PMN Apoptosis Research Articles

Aspirin and Salicylate Suppress Polymorphonuclear Apoptosis Delay Mediated by Proinflammatory Stimuli

During inflammation, polymorphonuclear leukocyte (PMN) apoptosis can be delayed by different proinflammatory mediators. Classically, it has been accepted that the widely used anti-inflammatory drug acetyl salicylic acid (ASA) exerts its action through inhibition of cyclooxygenases and subsequent prostaglandin synthesis. We hypothesized that another anti-inflammatory action of ASA could be the shortening of PMN survival. We found that at therapeutic concentrations (1-3 mM), ASA and its metabolite salicylate (NaSal), but not indomethacin or ibuprofen, counteracted the prolonged PMN survival mediated by lipopolysaccharide (LPS) through inhibition of nuclear factor-kappaB (NF-kappaB) activation. Both salicylates also inhibited interleukin (IL)-1alpha or acidic conditions antiapoptotic activity. Higher concentrations of both drugs had a direct apoptotic effect. Salicylates were not effective when PMN apoptosis delay was induced by granulocyte macrophage-colony-stimulating factor (GM-CSF), a NF-kappaB-independent cytokine. Promotion of PMN survival by the combination of IL-1alpha and LPS was also reversed by salicylates, but higher concentrations were required. ASA concentrations that did not trigger PMN death increase the zymosan- or tumor necrosis factor-alpha-mediated proapoptotic effect. The LPS- and IL-1alpha- but not GM-CSF-mediated antiapoptotic effect was markedly reduced in PMNs from donors who had ingested ASA. Using a thioglycolate-induced peritonitis model, we showed that in ASA- or NaSal-treated mice there was not only a decrease in the number of cells recruited but also an increase in the percentage of apoptotic PMNs as well as an enhancement of phagocytosis compared with controls. Our findings demonstrate that acceleration of PMN apoptosis by turning off the NF-kappaB-mediated survival signals elicited by proinflammatory stimuli is another anti-inflammatory action of ASA and NaSal.

INTERFERON-?? INHIBITS CASPASE-3 ACTIVITY IN POST-INJURY NEUTROPHILS

Interferon-γ (IFN) is known to enhance several neutrophil (PMN) functions as well as exert an antiapoptotic effect through the inhibition of specific caspases. Trauma has been reported to alter PMN cytokine responses and apoptosis. We hypothesize that trauma alters IFN inhibited PMN apoptosis and Caspase-3 activity. To test this PMN's from control or critically injured subjects after traumatic shock were incubated in medium alone or medium with IFN (100 units/ml). PMN apoptosis was assayed with Annexin V staining and Caspase-3 activity was detected by flow cytometry using a FITC-labeled antibody. Data are presented as net mean flouroescent intensity.TableIFN inhibited PMN apoptosis in healthy controls. (LPS was used a positive control) IFN further inhibited PMN apoptosis in trauma patients. Caspase 3 activity was significantly inhibited in IFN stimulated normal subject PMN. In addition, caspase 3 activity was inhibited in trauma patient PMN and further inhibited in IFN treated trauma patient PMN. These data demonstrate that the apoptosis of circulating PMN following major injury is responsive to IFN. Furthermore, IFN specifically inhibits caspase-3 in human PMN. The inhibited activity of caspase-3 may contribute to IFN mediated antiapoptotic effects in both control and post-injury PMN. IFN may induce the production of intracellular proteins which up or down regulate PMN apoptosis such as Bcl-XL, inhibitor of apoptosis family members, or survivin. The results suggest that IFN-γ may contribute not only to augmented host defense against microorganisms but also increased tissue damage at inflammatory sites after shock by delayed PMN apoptosis.

THE SIMILARITY AND DIFFERENCE OF THE ROLE(S) IN HUMAN NEUTROPHIL APOPTOSIS BETWEEN IL-18 AND LIPOPOLYSACCHARIDE (LPS)

Background: Decreased neutrophil (PMN) apoptosis is implicated in persistent inflammation. The release of IL-18 into sera during systemic inflammation corresponds to disease severity. IL-18 receptor and toll-like receptors (TLRs) including TLR4, which is the receptor for apoptosis-inhibiting agent, LPS, possess a highly conserved intracellular domain called "Toll-IL- 1R domain" and activate overlapping signaling pathways. Purpose: To assess whether IL-18 can modulate PMN apoptosis and elucidate the similarity and difference in the role(s) of IL-18 and LPS in PMN apoptosis. Method: Blood was obtained from healthy volunteers. Purified PMNs were pre-treated for 0.5 hours with either PI3-kinase inhibitor (LY2940, 20 μM), ERK inhibitor (PD98059,50μM), p38 MAPK inhibitor (SB203580,25 μM),or vehicle and then incubated for 5.5 hours with or without recombinant human (rh) IL-18 (100 μg/ml) or LPS (1 ng/ml). Apoptosis was determined as the percent of PMN expressing hypodiploid DNA by propidium iodide staining and one-color flow cytometry. Results: Both IL-18 and LPS decreased PMN apoptosis. Pretreatment with LY294 significantly increased apoptosis in all the culture groups, but apoptosis rate was significantly lower in the group cultured with LPS than other two culture groups. Pretreatment with PD98059 also increased apoptosis in all the culture groups and there was no difference in apoptosis rate among the groups. SB203580 had no influence on apoptosis in all the culture groups. Conclusion: Both IL-18 and LPS similarly inhibit PMN apoptosis by activating PI3-kinase and ERK pathways. However, PI3-kinase plays more important role(s) in IL-18- induced inhibition of apoptosis than in LPSinduced apoptosis inhibition.Table: The effects of various kinase inhibitors on PMN apoptosis in the presence of rhlL-18 or LPS

NEUTRALIZATION OF KC AND MIP-2; DIVERGENT EFFECTS ON ACTIVATION PATHWAYS IN MOUSE PMN CELL LINE -CONSISTENT WITH MOUSE BLOOD PMN

Acute lung injury (ALI) is associated with the targeting and sequestration of PMN in the lung. Chemokines, produced by resident and non-resident pulmonary cells in response to inflammatory stimuli, activate pathways that regulate PMN chemotaxis, apoptosis and cytolytic activity. We have shown that systemic antibody blockade of murine PMN chemokines keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2) and local pulmonary silencing with KC or MIP-2 siRNA differentially effect lung tissue injury and inflammation in a mouse model of hemorrhage (Hem) induced PMN priming for ALI following septic challenge. To further investigate the divergent roles KC and MIP-2 play while sharing affinity for a common CXCR2-like receptor, we examined phosphorrylation patterns for proteins associated with PMN activation. Previously we reported differences in the %-phospho-AKT, ERK1/2 and p38, 12-14H-post systemic antibody neutralization of KC or MIP-2 following Hem. This data suggested that KC and MIP-2 function through different downstream signaling pathways. In order to further test our hypothesis of divergent activation pathways, an inducible, terminally differentiated PMN cell line (MPRO) was used. MPRO cells, 3 × 104 cells/well in 96 well plates, were stimulated with 50 nM platelet activating factor and treated with either 100 ng/ml anti-KC or 25 ng/ml anti-MIP-2 or IgG. Phospho- vs. total protein was assessed for pathway activation proteins AKT, ERK1/2 and p38 by ELISA method (SuperArray CASE Kit). Phosphorylation profiles for antibody vs. IgG treated MPRO cells for AKT, ERK1/2 and p38 and were consistent with those obtained from mouse blood PMN. The %-phospho-p38 and AKT decreased below IgG levels in the anti-MIP-2 treated cells. While %-phospho-ERK1/2 was decreased below IgG, there was no difference between antibody treatments. These findings support our hypothesis that murine PMN chemokines, KC and MIP-2, differentially stimulate activation pathways that regulate PMN activity. (NIH HL73525)

Hydrogen sulfide prevents apoptosis of human PMN via inhibition of p38 and caspase 3

Hydrogen sulfide, together with carbon monoxide and nitric oxide, is now considered a gasotransmitter able to induce specific cellular responses. As hydrogen sulfide is a component of several natural compounds known to be effective in many inflammatory pathologies, particularly of the respiratory tract, we studied its effects in vitro on the survival and bactericidal activity of purified human neutrophils. We found that (1) HS− ions promote the survival of granulocytes, but not that of lymphocytes or eosinophils, cultured in serum-free medium; (2) the pro-survival effect of HS− is due to inhibition of caspase-3 cleavage and p38 MAP kinase phosphorylation; (3) the bactericidal activity of neutrophils is not impaired by hydrogen sulfide. We conclude that HS− promotes the short-term survival of neutrophils potentially accelerating the resolution of inflammatory processes and preventing the occurrence of new ones.

Role of TNF-alpha and GM-CSF on apoptosis and adhesion in PMN of patients with Crohn's disease

Role of TNF-alpha and GM-CSF on apoptosis and adhesion in PMN of patients with Crohn's disease

Toll-like receptors types 2 and 6 and the apoptotic process in human neutrophils

Neutrophils (PMN) apoptosis plays an important role in limiting the last phase of inflammatory processes. It is unknown whether Toll-like receptor (TLR)2 acts independently or together with TLR6 in this process. The aim of this study was to estimate the relationship between the expressions of TLR2 and TLR6 and the apoptosis of human neutrophils in physiological conditions. We investigated the influence of recombinant human interleukin (IL)-18 and N-formyl-metionyl-leucyl-phenylalanine (fMLP) on the relationships between these receptors and neutrophil apoptosis. Our results showed that after 4-h incubation, the percentage of apoptotic PMNs significantly increased compared with PMN counts before incubation. The stronger expression of TLR2 on the neutrophils suggests that this receptor contributes more significantly to the induction of PMN apoptosis than does TLR6. We also demonstrated an influence of recombinant human IL-18 (rhIL-18) on the expression of TLR6, whereas this effect was not observed in the expression of TLR2. We observed that both rhIL-18 and fMLP inhibited the apoptosis of PMNs and that rhIL-18 had a stronger effect than fMLP. The obtained results suggest that not only TLR2, but also TLR6 plays an important role in the regulation of the apoptosis of PMNs. Changes in the expression of TLR6 and inhibition of apoptosis of PMNs by rhIL-18 seem to confirm the vital role this receptor and of rhIL-18 in regulating the survival of these cells. These data can be useful in developing methods to regulate PMN apoptosis in conditions associated with their excessive and unfavorable activation.

In Vitro Effects of Nonesterified Fatty Acids on Bovine Neutrophils Oxidative Burst and Viability

An in vitro study was conducted to examine the influence of nonesterified fatty acids (NEFA) on bovine polymorphonuclear leukocytes (PMN). Eight healthy, midlactating Holstein cows were used as blood donors. Blood PMN were isolated and incubated with a mixture of NEFA, reflecting composition of bovine plasma NEFA at concentrations that were intended to mimic those found in blood of cows undergoing high, moderate, or low lipomobilization intensity (2, 1, 0.5, 0.25, 0.125, and 0.0625mM). Control samples were incubated in absence of NEFA. Phagocytosis and oxidative burst activities were assessed by a 2-color flow cytometric method, which was based on oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123. Oxidative burst products were generated by incubating PMN with Staphylococcus aureus labeled with propidium iodide. A flow cytometric technique was used to detect PMN viability, necrosis, and apoptosis using fluorescein isothiocyanate-labeled annexin-V and propidium iodide. Phagocytic activity was not affected by NEFA. The highest concentration of NEFA (2mM) was associated with a dramatic increase of phagocytosis-associated oxidative burst activities with a reduction in cell viability (48.0 vs. 97.5% in control samples) and with a marked increase of necrosis (49.4 vs. 0.5% in control samples). Conversely, the mixture of NEFA did not affect the occurrence of apoptosis. Enhancement of the oxidative burst associated with the highest concentration of NEFA might explain the reduced viability and higher percentage of necrosis observed under the same conditions. This study demonstrated a substantial resistance of bovine PMN to an overload of fatty acids. However, observation that the highest concentration of NEFA regulated some PMN functions encourages the possibility of in vivo studies to assess the relationships between intensity of lipomobilization, plasma NEFA, and bovine PMN functions.

Neutrophil apoptosis: a marker of disease severity in sepsis and sepsis-induced acute respiratory distress syndrome

IntroductionApoptosis of neutrophils (polymorphonuclear neutrophils [PMNs]) may limit inflammatory injury in sepsis and acute respiratory distress syndrome (ARDS), but the relationship between the severity of sepsis and extent of PMN apoptosis and the effect of superimposed ARDS is unknown. The objective of this study was to correlate neutrophil apoptosis with the severity of sepsis and sepsis-induced ARDS.MethodsA prospective cohort study was conducted in intensive care units of three tertiary hospitals in Porto Alegre, southern Brazil. Fifty-seven patients with sepsis (uncomplicated sepsis, septic shock, and sepsis-induced ARDS) and 64 controls were enrolled. Venous peripheral blood was collected from patients with sepsis within 24 hours of diagnosis. All surgical groups, including controls, had their blood drawn 24 hours after surgery. Control patients on mechanical ventilation had blood collected within 24 hours of initiation of mechanical ventilation. Healthy controls were blood donors. Neutrophils were isolated, and incubated ex vivo, and apoptosis was determined by light microscopy on cytospun preparations. The differences among groups were assessed by analysis of variance with Tukeys.ResultsIn medical patients, the mean percentage of neutrophil apoptosis (± standard error of the mean [SEM]) was lower in sepsis-induced ARDS (28% ± 3.3%; n = 9) when compared with uncomplicated sepsis (57% ± 3.2%; n = 8; p < 0.001), mechanical ventilation without infection, sepsis, or ARDS (53% ± 3.0%; n = 11; p < 0.001) and healthy controls (69% ± 1.1%; n = 33; p < 0.001) but did not differ from septic shock (38% ± 3.7%; n = 12; p = 0.13). In surgical patients with sepsis, the percentage of neutrophil apoptosis was lower for all groups when compared with surgical controls (52% ± 3.6%; n = 11; p < 0.001).ConclusionIn medical patients with sepsis, neutrophil apoptosis is inversely proportional to the severity of sepsis and thus may be a marker of the severity of sepsis in this population.

Spontaneous neutrophil apoptosis and regulation of cell survival by granulocyte macrophage-colony stimulating factor

Polymorphonuclear leukocytes (PMNs or neutrophils) are the most prominent cellular component of the innate immune system in humans and produce an array of potent cytotoxic molecules. It is important that neutrophils undergo constitutive (spontaneous) apoptosis as a mechanism to facilitate normal cell turnover and immune system homeostasis. Conversely, several proinflammatory cytokines, including granulocyte macrophage-colony stimulating factor (GM-CSF), prolong neutrophil survival. The molecular mechanisms that regulate PMN apoptosis or survival remain incompletely defined. To that end, we compared global gene expression in human neutrophils during spontaneous apoptosis with that in cells cultured with human GM-CSF. Genes encoding proteins that inhibit apoptosis, such as myeloid cell leukemia sequence 1, caspase 8 and Fas-associated via death domain-like apoptosis regulator (CFLAR), B cell chronic lymphocytic leukemia/lymphoma 2 (BCL2)/adenovirus E1B 19 kDa-interacting protein 2 (BNIP2), and serum/glucocorticoid-regulated kinase (SGK), were down-regulated coincident with neutrophil apoptosis. In contrast, those encoding apoptosis inhibitor 5, BCL2-like 1, BNIP2, CFLAR, SGK, and tumor necrosis factor alpha-induced protein 8 were up-regulated in PMNs cultured with GM-CSF. Correspondingly, GM-CSF delayed PMN apoptosis (P<0.03), increased cell viability (P<0.03), and prolonged neutrophil phagocytic capacity (P<0.05). Prolonged functional capacity was paralleled by striking up-regulation of proinflammatory genes and proteins, including CD14, CD24, CD66, and human leukocyte antigen-DR. In addition, expression of SGK protein diminished during PMN apoptosis but was restored by culture with GM-CSF, suggesting SGK is involved in leukocyte survival. These studies provide a global view of the molecular events that regulate neutrophil survival and apoptosis.

In VitroEffect of Nitrogen and He-Ne Laser on the Apoptosis of Human Polymorphonuclear Cells from Burn Cases and Healthy Volunteers

The aim of this study was to examine the effect of He-Ne and nitrogen lasers on the apoptosis of PMN in normal versus burn patients. Nitrogen and He-Ne laser exposure increases the apoptotic death rate for human macrophages. Inflammation is a major consequence of thermal injury, and polymorphonuclear cell (PMN) infiltration exacerbates inflammatory process through the release of proinflammatory cytokines. The apoptotic death instead of necrotic death of PMN under the situation may help to resolve inflammation. Ten healthy volunteers and 10 burn cases (30-50% burn surface) were included in the study. The PMN was separated by dextran sedimentation and density gradient centrifugation before suspending in RPMI-1640 medium supplemented with autologus serum. The cell suspension aliquoted in microwells was exposed to nitrogen (wavelength of 337 nm with power output of 3 mW) and He-Ne (LGN model no. 111, Russia, wavelength of 632.8 nm with power output of 3 mW) lasers for 10 and 5 min. The wells not exposed to laser were used as controls. After 24-36 h of incubation, the apoptotic rates were measured as percentage by morphological studies on acridine orange-ethidium bromide stained preparation using fluorescent microscope. Percentage of apoptotic death increases from 32.9% (SD +/- 4.14) in control PMN to 41.97% (SD +/- 14) in PMN exposed to nitrogen laser for 5 min and further increased to 62.7% (SD +/- 15.11) with nitrogen laser exposure for 10 min. He-Ne laser exposure for 10 min increased apoptotic cell percentage to 41.9%. Increased apoptosis in PMN exposed to nitrogen laser was statistically significant (p < 0.03) both for PMN from healthy subjects and burn cases. It was significantly elevated (p = 0.005) only for PMN from healthy volunteers exposed to He-Ne laser for 10 min but not among He-Ne exposed PMN from burn cases. These observations support the therapeutic application of nitrogen laser to reduce inflammation and improve wound healing for burn cases.

Effects of uraemia and dialysis modality on polymorphonuclear cell apoptosis and function

Previous studies have reported that incubation of polymorphonuclear cells (PMN) in uraemic plasma or with different haemodialysis membranes and peritoneal dialysis solutions increases apoptosis in this cell type. In addition, PMN harvested from uraemic patients show a reduced ability to generate superoxide in response to stimuli as well as impaired phagocytosis, chemotaxis and degranulation. The aim of the current study was to investigate the effect of uraemia and dialysis modality on apoptosis and function in freshly harvested non-incubated PMN. Polymorphonuclear cells were harvested from 14 chronic haemodialysis (HD) patients, from 14 continuous peritoneal dialysis patients (CAPD), 28 chronic kidney disease (CKD), pre-dialysis patients and from 14 healthy subjects (Controls). In these in vivo experiments, PMN apoptosis was studied by means of flow cytometric analysis of annexin V binding to freshly isolated cells. Polymorphonuclear cell phagocytosis and production of reactive oxygen species by unstimulated or stimulated (S.aureus, fMLP, PMA) cells were also studied by flow cytometry using whole blood. We observed increased PMN apoptosis in CKD patients. CAPD and HD patients displayed PMN apoptosis rates similar to controls. In the HD group, PMN exhibited decreased phagocytosis rates. In contrast, phagocytosis rates in PMN from CAPD were not significantly different from controls. In the CKD and HD groups, apoptosis was inversely correlated with respiratory burst activity and phagocytosis. Our results suggest that both uraemia and treatment modality may interfere with PMN apoptosis and function. Dialysis appears to normalize the increased PMN apoptosis rates observed in CKD patients.

In vitro effect of recombinant human tumor necrosis factor-α on canine neutrophil apoptosis

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-α (TNF-α) is suggested to play an important role. Since an effect of TNF-α on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-α in the present study to investigate the effect of TNF-α on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-α at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-α combined with cycloheximide. Therefore, it is indicated that TNF-α is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-α requires protein synthesis in the PMNs.

In vitro effect of recombinant human granulocyte colony-stimulating factor on canine neutrophil apoptosis

Apoptosis is essential in eliminating neutrophils (polymorphonuclear leukocytes: PMNs) in animals. The suppression of PMN apoptosis is believed to be beneficial in eradicating pathogens and is implicated in the pathogenesis of human inflammatory diseases. In the present study, canine PMNs were stimulated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to investigate the in vitro effect on the apoptosis of canine PMNs. Apoptotic cell rates were assessed by flow cytometry in relation to the ability of PMNs to produce reactive oxygen species (ROS). Canine PMN apoptosis was markedly suppressed by rhG-CSF treatment, in association with the retention of the PMN ability to produce ROS. The addition of cycloheximide abolished this suppression by rhG-CSF. Moreover, canine PMNs, which were stimulated by rhG-CSF, expressed high levels of anti-apoptotic mcl-1 gene mRNA, as quantified by real-time polymerase chain reaction method. The results suggest that PMNs, stimulated by G-CSF, could work effectively over a longer period to eliminate pathogens, and that the prolongation of the PMN life-span might occasionally aggravate tissue injuries in dogs. In addition, the suppression of PMN apoptosis seems to be mediated by the induction of anti-apoptotic mcl-1 gene expression.

Increased chemoattractant induced neutrophil oxidative burst, accelerated apoptosis, and dysregulated tyrosine phosphorylation associated with lifelong bacterial infections

A boy with lifelong recurrent bacterial infection at cutaneous and mucosal sites was investigated. PMN oxidative burst to phorbol myristate acetate (PMA) and zymosan was normal but was increased 20- to 50-fold upon C5a or formyl-met-leu-phe (fMLP) chemoattractant stimulation, accompanied by accelerated PMN apoptosis. His PMNs showed increased constitutive tyrosine phosphorylation of 21-, 25-, and 44-kDa proteins, and of src-family kinases (p59 hck, p58 fgr, and p53/56 lyn). Phosphorylation was abnormally enhanced following fMLP stimulation. Expression and activity of the major PMN tyrosine phosphatases, i.e., CD45, CD148, and SHP-1 and -2, was normal. However, dephosphorylation of phospho-p58 fgr and phospho-p53/56 lyn by lysates of patient's PMNs was enhanced. Thus, another phosphatase may be overactive, perhaps dephosphorylating a regulatory (inhibitory) site on a protein tyrosine kinase, accounting for the abnormal PMN tyrosine phosphorylation and function. With age (now 13 years), T-cell lymphopenia and loss of T-cell responses developed. This appears to be a unique primary immunodeficiency with abnormal PMN oxidative and apoptotic responses to chemoattractants, dysregulated protein tyrosine phosphorylation, serious bacterial infection, and T-lymphocyte attrition.

Intestinal Epithelial Cells Modulate PMN Activation and Apoptosis Following Bacterial and Hypoxic Challenges

The post-ischemic gut may serve to prime and activate neutrophils which may lead to the subsequent development of the systemic inflammatory response syndrome (SIRS) and multiple organ failure. However, the initiating event which may trigger this immunoinflammatory cascade from the gut is unknown. Recent studies have indicated that intestinal epithelial cells (IEC) play an integral role in generating and transmitting signals between luminal bacteria and the host cells in the underlying gut tissues. The purpose of this study was to investigate the ability of IEC to modulate PMN responses to bacteria and/or hypoxia/reoxygenation (H/R) challenges in vitro. Caco2 cell monolayers were established in a two-chamber cell culture system. Neutrophils from normal human volunteers were placed in the basal chamber and the cell co-culture exposed to either apical bacteria (E. coli) and/or H/R challenge. PMN apoptosis, and percentage of CD11b expression, superoxide anion production, and elastase release were subsequently quantitated. Coculture of PMNs with Caco2 cells led to a significant reduction in neutrophil apoptosis in both normoxic and H/R conditions. CD11b expression was increased in PMNs exposed to bacteria but the greatest expression was noted with PMN cocultured with Caco2 cells and H/R. Superoxide anion production was increased in all groups following either H/R or bacterial challenge and H/R. Elastase release was highest in neutrophils following H/R and exposure to E. coli. IEC modulate PMN response to bacteria and H/R insults. This results in the production of activated neutrophils with an exaggerated lifespan which may promote remote organ failure. Attempts to modulate this response may be useful in preventing multiple organ failure following severe traumatic shock.

Fracture Hematoma Is a Potent Proinflammatory Mediator of Neutrophil Function

Patients with multiple skeletal injuries are susceptible to acute respiratory distress syndrome and multiple organ failure, which result from hyperactivation of the immune system. This study was designed to evaluate in vitro the proinflammatory properties of fracture hematoma (FH). FH was isolated from patients undergoing emergent open reduction and internal fixation for isolated closed fractures. Neutrophils (PMNs), isolated from healthy volunteers, were exposed to the FH supernatant and activation was examined (CD11b and CD18 adhesion receptor expression and respiratory burst). PMN phagocytosis, apoptosis, and transmigration across an endothelial barrier were also assessed. FH increased PMN respiratory burst (control, 100; FH-treated, 186) and phagocytosis (control, 100; FH-treated, 172) but had no effect on adhesion receptor expression. Transendothelial migration of PMNs was unaffected, although FH was toxic to endothelial cells. In contrast, apoptosis of FH-treated PMNs was delayed (control, 46; FH-treated, 8). These effects, although beneficial at the site of injury in the context of antibactericidal function, may cause PMN-mediated tissue injury systemically.

Insights into Pathogen Immune Evasion Mechanisms: Anaplasma phagocytophilum Fails to Induce an Apoptosis Differentiation Program in Human Neutrophils

Polymorphonuclear leukocytes (PMNs or neutrophils) are essential to human innate host defense. However, some bacterial pathogens circumvent destruction by PMNs and thereby cause disease. Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, survives within PMNs in part by altering normal host cell processes, such as production of reactive oxygen species (ROS) and apoptosis. To investigate the molecular basis of A. phagocytophilum survival within neutrophils, we used Affymetrix microarrays to measure global changes in human PMN gene expression following infection with A. phagocytophilum. Notably, A. phagocytophilum uptake induced fewer perturbations in host cell gene regulation compared with phagocytosis of Staphylococcus aureus. Although ingestion of A. phagocytophilum did not elicit significant PMN ROS, proinflammatory genes were gradually up-regulated, indicating delayed PMN activation rather than loss of proinflammatory capacity normally observed during phagocytosis-induced apoptosis. Importantly, ingestion of A. phagocytophilum failed to trigger the neutrophil apoptosis differentiation program that typically follows phagocytosis and ROS production. Heat-killed A. phagocytophilum caused some similar initial alterations in neutrophil gene expression and function, which included delaying normal PMN apoptosis and blocking Fas-induced programmed cell death. However, at 24 h, down-regulation of PMN gene transcription may be more reliant on active infection. Taken together, these findings suggest two separate antiapoptotic processes may work concomitantly to promote bacterial survival: 1) uptake of A. phagocytophilum fails to trigger the apoptosis differentiation program usually induced by bacteria, and 2) a protein or molecule on the pathogen surface can mediate an early delay in spontaneous neutrophil apoptosis.

Physiology of the periparturient period and its relation to severity of clinical mastitis

Incidence of clinical mastitis is highest at drying off and during the periparturient period. Intramammary Escherichia coli infection in high-yielding cows can show a severe clinical response during the early post-partum period. Severe clinical mastitis is mainly determined by cow factors, in particular the functionality of the circulating polymorphonuclear leukocytes (PMN) which are recruited to the mammary gland during the inflammatory reaction. There is a co-incidence between the periods of highest incidence of clinical mastitis and specific structural changes in the mammary gland. During the periparturient period, marked changes in various systemic and local hormones are related to the secretory state of the mammary gland epithelium (lactogenesis). Estrogen and progesterone induce proliferation of the mammary epithelium throughout gestation and act as survival factors in different tissues, although conflicting data have been reported on their effect on PMN oxidative burst. Somatotropin (STH), responsible for maintenance of lactation in ruminants, has been shown to positively influence innate immunity and a more rapid recovery in milk production of severely affected animals. The concentration of STH, and as a result also IGF-I levels is, however, quite low during early lactation. IGF-I and its regulating binding proteins are associated with cell survival, modulation of apoptosis and functionality of PMN in humans. During early lactation, bio-availability of IGF-I is decreased, which might reduce its stimulating effects on PMN quality and functionality. PRL, concomitantly known as a lactogenic hormone and an immunoregulatory cytokine, has also been associated with modulation of the immune system. It is expected that in periparturient animals, hormone changes could interfere with the immune response and the clinical response of mastitis.

The role of the MAPK pathway alterations in GM-CSF modulated human neutrophil apoptosis with aging

BackgroundNeutrophils represent the first line of defence against aggressions. The programmed death of neutrophils is delayed by pro-inflammatory stimuli to ensure a proper resolution of the inflammation in time and place. The pro-inflammatory stimuli include granulocyte-macrophage colony-stimulating factor (GM-CSF). Recently, we have demonstrated that although neutrophils have an identical spontaneous apoptosis in elderly subjects compared to that in young subjects, the GM-CSF-induced delayed apoptosis is markedly diminished. The present study investigates whether an alteration of the GM-CSF stimulation of MAPKs play a role in the diminished rescue from apoptosis of PMN of elderly subjects.MethodsNeutrophils were separated from healthy young and elderly donors satisfying the SENIEUR protocol. Neutrophils were stimulated with GM-CSF and inhibitors of the MAPKinase pathway. Apoptosis commitment, phosphorylation of signaling molecules, caspase-3 activities as well as expression of pro- and anti-apoptotic molecules were performed in this study. Data were analyzed using Student's two-tailed t-test for independent means. Significance was set for p ≤ 0.05 unless stated otherwise.ResultsIn this paper we present evidence that an alteration in the p42/p44 MAPK activation occurs in PMN of elderly subjects under GM-CSF stimulation and this plays a role in the decreased delay of apoptosis of PMN in elderly. We also show that p38 MAPK does not play a role in GM-CSF delayed apoptosis in PMN of any age-groups, while it participates to the spontaneous apoptosis. Our results also show that the alteration of the p42/p44 MAPK activation contributes to the inability of GM-CSF to decrease the caspase-3 activation in PMN of elderly subjects. Moreover, GM-CSF converts the pro-apoptotic phenotype to an anti-apoptotic phenotype by modulating the bcl-2 family members Bax and Bcl-xL in PMN of young subjects, while this does not occur in PMN of elderly. However, this modulation seems MAPK independent.ConclusionOur results show that the alteration of p42/p44 MAPK activation contributes to the GM-CSF induced decreased PMN rescue from apoptosis in elderly subjects. The modulation of MAPK activation in PMN of elderly subjects might help to restore the functionality of PMN with aging.