Untreated intra-abdominal sepsis: lack of synergism between polymorphonuclear leukocyte (PMN) complement receptors CR1/CR3 and IgG receptor FcRIII

We have examined the effects of untreated intra-abdominal sepsis on polymorphonuclear leukocyte (PMN) phagocytosis. Phagocytosis was studied in a receptor-specific fashion looking at the interrelationship between FcRIII-, complement receptor (CR1)-, and complement receptor 3 (CR3)-mediated phagocytosis. Twelve swine underwent either sham laparotomy (n = 5) or laparotomy with cecal ligation and incision (n = 7) to induce intra-abdominal sepsis. PMN phagocytosis was assayed on POD 1, 4, and 8. In animals undergoing sham laparotomy, FcRIII-mediated phagocytosis was less than 10% on all days and was augmented with the addition of C3b or C3bi to the target particles (FcR + CR1 or FcR + CR3 greater than FcR alone). In animals undergoing cecal ligation and incision, baseline FcRIII-mediated phagocytosis increased between POD 1 and 4 and fell between POD 4 and 8. No increase in phagocytosis was seen on POD 4 or 8 with the addition of C3b or C3bi to the target particles (FcR + CR1 or FcR + CR3 = FcR alone). Preligation of the FcRIII but not FcRII or FcRI receptor with a monoclonal antibody (3G8) markedly reduced phagocytosis in the animals with intraabdominal sepsis.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Priming of neutrophil [Ca2+]i signaling and oxidative burst by human fracture fluids

BACKGROUND: Patients with major fracture/soft-tissue injuries are at risk for adult respiratory distress syndrome after secondary infection. Fracture fluids (FF) are rich in neutrophil (PMN) -specific chemokines such as interleukin-8. PMN respond to both interleukin-8 and bacterial stimuli with calcium ([Ca2+]i) fluxes, which can initiate respiratory burst (RB). We hypothesize that small amounts of FF entering the circulation could exaggerate PMN [Ca2+]i and RB responses, potentially increasing the risk of adult respiratory distress syndrome. METHODS: FF were obtained from 10 patients at open fixation of the femur 2 to 5 days postinjury. Volunteer PMN were isolated and loaded with fura dye. PMN were preincubated either in 30% autologous plasma (AP)/70% buffer, or in 5% FF/25% AP/70% buffer. Cells were resuspended in buffer with 1,2,3-dihydrorhodamine and stimulated with low-dose n-formyl-methionyl-leucyl-phenylalanine (fMLP). [Ca2+]i was assayed by fura fluorescence at 505 nm after excitation at 340/380 nm. RB was assessed by 1,2,3-dihydrorhodamine fluorescence at 530 nm after 488 nm excitation. RESULTS: PMN basal [Ca2+]i was higher after FF incubation than AP incubation (94+/-12 vs. 61+/-9 nmol/L, p = 0.0002). Peak [Ca2+]i response to fMLP was 475+/-47 nmol/L after FF but only 356+/-22 nmol/L after AP (p = 0.01). Two hundred seconds after fMLP, [Ca2+]i remained higher after FF (172+/-17 vs. 145+/-9 nmol/L, p = 0.04). Basal RB was slightly higher after FF than AP (13.4+/-0.3 vs. 11.3+/-0.3 units, p = 0.051) as was the maximal rate of extracellular oxidant release (1.10+/-0.17 vs. 0.76+/-0.16 units/s, p = 0.004) and total oxidant production (42.5+/-0.8 vs. 31.7+/-0.8 units, p = 0.006). CONCLUSION: Small amounts of FF in plasma can exaggerate PMN [Ca2+]i flux and RB responses to subsequent bacterial stimuli. These findings are consistent with the hypothesis that release of FF into the circulation primes PMN and, thus, may predispose to adult respiratory distress syndrome. Such PMN priming events might have important implications for both the operative and medical management of patients with major fractures.     

Store-operated calcium channel inhibition attenuates neutrophil function and postshock acute lung injury

BACKGROUND: A wide variety of neutrophil (PMN) functions are regulated by cytosolic calcium concentration. Calcium channel blockade might therefore decrease postshock inflammation but could also limit important cardiovascular compensations. PMN Ca2+ entry occurs, however, through store-operated calcium entry (SOCE) channels rather than the voltage operated (L-type) channels that regulate cardiovascular tone. We hypothesized that SOCE inhibition might suppress postshock PMN activation, lessening lung injury without compromising cardiovascular performance. METHODS: Human PMNs were treated in vitro with N-propargyl-nitrendipine (MRS1845 [MRS]) a dihydropyridine Ca2+ channel blocker with relative specificity for SOCE channels. Calcium flux was measured by fura fluorescence. Chemotaxis was studied in modified Boyden chambers. Respiratory burst was studied by dihydrorhodamine fluorescence. Exploratory studies were then performed where rats were subjected to trauma and hemorrhagic shock (T/HS) (laparotomy, then hemorrhage to a mean arterial pressure of 30-40 mm Hg for 90 minutes) after pretreatment with MRS or vehicle given intraperitoneally at laparotomy. In vivo PMN CD11b expression was then assayed by flow cytometry and lung injury was assessed as percentage Evans blue dye leak 3 hours after resuscitation. The shed blood volume required to achieve standardized hypotension was measured. RESULTS: In vitro, MRS suppressed human PMN SOCE without affecting calcium store release; it suppressed chemotaxis (60 +/- 6 vs. 150 +/- 15 x 10(3) PMNs/well, p = 0.002) and suppressed respiratory burst (62 +/- 11% vs. 100%, p < 0.05) at IC50 concentrations similar to those needed to suppress SOCE. In subsequent in vivo rat studies, MRS decreased postshock PMN CD11b expression from 397 +/- 93 to 268 +/- 39 MFU mean flourescent units (p < 0.05) and decreased lung Evans blue dye permeability from 8.1 +/- 1.9% to 3.4 +/- 0.1% (p < 0.05). MRS had no noticeable effect on the relationship between blood pressure and blood loss, with shed blood volume remaining almost identical (26 +/- 2 mL/kg vs. 27 +/- 3 mL/kg, p = not significant). CONCLUSION: Modulation of PMN Ca2+ entry by means of selective SOCE channel inhibition attenuates PMN inflammatory responses in vitro. In vivo, SOCE channel blockade attenuates trauma and hemorrhagic shock-induced PMN priming and lung injury without gross evidence of hemodynamic side effects. The relative specificity of SOCE channel blockade for "nonexcitable" cells such as PMNs may make it a valuable form of chemoprophylaxis for the inflammatory consequences of hemorrhagic shock in trauma patients.     

Fracture hematoma is a potent proinflammatory mediator of neutrophil function

BACKGROUND: 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). METHODS: 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. RESULTS: 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). CONCLUSION: These effects, although beneficial at the site of injury in the context of antibactericidal function, may cause PMN-mediated tissue injury systemically.     

Delayed cyclo-oxygenase blockade reduces the neutrophil respiratory burst and plasma tumor necrosis factor levels in sepsis-induced acute lung injury.

Ibuprofen pretreatment attenuates the enhanced neutrophil (PMN) respiratory burst and reduces increased plasma tumor necrosis factor (TNF) activity in porcine sepsis-induced acute lung injury (ALI). These septic responses have been linked to increased alveolar-capillary membrane (ACM) permeability. This study was designed to establish whether delayed ibuprofen treatment would have the same effect and to examine the relationship between PMN oxidant generation and TNF. Three groups of anesthetized, ventilated pigs (15-25 kg) were used. Group Ps received Pseudomonas aeruginosa (5 x 10(8) CFU/mL at 0.3 mL/20 kg/min) for one hour IV; The control group (Con) received 0.9% NaCl. Group D-Ibu received ibuprofen 12.5 mg/kg as a delayed bolus at 30 minutes and again at 120 minutes after Ps. Protein (BAL-P, microgram/mL) in harvested bronchoalveolar lavage fluid and extravascular lung water (EVLW, mL/kg) were used to estimate the integrity of the ACM. Superoxide anion (O2-) generation (ferricytochrome c reduction) from circulating PMNs and plasma TNF activity (L929 fibroblast bioassay) were measured. The EVLW increased significantly (p less than 0.05), as did BAL-P (p less than 0.01), in the P. aeruginosa-treated animals at 300 minutes. These increases were abolished in Group D-Ibu: EVLW, 6.6 +/- 1.0 baseline vs. 14.6 +/- 2.6 Ps 300 vs. 6.8 +/- 0.9 D-Ibu 300; BAL-P, 175 +/- 28 baseline vs. 984 +/- 186 Ps 300 vs. 284 +/- 42.8 D-Ibu 300. Both enhanced PMN oxidant activity and increased plasma TNF activity were significantly attenuated by delayed ibuprofen treatment. These data support the efficacy of the nonsteroidal anti-inflammatory drug, ibuprofen, when used after the onset of a septic stimulus.     

The preischemic combination of the sodium-hydrogen exchanger inhibitor cariporide and the adenosine agonist AMP579 acts additively to reduce porcine myocardial infarct size

BACKGROUND: We tested whether the combination of two known cardioprotective agents, the type-1 sodium-hydrogen exchanger inhibitor cariporide plus the adenosine A(1)/A(2a) receptor agonist AMP579 ([1S-[1a,2b,3b, 4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methylpropyl]amino]-[(3)H]-imidazo[4,5-b]pyridyl-3-yl]cyclopentane carboxamide), acted additively to reduce myocardial infarct size. STUDY DESIGN: Pigs underwent 1 hour of coronary artery occlusion and 3 hours reperfusion. Vehicle-treated controls were compared with animals treated before ischemia with low-dose and high-dose cariporide and AMP579, and low-dose cariporide plus AMP579. The effects of both agents, alone and in combination, were also tested in isolated porcine polymorphonuclear neutrophils (PMNs). The PMN respiratory burst was induced with phorbol 12-myristate 13-acetate and quantified by the increase in 2',7'-dichlorofluorescein fluorescence, measured by flow cytometry. RESULTS: Infarct size in the control pigs was 65 +/- 1% of the area at risk. Cariporide dose-dependently reduced infarct size to 39 +/- 2% and 24 +/- 3% in the low- and high-dose groups, respectively. Infarct size was 54 +/- 3% in the low-dose AMP579 group and 47 +/- 3% with high dose. The combination of low doses of cariporide and AMP579 reduced infarction to 25 +/- 6% of the area at risk. In the PMN studies, cariporide and AMP579 alone had no effect on 2',7'-dichlorofluorescein fluorescence, but the combination of the two agents reduced the PMN 2',7'-dichlorofluorescein increase to 79 +/- 5% of the vehicle control response. CONCLUSIONS: The preischemic combination of low doses of cariporide and AMP579 decreased myocardial infarct size more than either agent used alone in low concentration, indicating an additive effect of the two agents. Given the effects that cariporide plus AMP579 combination exerted on PMN activity, it appears that this combination has the potential to reduce PMN-mediated effects during myocardial reperfusion.     

Xenon preserves neutrophil and monocyte function in human whole blood

PURPOSE: Most volatile anesthetics are known to inhibit the oxidative and phagocytic function of neutrophils. In the present study, we investigated the effect of xenon on phagocytosis and respiratory burst activity of neutrophils and monocytes in human whole blood. METHODS: Heparinized whole blood from 22 healthy volunteers was incubated for 60 min in the presence of 65% xenon. Sixty-five percent nitrous oxide was used as a positive control to prove the reliability of our in vitro system. Phagocytosis of fluorescein isothiocyanate labelled, opsonized Escherichia coli (E. coli) by neutrophils and monocytes was measured using flow cytometry. After induction with either N-formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol-12-myristate-13-acetate or opsonized E. coli, respiratory burst activity was assessed by measuring the oxidation of dihydrorhodamine 123 to rhodamine 123 with a flow cytometer. RESULTS: Exposure of human whole blood to xenon increased the percentage of neutrophils showing phagocytosis (94 +/- 3% vs 92 +/- 4%; P < 0.01), and the amount of ingested bacteria (P < 0.01). Respiratory burst activity in neutrophils and monocytes was not affected by xenon. Nitrous oxide significantly reduced the percentage of neutrophils showing respiratory burst after FMLP stimulation. Furthermore, E. coli-induced stimulation resulted in a decreased number of reacting neutrophils (84 +/- 15% vs 95 +/- 5%; P < 0.05) and monocytes (70 +/- 22% vs 83 +/- 11%; P < 0.05) as well as a reduced production of hydrogen peroxide in both cell lines compared to control. CONCLUSION: In contrast to nitrous oxide, xenon preserves neutrophil and monocyte antibacterial capacity in vitro.     

Expression of complement 3 receptors (CR1 and CR3) on neutrophils and erythrocytes in patients with IgA nephropathy

Expression of C3 receptors (CR1 and CR3) on neutrophils (PMN) was measured in 26 patients with IgA nephropathy (IgA N), 17 normal persons, and 8 patients with non-IgA glomerulonephritis (non-IgA GN) by fluorescence activated cell sorting after labeling with monoclonal antibodies. Mean channel fluorescence for both CR1 and CR3 on PMN was significantly higher in IgA N patients than in either control group (p less than 0.01). (CR1 mean +/- SD 42.5 +/- 10.4 in IgA N, 31.7 +/- 11.5 in normal controls, 27.8 +/- 9.0 in non-IgA GN; CR3 94.0 +/- 16.5, 75.0 +/- 16.6 and 76.7 +/- 15.6, respectively.) No differences were found between the two control groups or between IgA N patients with normal and impaired renal function. These results imply that PMN are activated in IgA N patients. The expression of CR1 and CR3 of PMN may be upregulated by immune complexes (ICs), enhancing both phagocytosis of C3b- or iC3b-coated particles, and absorptive pinocytosis of soluble ICs containing C3b or iC3b. Erythrocyte CR1 and CR3 expression was measured by ELISA and found to be slightly but significantly lower in IgA N patients than in the other 2 groups (CR1 85.3 +/- 28.4 in IgA N, 113.1 +/- 28.8 in normal controls, 109.4 +/- 28.2 in non-IgA GN, p less than 0.02; CR3 80.9 +/- 20.0, 100.4 +/- 19.9, 101.2 +/- 24.9, respectively, p less than 0.05).     

Polymorphonuclear leukocyte function during hemodialysis: relationship to complement activation

Phagocytosis, H2O2 production, and C3bi receptor (CR3) expression by polymorphonuclear leukocytes (PMN) obtained from patients before, during, and after a hemodialysis treatment were evaluated by flow microfluorometry. The results were compared to changes in plasma levels of C3ades Arg and C5ades Arg. Prior to hemodialysis C3ades Arg and C5ades Arg levels, CR3 expression and phagocytosis were not different from normal controls. However, both basal and phagocytosis-induced H2O2 production were increased. C3ades Arg and C5ades Arg were increased after 15 min of dialysis; this was accompanied by transient but significant reductions in PMN count and phagocytosis and increased CR3 expression. No changes in basal or stimulated H2O2 production were observed. We conclude that PMN of hemodialysis patients are primed for an enhanced respiratory burst before dialysis is initiated. Dialysis-induced complement activation after the initiation of dialysis does not further stimulate H2O2 production or enhance the response to phagocytosis. However, complement activation may cause leukopenia and CR3 expression.     

Intraabdominal sepsis: effects on polymorphonuclear leukocyte Fc receptor-mediated phagocytosis

In vitro studies have shown that phagocytic cells are capable of undergoing activation in response to inflammatory signals and that the activation process is quite complex. A relationship between polymorphonuclear leukocyte (PMN) Fc receptor-mediated phagocytosis and oxidative metabolism has been seen in humans. We have sequentially examined circulating polymorphonuclear leukocytes (PMNs) from a total of 13 postoperative swine with either no sepsis, untreated intraabdominal sepsis, or treated intraabdominal sepsis to determine phagocytic activity over 8 postoperative days (POD). Products of the oxidative burst (i.e., myeloperoxidase) reduced the phagocytic activity of nonseptic swine PMN. Phagocytic activity was augmented by inhibiting the nonseptic swine oxidative burst with 10 mM sodium azide (an inhibitor of myeloperoxidase). In swine with untreated intraabdominal sepsis, PMN Fc receptor-mediated phagocytosis exhibited a biphasic response. An initial (between POD1 and POD4) increase in PMN function was followed by a subsequent (between POD4 and POD8) decrease in PMN function. Partial preservation of phagocytic capability was seen when swine were reexplored on POD4 and had their intraabdominal sepsis treated. These results indicate that (1) as in humans, nonseptic swine PMN Fc receptor-mediated phagocytosis is augmented by inhibition of the PMN respiratory burst; (2) untreated intraabdominal sepsis produces an initial increase and subsequent decrease in PMN Fc receptor-mediated phagocytosis; (3) early treatment of intraabdominal sepsis results in partial restoration of PMN Fc receptor-mediated phagocytosis.     

A single dose of endotoxin activates neutrophils without activating complement

Both complement (C) and neutrophils (PMN) are activated in critically ill patients. To evaluate the role of endotoxin in this response, we studied C activation products and PMN cell surface receptors in seven normal subjects before and after endotoxin (USRef 20 U/kg) or saline solution administered on separate occasions. By 4 hours, with endotoxin only, all subjects had myalgia, headache, an increase in body temperature and heart rate, and leukocytosis that returned to normal by 24 hours. At the same time, PMN cell surface receptors for the complement opsonin C3b increased, as measured by indirect immunofluorescence, rising to 251 +/- 44% of baseline by 4 hours (p less than 0.01) and remaining elevated at 24 hours (237 +/- 16%, p less than 0.01). PMN receptors for iC3b increased to 308 +/- 49% of baseline by 4 hours (p less than 0.02) and returned to normal by 24 hours. There was no change in plasma of C3a desArg, C4a desArg, and C5a desArg (4 hours: mean C3a: 153.4 +/- 11.5 ng/ml versus 176.2 +/- 16.2 ng/ml for saline solution, p = ns; C4a: 159.6 +/- 32 ng/ml versus 151.4 +/- 21 ng/ml, p = ns; C5a: undetectable). To confirm the lack of C activation, we examined PMN chemotaxis (CTX) to C5a for any impairment caused by prior in vivo exposure to C5a. CTX to C5a was unaffected (4 hours: 109% +/- 22% of normal versus 114% +/- 10% for saline solution, p = ns). PMN CTX to formyl-methionyl-leucine-phenylalanine and PMN phagocytosis and killing of S. aureus were also unaffected by endotoxin. Thus, a single dose of endotoxin produced a subjective febrile illness and precipitated sustained PMN activation as indicated by increased PMN cell surface complement receptor number in the absence of C activation.     

Inhibitory effects of S-(-) and R-(+) bupivacaine on neutrophil function

BACKGROUND: Local anesthetics inhibit migration, enzyme release and superoxide anion generation of polymorphonuclear leukocytes (PMN). Due to their ability to phagocytose and kill bacteria PMN represent a major defense mechanism in the circulating blood. In this study we determined the influence of racemic bupivacaine and its enantiomers on neutrophil phagocytic activity, oxidative burst as well as surface expression of complement and Fcgamma receptors. METHODS: Venous blood was pre-incubated with different concentrations of either racemic bupivacaine, R-(+) or S-(-) bupivacaine. Fluoresceine isothiocyanate (FITC)-labeled antibodies against Fcgamma receptor III (CD16), complement receptor 1 (CD35) and complement receptor 3 (CD11b) were used to determine surface receptor expression. Phagocytic activity was measured by ingestion of FITC-labeled vital Staphylococcus aureus. Oxidative burst was determined by conversion of nonfluorescent dihydrorhodamine 123 into fluorescent rhodamine 123. Fluorescent intensity of each sample was determined by flow cytometry. RESULTS: Racemic bupivacaine inhibited surface receptor expression, phagocytosis, and oxidative burst in a time- and concentration-dependent manner. Although the S-(-) enantiomer exerted significantly less inhibitory action on neutrophil function compared to R-(+) and racemic bupivacaine, these effects were small compared to the overall changes. CONCLUSION: These findings suggest that bupivacaine impairs surface receptor expression and may thereby contribute to reduced phagocytic activity and oxidative burst. Enantiomer-specific effects of bupivacaine may play a minor role in the inhibition of these leukocyte functions.     

Phagocytic function of polymorphonuclear leukocytes and the RES in endotoxemia

The reticuloendothelial system (RES) and the polymorphonuclear leukocytes (PMNs) are thought to play a major role in defense against sepsis. Disturbances in the function of these two phagocytic systems during a septic event is associated with the development of lung capillary injury. Endotoxemia is said to lead to similar changes. Our study examined the function of the RES and PMNs after bolus injection of endotoxin (2 micrograms/kg BW) in a standardized sheep model. For up to 24 hr after endotoxin, blood samples were drawn and PMN function was followed by chemiluminescence, chemotaxis, and adherence as well as the phagocytosis and killing of bacteria. RES function was determined by the blood clearance of a labeled Tc99 colloid. We found an increase of RES clearance directly after endotoxin. Chemotaxis, phagocytosis, and killing were reduced. Adherence was increased. Chemiluminescence peak maximum (CLPM), representing the metabolic activity of the PMNs, was initially increased but shortly thereafter showed a significant decline (at 1 hr: 0.52 +/- 0.13 X 10(6) cpm with P less than 0.05 compared to baseline). The chemiluminescence peak time (CLPT), a measure of membrane receptor function, was significantly reduced (10.0 +/- 2.2 min with P less than 0.001 compared to baseline). Endotoxin led to a reduction of intracellular PMN functions (phagocytosis, killing, CLPM) within 1 hr. Membrane localized functions (adherence, CLPT) were increased. The changes in PMN function might be the reason for the development of lung capillary injury, in spite of undisturbed RES clearance.     

Neutrophil activation after burn injury: contributions of the classic complement pathway and of endotoxin

We attempt to elucidate the mechanisms of neutrophil (PMN) activation after burn injury. We previously reported prolonged elevations of PMN cell surface complement (C) opsonin receptor levels after burn trauma with a corresponding period of depressed PMN chemotaxis to C5a, which suggests that the C product, C5a, was responsible for PMN activation. However, a lack of direct correlation of C activation with C receptor levels soon after injury raised the possibility of a second PMN-activating substance. We therefore investigated the effect of endotoxin (LPS) on the expression of the C receptors (CR1 and CR3) by normal human PMNs. Concentrations from 0 to 50 ng/ml of LPS 026:B6 caused a dose response increase in the PMN surface expression of CR1 and CR3 as assessed by monoclonal antibody binding and indirect immunofluorescence. The relative CR1-dependent fluorescence rose from a mean of 50 to 385 and CR3 from 50 to 300. Chelation by ethylenediaminetetra acetic acid (EDTA) did not influence this dose response, thus ruling out the possibility of C activation by LPS--an inference supported by the lack of complement activation observed with these concentrations of LPS in normal serum. A similar dose response was obtained in the absence of other cell types or serum, which implies a direct effect that mimicked that of C5a. To determine the mechanism of the later, prolonged C activation after burn injury, we next examined C activation products in 22 patients with burn injuries. Elevations of plasma C3a desArg were present and persisted for 50 days. Elevations were at maximum levels on days 9 through 13 postburn (mean +/- standard error of mean [SEM], 496 +/- 47 ng/ml versus normal 113 +/- 32; p less than 0.01). These were accompanied by elevations of C4a desArg (917 +/- 154 ng/ml versus normal 424 +/- 50; p less than 0.01), which are indicative of classic pathway activation. Finally, we examined PMN function, phagocytosis and percentage killing of Staphylococcus aureus, and found PMN function to be unaltered in the 22 patients. Thus PMN activation after burn injury appears to be caused by LPS soon after injury and by C5a later after injury and affects only selected PMN functions.     

Administration of a tumor necrosis factor inhibitor at the time of myocardial infarction attenuates subsequent ventricular remodeling.

BACKGROUND: Tumor necrosis factor (TNF) causes myocardial extracellular matrix remodeling and fibrosis in myocardial infarction and chronic heart failure models. Pre-clinical and clinical trials of TNF inhibition in chronic heart failure have shown conflicting results. This study examined the effects of the administration of a TNF inhibitor immediately after myocardial infarction on the development of heart failure. METHODS: Lewis rats underwent coronary artery ligation and then received either intravenous etanercept (n = 14), a soluble dimerized TNF receptor that inhibits TNF, or saline as control (n = 13). Leukocyte infiltration into the infarct borderzone was evaluated 4 days post-ligation in 7 animals (etanercept = 4, control = 3). After 6 weeks, the following parameters were evaluated in the remaining animals: cardiac function with a pressure-volume conductance catheter, left ventricular (LV) geometry, and borderzone collagenase activity. RESULTS: Etanercept rats had significantly less borderzone leukocyte infiltration 4 days post-infarction than controls (10.7 +/- 0.5 vs 18.0, +/-2.0 cells/high power field; p < 0.05). At 6 weeks, TNF inhibition resulted in significantly reduced borderzone collagenase activity (110 +/- 30 vs 470 +/- 140 activity units; p < 0.05) and increased LV wall thickness (2.1 +/- 0.1 vs 1.8 +/- 0.1 mm, p < 0.05). Etanercept rats had better systolic function as measured by maximum LV pressure (84 +/- 3 mm Hg vs 68 +/- 5 mm Hg, p < 0.05) and the maximum change in left ventricular pressure over time (maximum dP/dt) (3,110 +/- 230 vs 2,260 +/- 190 mm Hg/sec, p < 0.05), and better diastolic function as measured by minimum dP/dt (-3,060 +/- 240 vs -1,860 +/- 230 mm Hg/sec; p < 0.05) and the relaxation time constant (14.6 +/- 0.6 vs 17.9 +/- 1.2 msec; p < 0.05). CONCLUSIONS: TNF inhibition after infarction reduced leukocyte infiltration and extracellular matrix turnover and preserved cardiac function.     

Inflammatory cell function in young rodents with experimental cholestasis: investigations of functional deficits, their etiology, and their reversibility

Children with cholestasis are susceptible to infective complications. This may be attributable to impaired host defense. We postulated that cholestasis affects systemic polymorphonuclear leukocyte (PMN) function by impeding chemotaxis, phagocytosis, and superoxide release, which are all critical in eliciting an adequate immune response. Sprague Dawley rats (225 g) were assigned to three groups: bile duct ligated (BDL), sham (SH), and normal control (NC). On day 21 after operation, PMN and sera were isolated. Chemotactic response to C5a and FMLP (formyl-methionyl-leucyl-phenylalanine), superoxide release, and phagocytic uptake of 14C-labeled Staphylococcus aureus were performed on pooled PMN samples. Results were expressed as mean +/- SD. Serum bilirubin at day 21 was 6.3 +/- 2.9 v 0.1 +/- 0.1 and 0.1 +/- 0 mg/dL (P less than .01) in BDL, SH, and NC groups, respectively. Kinetic studies of PMN phagocytosis demonstrated impaired 14C S aureus uptake by BDL neutrophils at 60 (P less than .05), 90 (P less than .05), and 120 minutes (P less than .05) compared with SH and NC groups. No differences in PMN chemotactic response to C5a and FMLP was observed in BDL, SH and NC groups (43 +/- 14 v 40 +/- 12 and 33 +/- 1, and 43 +/- 20 v 43 +/- 14 and 28 +/- 1 cell per field, respectively). Zymosan stimulated superoxide release did not differ between groups (14.3 +/- 3.6 (BDL) v 15.1 +/- 8.7 (SH) and 12 +/- 2.0 (NC) nmol/30 min/mg cell protein, respectively. Thus, cholestasis impairs neutrophil phagocytosis in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of polymorphonuclear leukocyte respiratory burst by elevated glucose concentrations in vitro

Although impaired polymorphonuclear leukocyte (PMN) function may be a cause of infectious complications in diabetic patients, the mechanisms of altered cell function are not understood. Our studies of PMN function in healthy subjects demonstrated significant reduction in the respiratory burst after 30 min of in vitro cell exposure to glucose concentrations greater than 11 mM (200 mg/dl). The respiratory burst was reduced 28 +/- 5 and 74 +/- 7% in PMNs incubated with 11 and 56 mM glucose, respectively. The impairment was independent of the cell stimulus (chemotactic peptide, calcium ionophore, or phorbol ester) and was not affected by sorbinil or myo-inositol. Because both D- and L-glucose had similar inhibitory effects, a nonenzymatic mechanism appeared to be the cause of impaired PMN function. Although mannitol and sorbitol did not affect cell function, monosaccharides (glucose, mannose, fructose) that form Schiff-base adducts with protein inhibited PMN function. These findings suggest a potential role for protein glycosylation in glucose-induced impairment of PMN function.     

Protein-calorie malnutrition impairs host defense against Candida albicans.

Protein-calorie malnutrition (PCM) impairs immune responsiveness predisposing to Candida albicans sepsis, but mechanisms are unclear. This study examined the effect of PCM on enteric-derived C. albicans intestinal translocation and the ability of in vivo interferon-gamma (IFN-gamma) to upregulate macrophage (MO) candidacidal mechanisms in PCM mice. Control (24% casein) and low protein (2.5%) diets were given for 4 weeks. Mice (n = 160) were fed C. albicans in their drinking water for 3 days and C. albicans translocation (mean colony-forming units (CFU)/g tissue +/- SEM) to the GI tract, liver, spleen, and kidney was assessed at 1 and 5 days following endotoxin challenge of 1, 5, and 10 mg/kg body wt. In a separate study (n = 100 mice), IFN-gamma (1000-10,000 U/day ip) vs saline was given for 3 days prior to harvesting peritoneal macrophages for assay of superoxide anion (O2-), percentage macrophage phagocytosis of C. albicans, and percentage killing of C. albicans. On Day 1, fungal translocation to the intestinal wall and systemic organs in the PCM group was significantly higher. On Day 5, mean CFU were significantly higher in the PCM group, indicating impaired organ clearance. Mean O2-, phagocytosis, and killing were significantly impaired in the PCM group (P less than 0.05), but IFN-gamma improved all functions. PCM significantly depressed host responses to C. albicans. IFN-gamma treatment enhanced candidacidal mechanisms, suggesting a therapeutic role in the malnourished host predisposed to C. albicans sepsis.     

CD18 adhesion receptors, tumor necrosis factor, and neutropenia during septic lung injury.

Sequestration of neutrophils (PMNs) in the pulmonary microvasculature and associated neutropenia are characteristic features of experimental models of septic lung injury. The etiology of altered PMN kinetics during septic lung injury is uncertain, but may be partially due to increased adhesiveness of activated PMNs to pulmonary endothelium. This study examines the relationship between the expression of PMN CD18 adhesion receptors, the evolving neutropenia, and plasma tumor necrosis factor (TNF) activity in a porcine model of septic lung injury. Acute lung injury was induced by infusion of live Pseudomonas aeruginosa (5 x 10(8) CFU/ml at 0.3 ml/20 kg/min) for 60 min (Group Ps, n = 6). Control animals (Group C, n = 3) received a 60-min infusion of sterile 0.9% saline. CD18 expression of circulating PMNs was measured by quantitative immunofluorescent flow cytometry. Plasma TNF activity was measured by L929 fibroblast cytolytic assay. Group Ps developed a significant neutropenia by 30 min (14.9 +/- 2.5 vs 23.4 +/- 3.3 x 10(3) cells/microliter at baseline, P less than 0.05, ANOVA) with circulating neutrophils exhibiting significantly increased CD18 expression by 60 min (6.34 +/- 0.72 vs 5.01 +/- 0.52 equivalent soluble fluorescence molecules (ESFM) x 10(3) at baseline, P less than 0.05, ANOVA). Group Ps demonstrated a significant increase in plasma TNF activity by 30 min (2.5 +/- 0.9 vs 0.7 +/- 0.3 U/ml at baseline). There was no significant change in PMN count, PMN CD18 expression, or plasma TNF activity in Group C. In complimentary in vitro studies, porcine PMNs stimulated with recombinant human TNF-alpha (n = 5) demonstrated a time- and dose-dependent increase in CD18 expression.(ABSTRACT TRUNCATED AT 250 WORDS)