Biomedical subjects

P Bankey

Publications and source records attributed to P Bankey.

14 recordsLinked to original sources

PubMed2001

Heat shock protein 27 inhibits apoptosis in human neutrophils.

BACKGROUND: Prolonged neutrophil(PMN) survival has been implicated in tissue injury following sepsis. A variety of bacterial products have been identified which inhibit PMN apoptosis including lipopolysaccharide(LPS). Extracellular heat shock proteins(Hsp) have recently been identified as potent regulatory signals for the innate immune system during the inflammatory response. We hypothesized that Hsp 27 can affect PMN phenotype with respect to apoptosis and cytokine profile. MATERIALS AND METHODS: PMN were isolated from the peripheral blood of healthy human volunteers by red blood cell sedimentation and gradient centrifugation. Cells were placed in media and cultured for 18 h with and without recombinant human Hsp 27 at various concentrations. In parallel experiments, PMN were stimulated with LPS, a known inhibitor of PMN apoptosis, for comparison. Apoptosis was quantified using annexin V and propidium iodide staining with flow cytometric analysis. Culture supernatants were assayed for secretion of TNF-alpha, IL-10, and IL-12. RESULTS: Hsp 27 significantly inhibits PMN apoptosis [control; 81.8 +/- 3.6%, vs Hsp 27, 60.4 +/- 4.1% p < 0.05]. The reduction is similar to that signaled by LPS, alone. Together their effect is not synergistic. The Hsp 27 response is dose-dependent. Hsp 27 does not induce secretion of TNF-alpha, IL-10, or IL-12, whereas LPS does signal IL-12 and TNF-alpha secretion. CONCLUSION: These data demonstrate that exogenous Hsp 27 may play a role in neutrophil-mediated tissue injury during trauma and sepsis via its ability to inhibit neutrophil apoptosis. However, Hsp 27 does not significantly alter neutrophil phenotype with respect to cytokine production profile.

Apoptosis↗

PubMed2001

Inhibition of p38 mitogen activated protein kinase increases lipopolysaccharide induced inhibition of apoptosis in neutrophils by activating extracellular signal-regulated kinase.

BACKGROUND: Prolonged polymorphonuclear neutrophil (PMN) survival has been implicated in tissue injury after sepsis. Previously we reported that lipopolysaccharide (LPS) inhibits PMN apoptosis via the activation of the extracellular signal-regulated kinase (ERK). Conversely, the p38 mitogen activated protein kinase (MAPK) pathway is involved in the spontaneous apoptosis of PMNs. The interaction between these 2 pathways and their ability to regulate apoptosis during sepsis remain largely undefined. We hypothesize that there is interaction between the ERK and p38 pathways during sepsis. METHODS: PMNs were isolated from healthy volunteers by Ficoll gradient centrifugation and red blood cell sedimentation. Cells were then pretreated for 1 hour with the ERK inhibitor (PD98059, 10 micromol/L), p38 inhibitor (SB203580, 1 micromol/L), or vehicle. After treatment with LPS, apoptosis and MAPK activity were correlated. RESULTS: LPS stimulation significantly inhibits PMN apoptosis compared with unstimulated cells. Furthermore, inhibition of ERK significantly abrogates this effect, whereas inhibition of p38 augments LPS induced inhibition of apoptosis. Elk-1 phosphorylation (ERK target) is significantly increased by LPS alone and by inhibition of the p38 pathway during LPS stimulation. This correlates with ERK phosphorylation by Western blot. CONCLUSIONS: These data show that p38 inhibition enhances ERK activity during endotoxemia. Furthermore, these data suggest that cooperation between ERK and p38 MAPK pathways dictates the apoptotic potential of PMNs during inflammatory states.

Adult↗

PubMed2001

The liver as an immune organ.

Systemic inflammation contributes to significant morbidity in the ICU. With its ability to generate antiinflammatory acute-phase proteins, cytokines via Kupffer cells, and recently acknowledged resident lymphocytes, the liver provides a central regulatory role in inflammation. The liver has constant exposure to foreign material as a result of gut translocation and first-pass metabolism from the bloodstream. Consequently, the balance between hepatic activation and tolerance becomes an important factor in the host response to inflammation. Interventions and therapies that can assess and modulate these hepatic functions can improve outcomes for ICU patients.

Acute-Phase Reaction↗

PubMed2000

Induction of global anergy rather than inhibitory Th2 lymphokines mediates posttrauma T cell immunodepression.

Depressed mitogen-induced IL-2 and IFN-gamma responses after severe mechanical or thermal injury are postulated to result from an expansion of Th2 lymphocytes with concomitant excessive production of IL-4 and/or IL-10. Here, we simultaneously assessed proliferation and Th1 (IFN-gamma) versus Th2 (IL-10, IL-4) lymphokine production in trauma patients' isolated T cells stimulated in a costimulation sufficient, antigen presenting cell independent system (anti CD3 + anti-CD4). T cells with depressed proliferation and IL-2 production simultaneously lost IL-4, IL-10, and IFN-gamma protein and mRNA responses. Exogenous IL-12 addition did not restore IFNgamma responses, but exogenous IL-2 partially restored IL-4, IFN-gamma, and IL-10 production. Although initially partially restored by exogenous IL-2 or stimulation with PMA + ionomycin, patient T cells with persisting anergy progressively lost even these lymphokine and proliferative responses. Development of global T cell anergy was not a result of lost T cell viability or protein synthesis, since it corresponded to predominance of anergic T cells with upregulated expression of CD11b, but downregulated CD28 and CD3 expression. Thus, the subset of posttrauma patients whose isolated T cells become unresponsive experienced progressively worsening global anergy, mediated not by an increased production of Th2 lymphokines, but possibly by T cell incapacity to be activated through TCR triggering or Ca(2+) mobilization.

Adult↗

PubMed2000

Inhibition of neutrophil apoptosis after severe trauma is NFkappabeta dependent.

BACKGROUND: Systemic inflammation may inhibit neutrophil (PMN) apoptosis and promote multiple organ dysfunction syndrome. We hypothesize that severe trauma causes dysregulation of PMN apoptosis. METHODS: Neutrophils were isolated from trauma patients (24-72 hours after injury; n = 16) and controls (healthy volunteers) and incubated for 18 hours. In separate experiments, control cells were treated +/- the nuclear factor kappa beta (NFkappabeta) inhibitor pyrrolidinithiocarbamate then incubated with 25% patient or control plasma. Apoptosis was quantified by enzyme-linked immunosorbent assay for histone-associated DNA and annexin V fluorescence-activated cell sorter. NFkappabeta activation was determined by Western blot for phosphorylated I kappabeta. RESULTS: Apoptosis was inhibited in trauma patient PMN. Neutrophil apoptosis correlated with multiple organ dysfunction syndrome score, Acute Physiology and Chronic Health Evaluation II, and platelet count. Patient plasma inhibited apoptosis and induced phosphorylation of I kappabeta in control cells. Inhibition of PMN apoptosis by patient plasma was blocked by pretreatment with pyrrolidinithiocarbamate. CONCLUSION: NFkappabeta-dependent inhibition of neutrophil apoptosis occurs after trauma. Early inhibition of PMN apoptosis is dependent on the magnitude of injury.

Apoptosis↗

PubMed2000

Inhibited neutrophil apoptosis: proteasome dependent NF-kappaB translocation is required for TRAF-1 synthesis.

Neutrophil (PMN) apoptosis regulates local and systemic inflammation during sepsis. Tumor necrosis factor receptor-associated factors (TRAFs) have been implicated as mediators of apoptosis; however, the signaling pathways for their production in stimulated PMN are unclear. We hypothesize that NF-kappaB translocation is necessary for the induction of TRAF-1 in PMNs with prolonged survival. Neutrophils were isolated from the blood of healthy volunteers by Ficoll gradient centrifugation and red blood cell sedimentation. Neutrophil NF-kappaB was inhibited with a proteasome inhibitor, PSI-I. Cells were treated with PSI-I (30 microM) or vehicle (DMSO 0.2%) for 50 min then incubated over an 18-h time course with LPS (10 to 1000 ng/mL), tumor necrosis factor alpha (TNFalpha) (2 to 20 ng/mL) or control media. In vitro apoptosis was quantified by propidium iodide FACS analysis. Total cellular TRAF-1 was detected by Western blot analysis of cell lysates. Steady state TRAF-1 mRNA was detected by RPA. NF-kappaB activity was determined by Western blot analysis for nuclear p65. Means and standard errors were calculated; data were analyzed by ANOVA. Lipopolysaccharide (LPS) and TNFalpha increased PMN nuclear p65 and steady state TRAF-1 mRNA. Apoptosis was inhibited by TNFalpha and LPS at 12 and 18 h (P < 0.01). Incubation of cells in the NF-kappaB inhibitor PSI-I blocked LPS and TNFalpha-induced inhibition of apoptosis (P < 0.05) and the induction of both nuclear p65 and TRAF-1 mRNA. These data demonstrate that inhibition of PMN apoptosis and TRAF-1 induction by LPS and TNFalpha is NF-kappaB dependent.

Apoptosis↗

PubMed2000

Activated neutrophils induce nitric oxide production in Kupffer cells.

Neutrophils (PMN) are proposed to contribute to hepatic dysfunction during sepsis. Transmigrating PMN have been demonstrated to adhere to and injure parenchymal cells (hepatocytes); however, the effect of sepsis-activated PMN on hepatic macrophages or Kupffer cells (KC) is poorly characterized. We hypothesize that PMN influence KC inflammatory mediator production, including nitric oxide. Rodent KC were co-cultured with PMN obtained from controls (Norm-PMN) or endotoxemic rats [lipopolysaccharide (LPS)-PMN] for 18 h. After an 18-h incubation, supernatants and cell lysates of the KC were analyzed for nitric oxide (NO) production. Co-cultures with LPS-PMN/KC demonstrated significantly increased production of nitrite and up-regulation of inducible nitric oxide synthase (iNOS) protein compared to KC alone or Norm-PMN/KC co-cultures. Immunohistochemistry revealed preferential iNOS protein staining in the cytoplasm of KC cultured with LPS-PMN compared to controls. Nitrite production in co-cultures of KC and LPS-PMN where cell contact was inhibited by a cell impermeable but diffusable membrane was significantly reduced. These data provide evidence that KC can be stimulated directly by activated PMN for production of NO. Further, they suggest another mechanism by which PMN modulate hepatic function during sepsis.

Animals↗

PubMed1999

Mitogen-activated protein kinases signal inhibition of apoptosis in lipopolysaccharide-stimulated neutrophils.

BACKGROUND: Neutrophil (PMN) apoptosis is critical to the resolution of infection and the limitation of inflammation. Bacterial endotoxin (lipopolysaccharide [LPS]) inhibits PMN apoptosis and activates the p38 mitogen-activated protein kinase (MAPK) signal cascade. The role of p38 and other MAPKs (ERK and SAPK/JNK) in regulating PMN apoptosis after LPS stimulation is unknown. We hypothesize that MAPK activation by LPS signals inhibition of PMN apoptosis. METHODS: PMNs were isolated from the blood of healthy human volunteers and incubated with PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), or 0.1% dimethyl sulfoxide (vehicle) for 1 hour before treatment with LPS (0, 10, or 1000 ng/mL). Neutrophil MAPK activation was determined by Western blot analysis for phosphorylated p38, ERK, and SAPK/JNK. Apoptosis was quantified by flow cytometry with use of propidium iodide and annexin V. RESULTS: LPS inhibited PMN apoptosis and activated p38 and ERK in a dose- and time-dependent fashion. SAPK/JNK was not activated by LPS. Treatment of cells with ERK inhibitor before LPS stimulation abrogated LPS signaled inhibition of PMN apoptosis. Conversely, p38 inhibition with SB203580 augmented inhibition of apoptosis by LPS. CONCLUSIONS: These data demonstrate opposing roles of MAPKs in mediating PMN apoptosis after LPS stimulation. We conclude that LPS signal transduction by ERK inhibits PMN apoptosis while activation of p38 promotes apoptosis.

Apoptosis↗

PubMed1998

Hepatocyte polyunsaturated fatty acid enrichment increases acute phase protein synthesis.

BACKGROUND: The production of acute phase proteins by the liver is critical for homeostasis and recovery after injury. Polyunsaturated fatty acids, specifically of the n-3 family, have demonstrated anti-inflammatory properties that promote recovery; however, the effects of these fatty acids on acute phase protein synthesis have not been fully evaluated. METHODS: The synthesis of the acute phase proteins alpha-2 macroglobulin and lipopolysaccharide binding protein was studied in hepatocyte-Kupffer's cell cocultures from Wistar rats. Cultures were endotoxin stimulated after enrichment with albumin complexed arachidonic acid (20:4n-6) or docosahexaenoic acid (22:6n-3). Protein synthesis was analyzed by [35S]-methionine labeling or Western blotting. Culture interleukin-6 levels were determined. RESULTS: Both polyunsaturated fatty acids increase hepatocyte synthesis of acute phase proteins alpha 2-macroglobulin and lipopolysaccharide binding protein compared with controls; however, the response in the docosahexaenoic acid (22:6n-3) treated cultures was significant (P < .05 vs control). Interleukin-6 was also increased in the polyunsaturated fatty acid cultures compared with controls (P < .05) vs control). Cellular phospholipids were significantly enriched with the individual supplemented fatty acids (P < .05 vs bovine serum albumin). CONCLUSIONS: Polyunsaturated fatty acids have the capability to increase in vitro acute phase protein synthesis. This may contribute to the observed anti-inflammatory effect of n-3 polyunsaturated fatty acid enrichment.

Acute-Phase Proteins↗

PubMed1995

Liposomes modulate Kupffer cell endotoxin response.

OBJECTIVES: To test the hypothesis that pretreatment with liposomes enriched with the omega 3 fatty acid docosahexaenoic acid (22:6 omega 3) will alter the Kupffer's cell and systemic cytokine (tumor necrosis factor and interleukin-6) response to endotoxin challenge, and to demonstrate alterations in Kupffer's cell phospholipid fatty acid composition after in vivo liposome treatment. DESIGN: Nonrandomized controlled laboratory investigation in Wistar rats. INTERVENTIONS: Animals were assigned to three pretreatment groups: no liposomes; liposomes, 100 mg/kg; or liposomes, 400 mg/kg given by bolus intravenous injection with the animals under inhalation anesthesia. Eighteen hours after liposome treatment, each group was challenged with Escherichia coli lipopolysaccharide (3 mg/kg intraperitoneally in 10 mL of lactated Ringer's solution) or lactated Ringer's solution only. In a separate set of experiments, Kupffer's cells were obtained from animals pretreated with liposome, 400 mg/kg, or controls and challenged with lipopolysaccharide (1, 100, or 10(4) ng/mL) in vitro. OUTCOME MEASURES: Serum and Kupffer's cell supernatant tumor necrosis factor and interleukin-6 bioactivity, Kupffer's cell phospholipid fatty acid composition, survival, and liver histologic findings. RESULTS: In vivo liposome pretreatment (400 mg/kg) resulted in significant increases in serum tumor necrosis factor and interleukin-6 levels 90 minutes after intraperitoneal lipopolysaccharide challenge (P < .05 vs no liposomes). Kupffer's cells isolated from liposome-treated animals (400 mg/kg) compared with untreated controls release significantly more tumor necrosis factor and interleukin-6 after lipopolysaccharide stimulation in vitro in a dose-dependent response (P < .05). Liposome treatment increased total polyunsaturated fatty acid, total omega 3, and docosahexaenoic acid 22:6 omega 3 content in Kupffer's cell phospholipids compared with untreated controls. Survival 24 hours after lipopolysaccharide challenge was reduced by liposome (400 mg/kg) pretreatment (P < .05 by chi 2 test). Livers from each treatment group demonstrated focal areas of hepatocyte necrosis and inflammatory cells. CONCLUSION: Liposome pretreatment increases the circulating and Kupffer's cell cytokine response to endotoxemia, increases Kupffer's cell polyunsaturated fatty acid content, and is associated with reduced survival.

Animals↗

PubMed1994

Acute-phase hepatocytes regulate liver sinusoidal cell mediator production.

BACKGROUND: Overproduction of liver sinusoidal cell (LCS) mediators in response to endotoxemia or gram-negative infection that follows tissue injury may contribute to hepatic dysfunction. OBJECTIVE: To better define the role of hepatocyte-derived acute-phase reactants in the regulation of sinusoidal cell mediator production following sequential insults, we tested the hypothesis that interleukin-6 (IL-6) prestimulation alters hepatocyte regulation of lipopolysaccharide (LPS)-stimulated sinusoidal cell tumor necrosis factor (TNF), IL-6, and nitric oxide production. METHODS: Hepatocytes and LSCs were isolated from Wistar rats, and in vitro responses were compared between LSCs alone and hepatocyte-LSC cocultures. Cocultures and LSCs alone were sequentially stimulated with IL-6 (5000 U/mL) then LPS (dose-response), and culture supernatants were analyzed for TNF (L929 cytolysis), IL-6 (7TD1 proliferation), and nitric oxide (Griess reaction). Induction of acute-phase protein synthesis by the stimulation of hepatocytes with IL-6 and dexamethasone (0.1 mumol/L) was assayed by methionine radiolabeling and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Coculture levels of messenger RNA for TNF-alpha and IL-6 were examined by RNA extraction and reverse transcriptase polymerase chain reaction with specific primers. RESULTS: Interleukin-6 and dexamethasone signal hepatocyte acute-phase protein synthesis. Prestimulation of cocultures, but not of LSCs alone, with IL-6 inhibits LPS-stimulated IL-6 and nitric oxide production significantly. Bioactivity of TNF is reduced to a lesser extent. Polymerase chain reaction analysis demonstrated similar levels of TNF and IL-6 message following sequential stimulation. CONCLUSIONS: Interleukin-6-stimulated acute-phase hepatocytes limit LPS-stimulated coculture cytokine bioactivity and nitric oxide production. This hepatocyte response may provide a local counterregulatory mechanism to limit LSC-mediated injury.

Acute-Phase Proteins↗

PubMed1990

Tumor necrosis factor production by Kupffer cells requires protein kinase C activation.

Tumor necrosis factor (TNF) has been proposed as a primary inflammatory mediator of septic shock. In vitro and in vivo studies indicate that endotoxin- or lipopolysaccharide (LPS)-activated macrophages are a principle source of TNF; however, membrane signal transduction and intracellular pathways by which LPS triggers TNF production in macrophages are unclear. Recent evidence indicates that specific protein phosphorylation via activation of protein kinase C (PKC) is an early, critical step in the signaling of macrophage TNF production by phorbol esters. We hypothesize that PKC activation is also required in LPS-signaled Kupffer cell (KC) TNF production. Murine KCs were obtained by liver perfusion and digestion and then stimulated with LPS (Escherichia coli O111:B4) or LPS in the presence of H-7, a selective PKC inhibitor. Conditioned media was collected at 3 hr for assay of TNF utilizing the L929 cytolysis bioassay standardized to murine-rTNF-alpha. We found that H-7 inhibited significantly LPS signaled TNF release at a concentration of 10 microM, while H-8 (a cyclic nucleotide specific inhibitor) had no effect. The effect of H-7 was dose dependent and present at varying concentrations of LPS. Down regulation of PKC activity by preincubation of KCs with phorbol myristate acetate (PMA, a direct activator of PKC) also resulted in significantly reduced TNF release after LPS stimulation. The inhibitor H-7 (10 microM) also significantly inhibited LPS signaled prostaglandin E2 release in Kupffer cells. Total and specific intracellular protein phosphorylation was determined by trichloroacetic acid precipitation and SDS-polyacrylamide gel electrophoresis after labeling stimulated Kupffer cells with 32Pi. Total protein phosphorylation was not significantly altered by LPS stimulation; however, autoradiograms from PMA- and LPS-stimulated KCs demonstrate enhanced phosphorylation of a 40-kDa protein (2.7 +/- 0.9-fold) and a 33-kDa protein (3.1 +/- 1.0-fold) which were inhibited by H-7. We conclude that activation of PKC and protein phosphorylation are required steps in the signal transduction pathway of LPS-stimulated TNF production in Kupffer cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1989

Hypoxia and endotoxin induce macrophage-mediated suppression of fibroblast proliferation.

Cellular mechanisms and environmental factors contributing to wound failure following shock and wound contamination are unclear. The activation of macrophages by exposure to hypoxia (pO2 less than 20) and/or lipopolysaccharide (10 micrograms/ml) in vitro was investigated for its effect on macrophage regulation of fibroblast proliferation. The effect on fibroblast proliferation of conditioned medium from activated murine macrophages or co-culture with activated macrophages was tested by measuring 3T3 fibroblast incorporation of 3H-thymidine and culture DNA content. Unstimulated macrophages produced growth factors that increase fibroblast proliferation (proliferation index (PI) = 1.4 +/- 0.15, p less than 0.05 vs. control). Activation by hypoxia alone had little effect on macrophage regulation of fibroplasia (PI = 1.55 +/- 0.28, N.S. vs. unstimulated macrophages). LPS activated macrophages suppressed fibroplasia and the combination of hypoxia with LPS augmented the suppression (PI = 0.5 +/- 0.11, LPS alone, p less than 0.05 and 0.25 +/- 0.05, LPS + hypoxia, p less than 0.01). In addition, hypoxia + LPS treated co-cultures had reduced DNA contents, suggesting reduced cell numbers (12.5 +/- 2.6 micrograms vs. 8.2 +/- 2.0 micrograms). We screened several macrophage cytokines for their direct effect on 3T3 proliferation and found that mr-Tumor Necrosis Factor-alpha (150 units) also suppressed proliferation. Conditioned supernatants from LPS activated macrophages contained 12 +/- 2 units of mrTNF as measured by L929 cytolysis; however, this was significantly less than required to induce suppression of proliferation by direct addition. The regulatory role of the macrophage appears to be dependent on its level of activation. Activation by hypoxia and LPS altered macrophage regulation of fibroblast proliferation from stimulation to suppression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Burn injury enhances alveolar macrophage endotoxin sensitivity.

Systemic activation of inflammatory cascades has been implicated in the pathogenesis of the multiple organ dysfunction syndrome. To begin to determine whether dysregulation of macrophage cytokine expression after burn plays a role in organ failure, we examined tumor necrosis factor alpha bioactivity of liver and lung macrophages under two conditions: (1) at 1, 3, 5, and 7 days after 40% scald burn, and (2) after sequential insult consisting of 40% scald burn followed by in vitro incubation with endotoxin. Burn alone did not significantly alter alveolar macrophage or Kupffer cell tumor necrosis factor alpha bioactivity at any of the timepoints examined. Sequential insult did not result in significant changes in Kupffer cell tumor necrosis factor alpha, but tumor necrosis factor alpha was increased 11.1 times in alveolar macrophages harvested on postburn day 3. Therefore macrophage cytokine responses to thermal injury are apparently both tissue-specific and time-dependent.

Animals↗