Pentoxifylline attenuates stored blood-induced inflammation: A new perspective on an old problem

BACKGROUND: Blood transfusion is a risk factor for many inflammatory processes. Its supernatant fraction has been proven to activate neutrophils. We hypothesized that pentoxifylline (PTX) would attenuate stored blood-induced neutrophil activation and pro-inflammatory mediator production. METHODS: Whole blood was incubated with HBSS, LPS (100 microg/mL), leukoreduced PRBC supernatant + LPS, or supernatant + LPS + PTX (2 mmol/L). TNF-alpha levels were measured by ELISA. MMP-9 was evaluated with zymography. Neutrophil CD66b expression was determined by flow cytometry in blood treated with HBSS, fMLP (1 micromol/L), supernatant + fMLP, or supernatant + fMLP + PTX. RESULTS: TNF-alpha levels were elevated in both the LPS and supernatant + LPS groups (100%; P < 0.01 and 120%; P < 0.01, respectively). PTX administration resulted in a 106% decrease in TNF-alpha (P < 0.0001). MMP-9 levels were increased in all groups. Administration of PTX to the supernatant + LPS group generated a 33% decrease in MMP-9 levels, which was not statistically significant (P < 0.4). Upregulation of CD66b expression was seen in LPS and supernatant + LPS groups. Significant attenuation was seen with PTX (47%; P < 0.01). CONCLUSIONS: PTX downregulates CD66b and TNF-alpha expression in supernatant-induced whole blood. Because blood transfusion can contribute to inflammatory injury, the adjunctive use of PTX may have therapeutic potential.     

Endotoxin suppresses matrix protein-induced upregulation of PMN candicidal activity: an effect reversed by low-dose TNF-alpha.

We investigated the relationship of polymorphonuclear leukocyte (PMN) candicidal activity, matrix proteins, and lipopolysaccharide (LPS) to determine how LPS modulates the normal enhancing effect of matrix proteins on PMN candicidal activity. LPS reduced PMN candicidal activity when PMN were adhered in the presence of either fibronectin or laminin. In the presence of fibronectin or laminin, LPS reduced CD11b/CD18 expression (the fibronectin receptor) as assessed using sheep erythrocytes coated with C3bi. Experiments with 125I-fibronectin and 125I-RGDS (Arg-Gly-Asp-Ser) demonstrated that LPS reduced both the binding of fibronectin and the bioavailability of the binding epitope on the PMN surface. Stimulating the PMN oxidative burst with PMA but not FMLP also reduced fibronectin and RGDS binding. Incubation of LPS-treated PMN with staurosporine blocked the decrease in fibronectin and RGDS binding. Exposure of PMN to LPS plus low-dose TNF-alpha restored both fibronectin and RGDS binding with a concomitant increase in CD11b/CD18 surface expression. Low-dose TNF-alpha restored PMN candicidal activity in the presence of LPS and was most effective if PMN were preadhered to fibronectin. These results demonstrate that: (1) matrix proteins enhance normal PMN candicidal activity, (2) LPS reduces PMN candicidal activity in the presence of matrix proteins, (3) stimulation of the PMN oxidative burst in particular via protein kinase c activation reduces the bioavailability of the fibronectin receptor, and (4) low-dose TNF-alpha may restore PMN candicidal activity in part by upregulating the surface receptor for fibronectin binding.     

Phosphodiesterase inhibition attenuates stored blood-induced neutrophil activation: a novel adjunct to blood transfusion

BACKGROUND: Activated neutrophils play a central role in the pathogenesis of ARDS and multiple organ failure (MOF). Transfusion of packed red blood cells (PRBCs) is an independent risk factor in the development of ARDS and MOF. It has been postulated that factors present in the supernatant of PRBCs activate neutrophils. The magnitude of neutrophil activation is dependent on the age of the stored blood. Our laboratory and others have reported that pentoxifylline (PTX), a nonspecific phosphodiesterase inhibitor, decreases neutrophil activation. We hypothesized that adding PTX to PRBCs would attenuate blood transfusion-induced neutrophil activation. STUDY DESIGN: Peripheral blood was obtained from healthy human volunteers. Oxidative burst, CD11b, and CD35 expression were measured by flow cytometry using a whole blood preparation. Whole blood was incubated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) (1 microM) alone and 42-day-old PRBC supernatant + fMLP with or without PTX (2 mmol/L). RESULTS: N-formyl-methionyl-leucyl-phenylalanine alone caused a significant increase in neutrophil oxidative burst (100%). The exposure of whole blood to PRBC supernatants + fMLP led to a 1.3-fold increase in neutrophil oxidative burst as compared with fMLP alone, indicating that PRBC supernatants prime neutrophils for oxidative burst by 75%. More importantly, PTX decreased neutrophil oxidative burst by 114% in supernatant + fMLP-stimulated whole blood (p < 0.001). PTX decreased CD11b expression in both fMLP (p < 0.01) and fMLP+supernatant-stimulated whole blood (p < 0.05). Supernatant from PRBCs did not have an additive effect to fMLP alone on CD11b expression. N-formyl-methionyl-leucyl-phenylalanine-induced CD35 expression was downregulated by PTX. The addition of PRBC supernatant did not increase the already upregulated fMLP-induced CD35 expression. CONCLUSIONS: Our results suggest that adding PTX to PRBC supernatant markedly decreases neutrophil activation. The lack of successful treatment strategies to effectively modulate the inflammatory response after blood transfusion indicates the need for novel therapies. Because the deleterious effects of blood transfusion on end-organ injury and MOF are associated with neutrophil activation, the adjunct use of PTX to blood transfusion may have therapeutic potential.     

Neutrophil degranulation and the effects of phosphodiesterase inhibition

BACKGROUND: Neutrophils play a major role as the first line in host defense after exposure to bacterial products. However, an exaggerated inflammatory response characterized by overwhelming neutrophil activation can be injurious to the host. Pentoxifylline (PTX), a nonspecific phosphodiesterase inhibitor, has been shown to attenuate neutrophil oxidative burst and decrease proinflammatory mediator synthesis. We hypothesized that PTX down-regulates neutrophil activation by decreasing the surface expression of both CD35 and CD66b, two markers of neutrophil degranulation. MATERIALS AND METHODS: Venous blood was obtained from three healthy volunteers. Whole blood was incubated with HBSS (control), f-methionyl-leucyl-phenylalanine (fMLP, 1 microM/L), PTX (2 mM/L), or fMLP + PTX. CD35 and CD66b expression were measured by flow cytometry. RESULTS: fMLP treatment caused a significant increase in CD35 and CD66b expression of when compared to controls (P < 0.01). PTX treatment revealed expression of both markers comparable to the control group. A 38% decrease in CD35 (64 +/- 12 versus 100; P < 0.01) and a 52% decrease in CD66b (48 +/- 7 versus 100; P < 0.01) expression were demonstrated in the fMLP + PTX group when compared to fMLP alone. CONCLUSION: In addition to the known effects of PTX on neutrophil oxidative burst, PTX also affects neutrophil degranulation, an essential step in enzyme release and subsequent tissue injury. These findings may have clinical relevance in the treatment of disease processes due to inflammation in which primed neutrophils play a role.     

Hypertonic resuscitation modulates the inflammatory response in patients with traumatic hemorrhagic shock

OBJECTIVE: To determine the effect of resuscitation with hypertonic saline/dextran (HSD) on the innate immune response after injury. SUMMARY OF BACKGROUND DATA: Hypovolemic shock causes a whole body ischemia/reperfusion injury, leading to dysregulation of the inflammatory response and multiple organ dysfunction syndrome. Hypertonicity has been shown to modulate the innate immune response in vitro and in animal models of hemorrhagic shock, but the effect on the inflammatory response in humans is largely unknown. METHODS: Serial blood samples were drawn (12, 24, 72 hours and 7 days after injury) from patients enrolled in a prospective, randomized, double-blind trial of traumatic hypovolemic shock, HSD (250 mL) versus lactated Ringer's solution (LR) as the initial resuscitation fluid. Neutrophil (PMN) CD11b/CD18 expression was assessed via whole blood FACS analysis with and without stimulation (fMLP 5 micromol/L or PMA 5 micromol/L). PMN respiratory burst was assessed using the nitro-blue tetrazolium assay. Monocytes stimulated with 100 ng LPS for 18 hours were assessed for cytokine production (TNF-alpha, IL-1Beta, IL-6, IL-10, IL-12). RESULTS: Sixty-two patients (36 HSD, 26 LR) and 20 healthy volunteers were enrolled. CD11b expression, 12 hours after injury, was increased 1.5-fold in patients resuscitated with LR compared with controls. Those resuscitated with HSD had a significant reduction in CD11b expression 12 hours after injury, compared with LR. There was no difference in respiratory burst early after injury. Monocytes from injured patients expressed lower levels of all cytokines in comparison to normal controls. Patients give HSD showed a trend toward higher levels of IL-1beta and IL10 production in response to LPS, 12 hours after injury. CONCLUSION: HSD resuscitation results in transient inhibition of PMN CD11b expression and partial restoration of the normal monocyte phenotype early after injury.     

TNF-alpha--accelerated apoptosis abrogates ANCA-mediated neutrophil respiratory burst by a caspase-dependent mechanism

BACKGROUND: Tumor necrosis factor (TNF)-alpha rapidly primes neutrophils (PMN) for an anti-neutrophil cytoplasmic antibody (ANCA)-induced respiratory burst and is thus proinflammatory. TNF-alpha also progressively accelerates apoptosis. We investigated the effect of TNF-alpha-mediated apoptosis on ANCA antigen expression and on ANCA-induced superoxide generation in human PMN. METHODS: PMN were brought to apoptosis by 10 ng/mL of TNF-alpha or a combination of TNF-alpha and 2.5 microg/mL cycloheximide, a protein synthesis inhibitor, or cycloheximide alone for three hours. Apoptosis and ANCA antigen expression were assessed by fluorescence-activated cell sorting (FACS) and microscopy. Superoxide was determined with the ferricytochrome C assay. RESULTS: TNF-alpha with cycloheximide for three hours caused apoptosis in 87% PMN compared to 2% in untreated controls (N=18; P < 0.01). Accelerated apoptosis was associated with an increase in ANCA-antigen expression for both proteinase 3 and myeloperoxidase (P < 0.05). Nevertheless, apoptosis was paralleled by a decreased proteinase 3 and myeloperoxidase ANCA-induced respiratory burst (P < 0.05). Furthermore, superoxide release in response to immune complexes, phorbol ester (PMA), and bacterial peptide (FMLP) was significantly decreased. Blocking caspase-3 activity prevented apoptosis in TNF-alpha with cycloheximide-treated cells (83% to 2%) and prevented compromised respiratory burst in response to ANCA. Caspase-3 inhibition abrogated apoptosis-mediated ANCA antigen up-regulation (PR3 141.6 +/- 34.1 MFI to 33.9 +/- 7.8; MPO 48.3 +/- 12.9 MFI to 11.9 +/- 3.2, N=6, P < 0.05). CONCLUSIONS: TNF-alpha-accelerated apoptosis was associated with increased ANCA antigen expression but with down-regulated respiratory burst activity in response to ANCA. Specific inhibition of apoptosis by caspase-3 blockade prevented the increase in ANCA-antigen expression and preserved the capability of generating superoxide, thereby establishing a causative role for apoptosis. We suggest that TNF-alpha exhibits dual actions by both priming and terminating ANCA-mediated activation of human PMN.     

LPS-Induced NF-kappaB activation and TNF-alpha release in human monocytes are protein tyrosine kinase dependent and protein kinase C independent

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is an important mediator of septic shock. Endotoxin (LPS) signal transduction in human monocytes leads to activation of nuclear factor-kappa B (NF-kappaB) and TNF-alpha release. Previous studies have implicated activation of both protein kinase C (PKC) and protein tyrosine kinases (PTK) in LPS-induced NF-kappaB activation and TNF-alpha production. We hypothesized that inhibition of either PKC or PTK would decrease LPS-induced NF-kappaB DNA binding and TNF-alpha release in human monocytes. MATERIALS AND METHODS: Human monocytes were stimulated with PMA (50 ng/ml) alone or LPS (100 ng/ml) with and without a nonspecific serine/threonine protein kinase inhibitor staurosporine (Stauro), a specific pan-PKC inhibitor bisindolylmaleimide (Bis), or an inhibitor of PTK genistein (Gen). TNF-alpha release in culture supernatants was measured by an ELISA. NF-kappaB DNA binding was evaluated by electrophoretic mobility shift assay. RESULTS: LPS increased NF-kappaB DNA binding and TNF-alpha release in human monocytes. Nonspecific protein kinase inhibition inhibited NF-kappaB activation and TNF-alpha release, while specific PKC inhibition with Bis had no effect on LPS-induced NF-kappaB DNA binding or TNF-alpha release. PTK inhibition with Gen attenuated both LPS-induced NF-kappaB DNA binding and TNF-alpha production in human monocytes. Direct activation of PKC with PMA induced both NF-kappaB activation and TNF-alpha production by human monocytes. CONCLUSIONS: These results suggest that LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes are independent of PKC activity. Furthermore, our results provide evidence that PTK plays a role in LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes and thus could be a potential therapeutic target in inflammatory states.     

Respiratory burst activity of polymorphonuclear cells is dependent on the cell preparation technique

BACKGROUND: Controversial results have been reported regarding the effect of anaesthetics on superoxide anion production during the respiratory burst (RB) of polymorphonuclear cells (PMN). The differences could be caused by the cell preparation methods and the aim of this study was to compare two techniques. METHODS: RB activity was measured in cell suspensions isolated with the single-step Ficoll procedure and in unfractionated whole blood. Two concentrations of propofol (therapeutic and 10-fold of this, 6 microg ml-1 or 60 microg ml-1) were investigated after cell preparation with both methods. RB was stimulated with Escherichia coli (E. coli), phorbol 12-myristate 13-acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) and measured by means of fluorescence intensity in a flow cytometer. RESULTS: The percentage of PMNs in whole blood which generate superoxide anions in response to fMLP was significantly lower (2.5 +/- 0.7%; mean +/- SEM) than that in Ficoll isolated cell suspensions (15.1 +/- 1.7%). Incubation with propofol led to a concentration-related decrease of RB activity in Ficoll separated PMNs after both PMA and fMLP stimulation. No significant effect of propofol was observed on the RB in PMA stimulated whole blood samples. CONCLUSION: The results suggest that the influence of cell preparation methods should be considered when the in vitro effects of anaesthetics on PMN functions are studied with flow cytometric methods.     

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.     

Intracellular monocyte cytokine production and CD 14 expression are up-regulated in severe vs mild chronic heart failure.

BACKGROUND: The role of circulating monocytes in the process of low-grade inflammation, characteristic of chronic heart failure (CHF), has recently been questioned. Lipopolysaccharide (LPS) desensitization has been proposed to mediate reduced monocyte cytokine elaboration in patients with severe CHF. METHODS: Intracellular monocyte production of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha, and monocyte CD 14 expression were measured flow-cytometrically without and after 8-hour LPS stimulation in 46 patients with CHF and in a healthy control group. RESULTS: Basal cytokine concentrations were similar for the control and the mild CHF groups (New York Heart Association [NYHA] Class I or II). After LPS stimulation, IL-6 (p=0.002) and TNF-alpha levels (p=0.001) were lower in the latter group, whereas IL-1 beta production was comparable. For the moderate-severe CHF patients, unstimulated IL-1 beta (p=0.04) was higher, whereas IL-6 (p=0.2) and TNF-alpha (p=0.1) levels were not different from the controls. Measurement of LPS-stimulated cytokine production showed no differences between the control group and patients with moderate-severe CHF (all p= 0.5). Upon comparing mild vs moderate-severe CHF patients, higher levels of unstimulated cytokine production (IL-1 beta, p=0.002; IL-6, p=0.01; TNF-alpha, p=0.003), stimulated IL-1 beta (p=0.002) and IL-6 (p=0.008) were found in the latter patients. CD 14 expression in the moderate-severe CHF group was higher than in the mild-CHF group (p = 0.03) and was strongly related to stimulated IL-1 beta (r=0.62, p<0.0001), IL-6 (r=0.56, p=0.0002) and TNF-alpha (r=0.41, p=0.006) production. CONCLUSIONS: CD 14 expression and monocyte cytokine production, both unstimulated and after LPS stimulation, are increased in moderate-severe CHF when compared with mild CHF. These data suggest that circulating monocytes, possibly via increased CD 14 expression, may play a significant role in the immunologic dysbalance observed in advanced CHF.     

Effects of spermidine and p-cresol on polymorphonuclear cell apoptosis and function

Polymorphonuclear leukocytes (PMNs) from chronic kidney disease (CKD) patients display accelerated apoptosis and dysfunction, which may predispose CKD patients to infections. In this study, we investigated the effect of spermidine and p-cresol on apoptosis and function on PMN from healthy subjects. We measured the effect of spermidine and p-cresol on apoptosis, ROS production unstimulated and stimulated (S. aureus and PMA) and expression of CD95, caspase 3, and CD11b on PMN. After incubation with p-cresol and spermidine, we did not observe any changes in apoptosis, viability or expression of caspase 3 and CD95 in PMN from healthy subjects. PMN incubated for 10 minutes with spermidine demonstrated a significant reduction in spontaneous, S. aureus and PMA-stimulated ROS production. p-cresol induced a decrease in PMA-stimulated ROS production. Spermidine and p-cresol also induced a decrease in the expression of CD11b on PMN. Spermidine and p-cresol decreased the expression of CD11b and oxidative burst of PMN from healthy subjects and had no effect on PMN apoptosis and viability.     

Effects of lung preservation solutions on PMN activation in vitro

Polymorphonuclear leukocyte (PMN) activation and PMN-endothelial cell interactions may cause graft failure due to ischemia-reperfusion injury after lung transplantation. We investigated the effects of Euro-Collins solution (EC), low-potassium dextran solution (LPD), and EC plus pentoxifylline (EC-PTXF) on adhesion molecule (CD11b/CD18 and L-selectin) expression, chemotaxis, and oxidative burst of PMN. PMN from healthy human volunteers were incubated with EC, LPD, and EC-PTXF, and, in controls, without preservation solution. LPD exerted a suppressive effect on PMN chemotaxis as compared to EC (P < 0.05), but had no attenuating effect on the increase of CD11b/CD18, the shedding of L-selectin, and intracellular oxidant generation. EC-PTXF attenuated the expression of CD11b/CD18 and the oxidative burst as compared to EC alone (P < 0.05). These effects of LPD and PTXF on PMN function may contribute to successful organ preservation in transplantation. Received 22 June 98 Received after revision: 30 December 1998 Accepted: 19 January 99     

The effects of a novel resuscitation strategy combining pentoxifylline and hypertonic saline on neutrophil MAPK signaling

BACKGROUND: The combination of hypertonic saline (HS) and pentoxifylline (PTX) has been shown to synergistically downregulate neutrophil oxidative burst in vitro. We investigated the effects of HS/PTX on human neutrophil mitogen-activated protein kinase (MAPK) signaling and the role of Protein kinase A (PKA) in this process. METHODS: Isolated neutrophils were treated with PTX (2 mmol/L), HS10 (10 mmol/L above isotonicity), and HS40 (40 mmol/L above isotonicity) alone or in combination for determination of intracellular cyclic adenosine monophosphate (cAMP) concentrations. Human neutrophils were stimulated with f-methionyl-leucyl-phenylalanine (fMLP) (1 micromol/L) before the treatments above in both the presence and the absence of PKA inhibition for Western blot analysis of MAPK p38 and extracellular signal-related kinase 1/2 (ERK 1/2) phosphorylation. RESULTS: Concomitant exposure to HS/PTX results in an additive increase in intracellular cAMP. fMLP-induced ERK 1/2 phosphorylation was synergistically attenuated by HS/PTX. Both PTX and HS reduced p38MAPK phosphorylation. No additive effect was observed with combined treatment. Although PKA inhibition abrogated the effects of PTX, HS retained some capacity to attenuate MAPK phosphorylation. CONCLUSION: HS/PTX is more effective in attenuating neutrophil ERK signaling than either component alone, whereas both components alone or in combination produced comparable results with p38MAPK. Although PTX functions primarily through PKA activation, HS may suppress neutrophils through a partially PKA-independent mechanism.     

Hypertonic saline resuscitation: efficacy may require early treatment in severely injured patients

BACKGROUND: Activation of polymorphonuclear neutrophils (PMN) is a critical event leading to host tissue injury and organ damage after trauma. Hypertonic saline (HS) resuscitation prevents PMN activation in vitro and in animal models. Here, we studied how clinical parameters and timing requirements influence the efficacy of HS in suppressing PMN activation. MATERIALS AND METHODS: Twenty-six injured patients and 16 healthy volunteers were included as study subjects. To study how clinical parameters affect the efficacy of HS, whole blood samples from patients were collected 24 hours after admission, treated with HS and N-formyl-methionyl-leucyl-phenylalanine (fMLP), and PMN oxidative burst and degranulation were measured using flow cytometry. We studied the effect of timing on the ability of HS to inhibit PMN function by exposing blood of healthy volunteers to plasma samples from trauma patients before or after the addition of fMLP and HS. RESULTS: Age and gender did not significantly influence the effect of HS on PMN function. The suppressive effect of clinically relevant HS concentrations (20 mmol/L) on PMN oxidative burst correlated weakly with Sepsis Severity Score (SSS) and Acute Physiology and Chronic Health Evaluation II (APACHE II) score but not with the Injury Severity Score (ISS) or Multiple Organ Failure score (MOF). There was no correlation between any of these clinical scores and degranulation. HS was significantly less effective in suppressing oxidative burst of PMN from patients with ISS >10, APACHE II >5, MOF >0, or SSS >1 compared with patients with ISS < or =10, APACHE II < or =5, MOF = 0, or SSS < or =1. HS more effectively suppressed PMN activation when PMN were pretreatment with HS, whereas it was less effective on PMN previously primed in vivo or in vitro by adding trauma plasma. HS was ineffective on PMN previously stimulated in vitro with fMLP. CONCLUSIONS: Our data suggest that HS resuscitation may prevent PMN activation most effectively when patients are treated with HS early in the field.     

Whole-Blood Assay to Measure Oxidative Burst and Degranulation of Neutrophils for Monitoring Trauma Patients

AbstractBackground and Purpose: Polymorphonuclear neutrophils (PMNs) protect the host from invading microorganisms, but excessive PMN activation after trauma causes tissue injury. Rapid monitoring of PMN function is critical for the assessment of the inflammatory state of trauma patients. Here, the authors adapted two simple and rapid methods to measure oxidative burst and degranulation of human PMNs in whole blood to avoid potential interference of cell isolation procedures with the assessment of PMN function.Material and Methods: Heparinized blood was drawn from healthy volunteers or trauma patients, preincubated at 37 °C for 5 min, and stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Four assays for oxidative burst were tested: (1) cytochrome C; (2) homovanillic acid (HVA); (3) Amplex^® Red; and (4) flow cytometry with dihydrorhodamine 123 (DHR). PMN degranulation was assessed with flow cytometry using antibodies to: (1) CD11b/Mac-1 (CD18); (2) CD63; and (3) CD66b (CD67).Results: With the exception of the DHR method, all methods to measure oxidative burst were found to be unsuitable in whole blood due to interference of plasma proteins and hemoglobin with the fluorimetric or photometric readouts. By contrast, all degranulation methods were suitable for whole-blood studies. However, for the assessment of formyl peptide-induced degranulation, anti-antibodies to CD11b/Mac-1 and CD66b were up to five times more sensitive than antibodies to CD63. Thus, the degranulation and DHR methods were optimized for increased sensitivity, speed, and specificity and their usefulness to measure PMN function in trauma patients was tested.Conclusion: The whole-blood methods based on flow cytometry with DHR, anti-CD11b/Mac-1, and anti- CD66b are rapid, simple, and reliable techniques to assess PMN function for trauma research.     

Effect of medium tonicity and dextran on neutrophil function in vitro

BACKGROUND: Multiple investigations have demonstrated that hypertonic saline (HS) diminishes the response of polymorphonuclear leukocytes (PMNs) to stimulation. Recent meta-analysis suggests that hypertonic saline in dextran (HSD) is clinically superior to HS. No work to date has examined the effect of added dextran on this immunomodulatory property. METHODS: Human PMNs were exposed to media of varying osmolarity (220-360 mOsm/L) and stimulated with f-met-leu-phe with or without dextran present in the medium. Cell volume, respiratory burst, PMN aggregation, and beta(2)-integrin (CD18) expression were measured. RESULTS: Stimulation with f-met-leu-phe increased cell volume, respiratory burst, aggregation, and CD18 expression. The increases in cell volume, respiratory burst, and aggregation were significantly attenuated by exposure to hypertonic medium. The addition of dextran to the media did not change the results. CONCLUSION: The alterations in PMN function associated with HS are not changed or attenuated by the addition of dextran, suggesting that the clinically superior HSD may have effects similar to HS in mitigating the tissue damage associated with activated PMNs.     

In vitro study of the potential role of guanidines in leukocyte functions related to atherogenesis and infection

BACKGROUND: The blunted immune response upon stimulation in chronic renal failure (CRF) is often coupled to a baseline inflammatory status which has been related to atherogenesis. Uremic biologic fluids and several specific uremic retention solutes alter cell-mediated immune responses, as well as the interaction of calcitriol with the immune system. METHODS: The present study evaluated the influence of different guanidino compounds on DNA synthesis, chemiluminescence production, and CD14 expression of undifferentiated and calcitriol-differentiated HL-60 cells. In a second setup, these guanidino compounds were evaluated for their specific effect on normal human leukocyte oxidative burst activity and tumor necrosis factor-alpha (TNF-alpha) expression. RESULTS: First, several guanidino compounds elicited proinflammatory effects on leukocytes. Methylguanidine and guanidine stimulated the proliferation of undifferentiated HL-60 cells and the antiproliferative effect of calcitriol (P < 0.05) was neutralized in the presence of methylguanidine (P < 0.05) and guanidinosuccinic acid (P < 0.05). The phorbol-myristate-acetate (PMA)-stimulated chemiluminescence production of the calcitriol differentiated HL-60 cells was enhanced in the presence of guanidine (P < 0.05). Methylguanidine and guanidinoacetic acid enhanced the lipopolysaccharide (LPS)-stimulated intracellular production of TNF-alpha by normal human monocytes (P < 0.05). Second, several guanidino compounds inhibited the function of leukocytes if they were activated. The PMA-stimulated chemiluminescence production of the calcitriol differentiated HL-60 cells was inhibited by the presence of methylguanidine (P < 0.05), guanidinoacetic acid (P < 0.05) and guanidinosuccinic acid (P < 0.05). After incubation of whole blood in the presence of methylguanidine, the Escherichia coli stimulated oxidative burst activity of the granulocyte population was significantly inhibited (P < 0.05). In addition, guanidinosuccinic acid had an inhibitory effect on the LPS-stimulated intracellular production of TNF-alpha by human monocytes (P < 0.01). CONCLUSION: Guanidino compounds exert proinflammatory as well as anti-inflammatory effects on monocyte/macrophage function. This could contribute to the altered prevalence of cardiovascular disease and propensity to infection in patients with CRF.     

Ischemia activates neutrophils but inhibits their local and remote diapedesis.

Hindlimb ischemia and reperfusion results in local limb and distant lung injury. This study tests whether the mechanism of injury is by ischemia mediated polymorphonuclear leukocyte (PMN) activation and diapedesis. Anesthetized rabbits were subjected to three hours of hindlimb ischemia (n = 8) or sham ischemia (n = 4). PMN derived solely from the reperfused ischemic limb and assayed flow cytometrically displayed an oxidative burst of 135 /- 8 fentamoles dichlorofluorescein (fmDCF)/cell compared to preischemc levels of 74 +/- 14 fmDCF/cell (p less than 0.05). Additional aliquots of isolated neutrophils were treated with phorbol myristate acetate (PMA) 10(-7) M. In contrast to a 162% increase in oxidative burst before ischemia, neutrophils at ten minutes of reperfusion had an enhanced response to PMA of 336% (p less than 0.05). Plasma collected from the ischemic hindlimb at ten minutes of reperfusion when introduced into an abraded skin chamber or intratracheally induced diapedesis in nonischemic animals. PMN accumulations in the skin chamber were 1636 +/- 258 PMN/mm3 after three hours (n = 8) compared to 63 +/- 18 PMN/mm3 induced by sham plasma (n = 4, p less than 0.05). Introduction of ischemic plasma intratracheally into a lobar bronchus (n = 4) induced PMN accumulations after three hours, measured by bronchoalveolar lavage fluid of 19 +/- 2 X 10(4) PMN/mm3 compared to 5 +/- 1 X 10(4) PMN/mm3 with sham plasma (n = 4, p less than 0.05). Diapedesis was completely prevented (0-3 PMN/mm3, p less than 0.05) by introducing ischemic plasma into skin chambers in animals whose hindlimbs had been made ischemic (n = 6) or into chambers located on skin regions that had been previously made ischemic (n = 6). Similarly after hindlimb ischemia, lavage of the lung with ischemic plasma yielded few PMN 0-3/mm3 (p less than 0.05). These data indicate that ischemia and reperfusion lead to generation of a circulating component in plasma that causes an oxidative burst in PMN and inhibits their diapedesis but promotes diapedesis when applied extravascularly to a naive animal.     

Phosphatidylinositol 3-kinase controls antineutrophil cytoplasmic antibodies-induced respiratory burst in human neutrophils

Antineutrophil cytoplasmic antibodies (ANCA) activate human polymorphonuclear neutrophils (PMN) primed with tumor necrosis factor alpha (TNF-alpha) in vitro. Phosphatidylinositol 3-kinase (PI3-K) and the protein-serine/threonine kinase Akt have been implicated in the control of the phagocyte respiratory burst. The hypothesis that PI3-K controls the ANCA-induced respiratory burst was tested. TNF-alpha-primed PMN were stimulated with a monoclonal antibody to myeloperoxidase (MPO) and with PR3- and MPO-ANCA, respectively. Akt activation was assessed with phospho-specific antibodies. Superoxide release was measured with ferricytochrome. ANCA antigen translocation was assessed by fluorescence-activated cell sorter. The effect of TNF-alpha and MPO-ANCA on Akt signaling was studied with immunoprecipitation and glutathione S-transferase pull-down assays. Western blotting revealed rapid transient Akt phosphorylation during TNF-alpha priming and a second phosphorylation after ANCA. PI3-K inhibition by LY294002 blocked both Akt phosphorylation and superoxide generation. A total of 20 +/- 3 nmol O(2)(-)/0.75 x 10(6) PMN/45 min was released after stimulation with PR3-ANCA. LY294002 (5 microM) decreased this amount to 0.3 +/- 2.6 nmol (n = 10, P < 0.05); the MPO-ANCA values were 23 +/- 3 versus 1.6 +/- 3.6 (n = 10, P < 0.05). p38 MAPK inhibition with 10 microM SB202190 that also decreased ANCA-induced superoxide generation prevented S473 phosphorylation of Akt in response to TNF-alpha and to ANCA. However, SB202190 but not LY294002 abrogated TNF-alpha-mediated ANCA antigen surface translocation, demonstrating that superoxide generation and ANCA antigen translocation proceed by separate mechanisms. Akt, PAK1, and Rac1 existed as cytosolic complex in resting PMN. TNF-alpha stimulation increased association of PAK1 with Akt. An MPO monoclonal antibody did not alter the Akt signaling complex further. The data demonstrate the importance of PI3-K for the ANCA-induced PMN oxidant production.     

Phosphodiesterase inhibition decreases nuclear factor-kappaB activation and shifts the cytokine response toward anti-inflammatory activity in acute endotoxemia

BACKGROUND: In sepsis, activation of inflammatory cells and excessive production of proinflammatory cytokines leads to tissue injury, multiple organ failure, and death. We postulated that attenuation but not complete abrogation of hyperinflammation is of clinical benefit in sepsis. Because pentoxifylline (PTX) is known to decrease tumor necrosis factor (TNF)-alpha production and to increase anti-inflammatory cytokine synthesis, we tested the hypothesis that PTX treatment would change the pro- and anti-inflammatory balance and decrease mortality in a murine model of acute endotoxemia. In addition, we investigated the effects of PTX on nuclear factor (NK)-kappaB activation using lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs) as a model. METHODS: Sprague-Dawley rats were treated with intravenous saline (sham), LPS (lipopolysaccharide, Escherichia coli serotype 0111:B4, 5 mg/kg), and concomitant injection of LPS + PTX (25 mg/kg). Four- and 24-hour plasma TNF-alpha and interleukin (IL)-10 levels, 4-hour white cell count, and 24-hour mortality rates were assessed. The IL-10/TNF-alpha ratio was also calculated. Human PBMCs were stimulated with LPS (10 microg/mL) and exposed to PTX (20 mM) concomitantly or 15 minutes after LPS stimulation. I-kappaB phosphorylation by Western blot and NF-kappaB nuclear translocation by electrophoretic mobility shift assay were assessed. RESULTS: PTX markedly down-regulates TNF-alpha production. IL-10 levels at 4 hours were up-regulated in both LPS and PTX + LPS-treated animals; however, levels were higher in the LPS groups, which paralleled high TNF-alpha levels. In contrast, IL-10 levels at 4 and 24 hours in PTX + LPS-treated animals remained constant, whereas in LPS-treated animals, IL-10 levels at 24 hours were markedly decreased. A shift in the internal milieu balance toward anti-inflammatory activity was confirmed by the calculation of the IL-10/TNF-alpha ratio. These changes were not related to changes in the number of circulating leukocytes. The 24-hour mortality rate was 50% in the LPS group and nil in PTX-treated animals. In LPS-stimulated PBMCs, PTX markedly decreases I-kappaB phosphorylation and NF-kappaB nuclear translocation. CONCLUSION: PTX enhances anti-inflammatory activity and decreases mortality in acute endotoxemia. PTX may be an important adjunct to therapies aiming to modulate the inflammatory response in sepsis.