days. The mechanisms underlying their short lifespan and spontaneous entry into apoptosis are poorly understood. Recently, the broad range cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to increase neutrophil apoptosis, implicating CDKs in the regulation of neutrophil lifespan. To determine which CDKs were involved in regulating neutrophil lifespan we first examined CDK expression in human neutrophils and found that only three CDKs: CDK5, CDK7 and CDK9 were expressed in these cells. The use of CDK inhibitors with differing selectivity towards the various CDKs suggested that CDK9 activity regulates neutrophil lifespan. Furthermore CDK9 activity and the expression of its activating partner cyclin T1 both declined as neutrophils aged and entered apoptosis spontaneously. CDK9 is a component of the P-TEFb complex involved in transcriptional regulation and its inhibition will preferentially affect proteins with short half-lives. Treatment of neutrophils with flavopiridol, a potent CDK9 inhibitor, increased apoptosis and caused a rapid decline in the level of the anti-apoptotic protein Mcl-1, whilst Bcl2A was unaffected. We propose that CDK9 activity is a key regulator of neutrophil lifespan, preventing apoptosis by maintaining levels of short lived anti-apoptotic proteins such as Mcl-1. Furthermore, as inappropriate inhibition of neutrophil apoptosis contributes to chronic inflammatory diseases such as Rheumatoid Arthritis, CDK9 represents a novel therapeutic target in such diseases.

Lactoferrin inhibits neutrophil apoptosis via blockade of proximal apoptotic signaling events.

Neutrophils are the most abundant leukocyte and have a short lifespan, dying by apoptosis approximately five days after leaving the bone marrow. Their apoptosis can be delayed at sites of inflammation to extend their functional lifespan, but inappropriate inhibition of apoptosis contributes to chronic inflammatory disease. Levels of the physiological iron chelator lactoferrin are raised at sites of inflammation and we have shown previously that iron-unsaturated lactoferrin inhibited human neutrophil apoptosis, but the mechanisms involved were not determined. Here we report that the anti-apoptotic effect of lactoferrin is dependent upon its iron saturation status as iron-saturated lactoferrin did not affect neutrophil apoptosis. We also show that the effect of lactoferrin is mediated at an early stage in apoptosis as it inhibited activation of sphingomyelinase, generation of ceramide, activation of caspase 8 and Bax and cleavage of Bid. Lactoferrin did not inhibit apoptosis induced by exogenous ceramide, supporting the proposal that it acts upstream of ceramide generation. We therefore conclude that raised lactoferrin levels are likely to contribute to chronic inflammation by delaying neutrophil apoptosis and that this is achieved by inhibiting proximal apoptotic signaling events.