TNFalpha-induced suppression of PMN apoptosis is mediated through interleukin-8 production.

Dysregulated neutrophil (polymorphonuclear PMN) apoptosis is thought to contribute to the onset of adult respiratory distress syndrome (ARDS) in critically ill patients. Tumor necrosis factor-alpha (TNFalpha), which is present in elevated levels in the bronchoalveolar lavage fluid in patients with ARDS, is thought to play a central role in regulating PMN function in the lungs. Studies have shown that short-term culture with TNFalpha increases apoptosis yet extended culture with TNFalpha suppresses apoptosis. However, it is unclear whether this latter effect of TNFalpha is directly or indirectly mediated through production of anti-apoptotic cytokines such as interleukin (IL)-8. To investigate the role of IL-8 in TNFalpha-induced apoptosis PMN were exposed to TNFalpha (100 ng/mL) in the presence or absence of antibodies to IL-8, and the extent of apoptosis was assessed. An enzyme-linked immunoassay was used to measure levels of the anti-apoptotic cytokine IL-8, induced by TNFalpha-stimulation. Because TNFalpha may mediate its effect through various cell-signaling pathways, we next assessed the effect of kinase inhibition on the ability of TNFalpha to effect apoptosis and IL-8 production. Treatment with TNFalpha had a biphasic effect: at 4-8 h, apoptosis was increased but was markedly suppressed at 24 h (P < 0.05). PMN cultured for 24 h with TNFalpha also showed markedly increased levels of IL-8. Neutralization of IL-8 inhibited the ability of TNFalpha to suppress apoptosis (P < 0.05). Incubation of TNFalpha + p38-mitogen-activated protein kinase (MAPK) inhibitor SB202190 increased apoptosis (P < 0.01) and decreased IL-8 production to PMN control. To a lesser extent, incubation of TNFalpha with inhibitors to NF-kappaB (SN50) and PI3K (LY294002) also increased apoptosis and decreased IL-8 production (P < 0.05). These data illustrate a novel mechanism by which TNFalpha can indirectly elicit an anti-apoptotic effect via p38-MAPK induced release of the anti-apoptotic chemokine IL-8. The exploitation of such a pathway represents a potential target for regulation of PMN-mediated acute lung injury.

Suppression of PMN apoptosis by hypoxia is dependent on Mcl-1 and MAPK activity.

BACKGROUND: Hypoxia has been shown to delay the onset of neutrophil (polymorphonuclear leukocytes [PMNs]) apoptosis. With the use of antisense oligonucleotides, we have previously demonstrated that Mcl-1 is necessary for this effect. We wanted to further characterize the expression of Mcl-1 and examine signaling pathways required for the delay in apoptosis that is mediated by hypoxia. METHODS: For kinase signaling inhibition, PMNs were incubated for 12 hours with the following inhibitors: PD98059 Mitogen Activated Protein Kinase Kinase (MEK), SB202190 (p38 mitogen-activated protein kinase [MAPK]), and LY294002 (phosphatidyl inositol-3-kinase [PI3K]). PMNs that were treated with inhibitors were assessed for apoptosis by morphologic features or were lysed for Western blot analysis. RESULTS: Western blot analyses, immunofluorescent staining, and quantification showed an upregulation of Mcl-1 expression after 12 hours of incubation in response to hypoxia. When inhibitors of either MEK or p38 MAPK were incubated with PMNs during hypoxia, apoptosis increased to similar levels as normoxia. We further wanted to determine whether signaling through p38 MAPK or MEK led to increased Mcl-1 expression. Western blot analysis confirmed that the inhibition of p38 MAPK led to a significant decrease in Mcl-1 expression. CONCLUSIONS: We have documented a novel mechanism by which hypoxia can modify PMN apoptosis in the wound site by the activation of p38 MAPK signaling, thereby inducing the anti-apoptotic protein Mcl-1.

The loss of Mcl-1 expression in human polymorphonuclear leukocytes promotes apoptosis.

The regulation of polymorphonuclear leukocyte (PMN) apoptosis can influence the duration of the inflammatory response. We have previously shown that PMN apoptosis is delayed by matrix adhesion and hypoxia; however, the mechanisms responsible for this delay are not well understood. Mcl-1, an antiapoptotic Bcl-2 family member, is present in neutrophils; therefore, we sought to characterize its localization and function as it relates to PMN apoptosis. We found that Mcl-1 localized to the nucleus and cytoplasm and that expression levels decreased as PMN were aged in culture. Reducing available Mcl-1 through the use of antisense oligonucleotides demonstrated that Mcl-1 is necessary to delay apoptosis during normal PMN aging and hypoxia but is not required for suppression of apoptosis by laminin adhesion. Our results demonstrate a distinct expression pattern of Mcl-1 and that Mcl-1 is crucial for the delay of apoptosis initiated by certain antiapoptotic factors.

Telomerase activity and expression of the telomerase catalytic subunit, hTERT, in meningioma progression.

OBJECT: In recent reports, 6 to 19% of meningiomas have been classified as atypical or anaplastic/malignant. Some atypical and anaplastic meningiomas appear to arise from benign tumors by progression. Telomerase activation has recently been associated with malignant progression of human tumors. The authors have investigated a series of benign, atypical, and anaplastic/malignant meningiomas for telomerase activity and expression of the telomerase catalytic subunit human telomerase reverse transcriptase (hTERT). METHODS: A quantitative telomeric repeat amplification protocol was used to detect telomerase enzyme activity in seven (21%) of 34 benign, but in nine (75%) of 12 atypical and in seven (100%) of seven anaplastic/malignant meningiomas. Very high levels of telomerase activity were observed only in highly aggressive tumors. Messenger (m)RNA expression of the catalytic subunit hTERT was found in 11 (33%) of 33 benign, 12 (92%) of 13 atypical, and all seven anaplastic/malignant tumors. All telomerase-positive lesions were also positive for hTERT mRNA, whereas no telomerase activity was detected in six (21%) of 29 hTERT-positive tumors. This indicates that upregulation of hTERT is the rate-limiting step for telomerase activation in the majority of meningiomas. Expression of telomerase and hTERT was seen in all four tumors with gross brain invasion. All recurrent tumors or meningiomas recurring during follow up expressed hTERT. CONCLUSIONS: The results are consistent with a role for telomerase activation during the development of malignancy in meningiomas. Hence, expression of telomerase activity and hTERT might prove to be potentially useful markers for the evaluation of these tumors.

Neutrophils regulate their own apoptosis via preservation of CXC receptors.

BACKGROUND: Dysregulated neutrophil (PMN) apoptosis is thought to contribute to an exaggerated inflammatory response in diseases such as acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS). The CXC chemokines, interleukin-8 (IL-8) and growth-related oncogene alpha (Gro-alpha), contribute to the inflammatory response and suppress PMN apoptosis. We hypothesized that PMN generation of CXC chemokines is an autocrine/paracrine mechanism for amplification of the PMN inflammatory response via suppression of apoptosis. METHODS: Freshly isolated human PMNs from healthy donors were incubated with IL-8 or Gro-alpha (100 ng/ml) for 0-12 h, and apoptosis was analyzed at 24 h. De novo synthesis of IL-8 or Gro-alpha was measured using an ELISA. To determine if receptors were available to bind these newly synthesized ligands (125)I radiolabeled monoclonal antibodies specific for each receptor (CXCRI, CXCRII) were used to determine PMN receptor density. Comparison was by one-way ANOVA. RESULTS: Significant suppression of apoptosis was seen at 24 h with only 4 h exposure to IL-8 or Gro-alpha (n = 5, P < 0.05). PMNs cultured with IL-8 for 4 h produced 31 +/- 4.3 ng/ml IL-8 by 24 h; PMNs cultured with Gro-alpha produced 19.7 +/- 4.0 ng/ml Gro-alpha (n = 6, P < 0. 05). Neither chemokine induced significant production of the other chemokine. The addition of either ligand promoted upregulation of CXCR1 (n = 4, P < 0.05) at 24 h. However, CXCR2 was downregulated by Gro-alpha and IL-8 to 71 +/- 7.5 and 79 +/- 6.3% of control, respectively (P < 0.05). CONCLUSION: IL-8 and Gro-alpha, which suppress apoptosis, stimulate their own production after short-term incubation with PMNs. PMNs maintain the ability to respond to these chemokines through expression of the CXC receptors which suggests that PMNs are active participants in the suppression of apoptosis at inflammatory sites. CXCRI remains upregulated after prolonged stimulation and may be an important target for mediating neutrophil responses to IL-8.

CXC chemokine suppression of polymorphonuclear leukocytes apoptosis and preservation of function is oxidative stress independent.

Interleukin 8 (IL-8) and growth-related oncogene alpha (Gro-alpha) delay neutrophil apoptosis, which is thought to be important for the resolution of inflammation. We hypothesized that (IL-8) and Gro-alpha interfere with extracellular death receptor signaling or intracellular caspase activation to suppress neutrophil apoptosis. In addition, we sought to determine if prolonged neutrophil half-life was associated with preservation of function. Polymorphonuclear leukocytes (PMN) were cultured with IL-8 or Gro-alpha (0-100 ng/mL) in normoxia or hypoxia, and the extent of apoptosis was assessed by histology and TdT-mediated dUTP nick end labeling (TUNEL). Subsequently, to determine the role of apoptotic-associated receptors, PMN were cultured with IL-8 and neutralizing monoclonal antibody to Fas (CD95), TNFR55, and TNFR75. To establish the effect of IL-8 or Gro-alpha on pro-apoptotic caspase activity, the cleavage of specific colorimetric substrates was assessed. Functional changes in PMN included the capacity to produce superoxide anion and phagocytosis of Escherichia coli. At the 100 ng/mL dose, the addition of IL-8 and Gro-alpha maximally suppressed PMN apoptosis from 54% (untreated) to 5% and 6%, respectively. The addition of neutralizing antibodies to Fas, TNFR55, or R75 caused no change in IL-8 suppression of apoptosis. Caspase 3 activity was markedly suppressed at 24 h by the inclusion of either IL-8 and Gro-alpha. IL-8 and Gro-alpha-stimulated PMN released more superoxide anion and had an increased phagocytic index vs. control PMN. IL-8 and Gro-alpha suppress neutrophil apoptosis to a similar level that is not influenced by oxygen tension at high doses. The effect of IL-8 and Gro-alpha does not depend on activation of the Fas, TNFR55, or R75 receptor pathways but involves suppression of caspase 3 activity. IL-8 or Gro-alpha extends the functional half-life of neutrophils and may explain their role in disease states such as acute respiratory distress syndrome.

Interleukin-8-induced suppression of polymorphonuclear leukocyte apoptosis is mediated by suppressing CD95 (Fas/Apo-1) Fas-1 interactions.

BACKGROUND: Neutrophil apoptosis is crucial in the resolution of inflammation. The role of interleukin (IL)-8 in neutrophil apoptosis has not been previously studied; we hypothesized that in addition to its role as a chemoattractant, IL-8 would regulate polymorphonuclear leukocyte (PMN) apoptosis. METHODS: PMNs were adhered to plastic during hypoxia or normoxia and treated with IL-8 dosages of 0 to 1000 ng/mL. Apoptosis was assessed by cellular histology and the TUNEL assay. For receptor inhibition, blocking antibodies to IL-8 receptors in the presence of IL-8 were added. Apoptosis of PMNs treated with anti-Fas antibody +/- IL-8 was also analyzed. RESULTS: After treatment with 100 ng/mL IL-8 apoptosis was decreased from an average of 39.1% 9.3%. Inhibition of IL-8RA was able to restore apoptosis to 59.4%. Western analysis showed that with IL-8, there was a marginal decrease of total Fas protein, whereas Fas ligand was increased. After incubation with an apoptosis inducing-Fas antibody plus IL-8 reduced apoptosis to 9.5%. CONCLUSIONS: IL-8 not only promotes the inflammatory response by recruiting PMNs but also acts to suppress apoptosis mainly through the IL-8RA in an oxygen tension independent manner. The reduction in apoptosis is associated with changes in Fas and FasL where the presence of IL-8 suppresses the proapoptotic function of Fas-FasL interactions.

Integrin regulation of polymorphonuclear leukocyte apoxis during hypoxia is primarily dependent on very late activation antigens 3 and 5.

BACKGROUND: Apoptosis is thought to be a central mechanism that leads to resolution of the inflammatory response. The regulation of polymorphonuclear leukocyte (PMN) apoptosis during hypoxia has not been previously characterized, and we hypothesized that integrin signaling by matrix proteins (laminin) would regulate PMN apoptosis. METHODS: PMNs at 1 x 10(5)/ml were adhered on plastic or laminin for 12 hours during normoxia or hypoxia. Apoptosis was determined both by cellular histologic evaluation and the TUNEL assays (Tdt). Phagocytosis in apoptotic PMNs was determined with two-color flow cytometric analyses with rhodamine-labeled heat-killed Escherichia coli (511 nm) and the Tdt reagent (563 nm). Western blot analyses were performed on nine apoptotic regulatory proteins with monoclonal antibodies directed against each protein, and tyrosine phosphorylation was assessed after integrin receptor cross-linkage. RESULTS: Adherence of PMNs to laminin reduced apoptosis by cellular histologic evaluation and the Tdt method (%apoptosis = 19 +/- 1.0 versus 63 +/- 4.2 by histologic evaluation, 38 +/- 3.8 versus 60 +/- 10.5 by flow cytometry +/- adherence to laminin). Apoptosis-positive PMNs exhibited significantly greater phagocytosis than apoptosis-negative PMNs +/- laminin. Western blot analyses demonstrated increased p53 expression after 2 and 4 hours of hypoxia. Cross-linkage of very late activation antigen-3 (alpha 3/beta 1) resulted in the phosphorylation of 53 kd, 44 kd, and 39 kd proteins at 30 seconds. CONCLUSIONS: (1) Chemotaxis of PMNs into the interstitium during hypoxia not only provides a means of ensuring PMN-pathogen contact but also provides a mechanism for improved survival by reducing apoptosis. (2) The reduction of apoptosis is mediated primarily by very late activation antigen-3, which leads to a subsequent increase in the intracellular expression of p53 and increased bacterial phagocytosis.