Characterization of a direct effect of phorbol myristate acetate on human neutrophil cell membrane using 31D8 monoclonal antibody.

We found that 4-beta-phorbol 12-myristate 13-acetate (PMA) caused decreased expression of the polymorphonuclear neutrophil (PMN) surface antigen 31D8. In contrast to the rapid initiation of the oxidative burst caused by PMA, the effect was slow to start but increased during incubation periods up to 50 min. To study this apparent protein kinase C-independent late effect of PMA, we measured 31D8 expression in PMNs after incubation with various concentrations of PMA. The maximum PMA-induced inhibition was 76 +/- 2%, with an ID50 of 3.9 +/- 0.4 ng/ml. Oxidants and prooxidants (hydrogen peroxide, hypochlorite, taurine-chloramine, and ferrous iron, with or without H2O2) had no direct effect on 31D8 antigen expression. The following substances were not protective against the inhibitory affect of PMA: (1) antioxidants (superoxide dismutase, catalase, azide, dimethyl sulfoxide, Desferal, and ascorbate, with the exception of alpha-tocopherol), (2) inhibitors of protein kinase C (H7 and W7), (3) inhibitors of 5-lipoxygenase (A-63162, MK886, and high-dose indomethacin) and (4) inhibitors of cyclooxygenase (low-dose indomethacin). Myeloperoxidase-deficient PMNs had normal 31D8 antigen expression and a decrease of 31D8 antigen expression by PMA, as did normal PMNs. The inactive analog of PMA, 4-alpha-phorbol didecanoate, had no effect on 31D8 antigen expression. alpha-Tocopherol (50 micrograms/ml) and betamethasone (150 micrograms/ml) protected against the PMA effect by 30.5 +/- 7.3 (P less than .0005) and 52 +/- 15 (P less than 0.004) channels, respectively. These results indicate that PMA has a protein kinase C-independent late effect on human neutrophils, which can be prevented by pretreatment with alpha-tocopherol or the steroid betamethasone. These compounds probably exert their protective effect by membrane stabilization.

Depressed neutrophil chemotaxis in children suffering blunt trauma.

OBJECTIVE: Impaired neutrophil (PMN) function, due in part to release of immature PMNs into the circulation, contributes to the increased rate of infection observed in adults suffering blunt trauma. The objective of this study was to determine whether similar events occur in children. METHODS: We assessed PMN chemotaxis and PMN maturation in 25 children (7 young children and 18 adolescents) and 25 adults 1 to 9 days after suffering blunt trauma, and in healthy adult control subjects. PMN chemotaxis was determined using a standard micropore filter assay, whereas PMN maturation was determined with 31D8, a novel monoclonal antibody that binds to mature PMNs more avidly than immature PMNs and band forms. RESULTS: In patients suffering blunt trauma, mean PMN chemotactic values were similar among children (44.6 +/- 2.3 microns) and adults (41.3 +/- 2.1 microns) and both were significantly less than among healthy adults (53.5 +/- 2.4 microns, P < .0005). PMN chemotactic values increased significantly in the 9 days after trauma for both children and adults (F = 13.8, df = 1, P < .0002). Mean PMN 31D8 binding among children with trauma (92.5 +/- 5.2) was significantly less than among healthy adults (117.6 +/- 5.4, P < .0009). CONCLUSIONS: Impairment in PMN chemotaxis occurs in children after blunt trauma and is due in part to release of immature PMNs into the circulation.

The influence of various factors on the accuracy of gallium-67 imaging for occult infection.

To examine whether the results and interpretation of gallium-67 citrate imaging may be adversely influenced by factors present in compromised patients, we reviewed our 1-year experience in 69 patients in intensive care units, renal transplants, and those on hemodialysis. Our results indicate that it is an inappropriate diagnostic procedure for acute pancreatitis since seven of nine had false-negative results. Using loglinear modeling and chi-square analysis we found that treatment with antiinflammatory steroids, severe liver disease, end-stage renal disease, and renal transplantation with immunosuppressive therapy did not interfere with gallium-67 uptake. Increased rate of true-negative results in patients with end-stage renal disease was due to a greater and earlier use of the test in the febrile transplant patient and in hemodialysis patients with infections not amenable to diagnosis with gallium-67 scan (transient bacteremia and bacteriuria). We conclude that gallium-67 imaging is a useful diagnostic tool that, with the exception of acute pancreatitis, has very few false-negative results.

Inhibition of human polymorphonuclear leukocyte cell responses by ibuprofen.

The stimulation of cell swelling, cell aggregation, polymorphonuclear leukocyte locomotion, and lysosomal enzyme release in response to chemoattractant were all inhibited by ibuprofen, a nonsteroidal anti-inflammatory agent. The dosages needed to induce 50% inhibition (ID50) were 5.9, 7.6, 60, and 95 micrograms/mL, respectively. Aside from the differences in ID50, there was also a difference in the degree of maximum inhibition (Imax) of the complement C5a-stimulated responses observed, so that at achievable serum drug concentrations of 20-50 micrograms/mL, inhibition of 67-78% for cell swelling, 69-82% for cell aggregation, 20-35% for migration response, and 17-38% for lysosomal enzyme release were demonstrated. Also observed were a minor stimulatory effect on nitroblue tetrazolium reduction and an inhibitory effect on the ability to kill Staphylococcus aureus, but only at very high concentrations (approximately 2 mg/mL).

Protective effects of ibuprofen and methylprednisolone on chemotactic factor-induced transcutaneous hypoxia.

We showed previously in vitro that ibuprofen, a nonsteroidal anti-inflammatory agent, at concentrations easily achievable in blood, inhibits polymorphonuclear leukocyte cell swelling and aggregation in response to chemotactic factor stimulation. To confirm this in vivo, we studied the ability of ibuprofen i.v. pretreatment to reverse the transcutaneous hypoxia induced by i.v. infusion of 1 nmol/kg of formyl-methionyl-leucyl-phenylalanine. This effect was compared with that of methylprednisolone. For ibuprofen and methylprednisolone, respectively, the maximum percentage of reversal of hypoxia was 85 and 106%; the dose required to produce 50% of maximum reversal was 2.7 and 4.6 mg/kg; and the serum drug concentration needed to achieve 50% of maximum reversal was 14 and 11 micrograms/ml. We conclude that ibuprofen could be a useful alternative to steroidal antiinflammatory agents for the prevention and treatment of complications of stimulated polymorphonuclear leukocytes.

The inhibitory effect of parenteral nutrition on recovery of neutrophil locomotory function in blunt trauma.

Twenty patients were investigated to determine whether total parenteral nutrition (TPN) influences the recovery of neutrophil (PMN) locomotory dysfunction in blunt trauma. Half were given TPN consisting of amino acids, glucose, electrolytes, and trace minerals, and half were given intravenous (I.V.) fluids consisting of 5% glucose in water or saline, electrolytes, and trace minerals. PMN locomotion was assayed using micropore filters. Analysis of the data by general linear modeling showed that PMN locomotion in TPN patients was significantly slower during the first 3 to 4 days postinjury. By sequential analysis, improved PMN function in the group not given TPN (NO TPN) occurred less than 95% of the time. TPN with amino acids and glucose may worsen and delay the recovery of PMN locomotory responses in blunt trauma, but the preference ratio of NO TPN:TPN for better PMN function was less than 95:5.

High teicoplanin uptake by human neutrophils.

The cellular/extracellular (C/E) concentration ratio of teicoplanin in polymorphonuclear leukocytes (PMNs) increased rapidly with time (C/E 60 +/- 13 at 20 min). The C/E ratio was time- and concentration-dependent. At 20 min and an initial concentration of 75 +/- 16 micrograms/ml the cellular drug concentration was 4,700 +/- 1,300 micrograms/ml. The mechanism of drug uptake was by an active process and transported (cellular) drug retained its antimicrobial activity. Washing removed 42% of cellular drug. Teicoplanin inhibited PMN chemotaxis at very high concentrations and PMN microbicidal activity at lower concentrations.

Pitfalls in testing drug uptake by leukocytes using radiolabeled drugs and an explanation of conflicting results with clindamycin.

Although studies of drug uptake by leukocytes use similar methods, the results reported are sometimes vastly different. To determine the possible reasons for this, we studied neutrophil uptake of [3H]-clindamycin using the most frequently used density gradient centrifugation method and the volume probes 3H2O and [3H]-polyethylene glycol. We found that the [3H]-clindamycin available to investigators had undergone radiolytic decomposition; thus, its microbiologic activity was only 20% of its original potency. By thin-layer chromatography and autoradiography, four extra label-bearing regions were observed with [3H]-clindamycin. Results of neutrophil uptake studies have shown a drop of cellular:extracellular concentration ratios from 40:1 in 1981 to 10:1 in 1982 and 1987.

Simplified micropore filter assay of neutrophil migration using whole blood.

A new method of micropore filter assay of neutrophil migration requiring only 0.1 ml of whole blood is described and compared with the standard separated polymorphonuclear neutrophil micropore filter assay. Whole blood was added to the upper compartment of a modified Boyden chemotactic chamber, and the neutrophils were allowed to migrate into a cellulose nitrate micropore filter. Acetic acid was used to remove erythrocytes and hemoglobin from the filter. Neutrophil chemotaxis was performed with cells from 19 neonates and 34 adults. The mean neonatal PMN migration was 40% of the adult value (48.1 +/- 12.6 vs 96.8 +/- 16.8 micron) with the whole blood assay and 39% of the adult value (44.7 +/- 10.1 vs 72.9 +/- 22.2 micron) with the separated polymorphonuclear neutrophil assay. The whole blood micropore filter assay, a simple and reliable method of determining neutrophil migration, is especially useful in studying patients when there are difficulties in obtaining large blood samples, such as with neonates and young children.

Partial characterization of a cell-directed inhibitor of leukotaxis in human serum.

A leukotactic defect is described in a man with a low cerebrospinal fluid leukocyte count in the presence of an acute listeria meningitis. On the basis of subsequent studies with his serum and normal human serum, a leukotactic inhibitor has been identified. This inhibitor, termed the cell-directed inhibitor (CDI), is relatively heat stable and nondialyzable. By ultracentrifugal analysis in sucrose density gradient, the inhibitor has been resolved into two activities with estimated sedimentation coefficients of 7 and 10 S. It interacts directly with neutrophils and monocytes to render them chemotactically defective. CDI also impairs the phagocytic function of neutrophils. Evidence is presented that an antagonist to the inhibitor is present in normal serum. CDI and its antagonist are probably normally occurring regulators of leukotaxis. In the patient studied, an elevated CDI serum level may be related to the development of the listeria infection and failure of cutaneous response to skin test antigens.

Additional evidence of autoxidation as a possible mechanism of neutrophil locomotory dysfunction in blunt trauma.

Previous studies in victims of blunt injury suggest that the observed neutrophil (PMN) locomotory dysfunction is, in part, due to autoxidation. To further clarify the occurrence and significance of autoxidation, we studied changes in levels of glutathione in PMN and of ascorbic acid and alpha-tocopherol in serum and blood cells of postsurgical and blunt trauma patients. Levels of total, reduced, and oxidized glutathione in PMN from trauma patients were similar to normal controls. Serum and cellular ascorbic acid and alpha-tocopherol levels dropped significantly after injury and remained below normal control levels during the 7 to 8-day study period. Low serum alpha-tocopherol was partially explainable on the basis of changes in serum lipids. When serum samples of trauma patients were thawed unprotected without pyrogallol, there was significant loss of recoverable alpha-tocopherol, whereas no significant losses occurred with unprotected thawed normal sera. Less total reducing capacity was observed in PMN of trauma patients compared with normal controls. These findings indicate that synthesis and regeneration capacity of glutathione are intact but that the levels of the consumable antioxidants, ascorbic acid, and alpha-tocopherol are compromised after injury. These results add further support to the hypothesis that autoxidation occurs in trauma.

Efficacy, toxicity, and pharmacokinetics of pentoxifylline and its analogs in experimental Staphylococcus aureus infections.

Pentoxifylline (PTX), a drug that improves neutrophil function in vitro, has been shown to protect neonatal mice against death from experimental staphylococcal infection in vivo at a dose of 50 mg/kg. Using a total of 774 neonatal mice, the effects of various doses of PTX were examined and compared with the effects of three analogs: HWA-448, HWA-285, and A81-3138. A subcutaneous abscess was induced with 10(8) Staphylococcus aureus, and drug or saline was given daily subcutaneously from 2 days before to 4 days after infection. Noninfected animals (given saline without S. aureus) had 0% mortality (0 of 66), and infected animals without drug (given saline) had a mortality of 70% (161 of 231). PTX and HWA-448 showed the greatest protection among the drugs tested at 15 mg/kg with mortality rates of 27 and 38%, respectively (Kaplan-Meier method, P = 0.0001 and 0.0004, respectively). HWA-285 was most protective at 25 mg/kg (mortality, 45%; P = 0.0046) and A81-3138 was most protective in animals at 15 mg/kg (mortality, 42%; P = 0.0045). PTX, HWA-448, HWA-285, and A81-3138 at doses of 200, 100, 100, and 50 to 75 mg/kg, respectively, were toxic as shown by worsened weight loss and increased mortality in animals when compared with infected animals without drug. PTX and its analogs decrease mortality from experimental infections at lower doses but are toxic at higher doses. Pharmacokinetic characteristics of the drugs were similar except that HWA-285 produced lower concentrations in serum and A81-3138 showed a dose-dependent kinetics (longer half-life at a higher dose).

A randomized trial of replacement antioxidant vitamin therapy for neutrophil locomotory dysfunction in blunt trauma.

Studies in patients with serious trauma indicate that the observed neutrophil (PMN) locomotory dysfunction is partly the result of auto-oxidation as shown by evidence of preactivation, diminished reducing capacity, and low serum and cellular ascorbic acid and alpha-tocopherol. To investigate whether replacement of the antioxidant vitamins ascorbic acid and alpha-tocopherol can improve the PMN locomotory defect, ascorbic acid, alpha-tocopherol, ascorbic acid and alpha-tocopherol, or placebo was administered to a total of 46 victims of blunt trauma. PMN locomotion was quantitated using a micropore filter assay. Locomotion data were analyzed by repeated measures analysis with a split plot design and data for days 2-6 after injury were compared. Compared with placebo, the antioxidants improved PMN locomotion. The mean differences in distance migrated (treated minus placebo) were ascorbic acid and alpha-tocopherol = 11.3 +/- 3.0 microns (one-tailed p = 0.001) (mean +/- SE); ascorbic acid = 4.7 +/- 3.4 microns (p = 0.19); and alpha-tocopherol = 3.3 +/- 2.9 microns (p = 0.27). Although both antioxidants given together produced the best results, a plot of the 95% confidence intervals indicates that ascorbic acid and alpha-tocopherol, either given alone, were also better than placebo. We conclude that antioxidant replacement therapy significantly improves the PMN locomotory abnormality in blunt trauma.

Polymorphonuclear leukocyte migration abnormalities and their significance in seriously traumatized patients.

Polymorphonuclear leukocyte (PMN) locomotory responses were studied in 24 patients who sustained serious blunt trauma, mostly from motor vehicle accidents. The results showed the presence of a combined cell- and serum-associated locomotory abnormality. The serum abnormality was due to a cell-directed inhibitor, and was present for an average of 3 days. The cell-associated abnormality persisted for approximately 1 week in uninfected patients, and 2 weeks in the infected group. Both mature and immature forms of PMNs contribute to the PMN locomotory dysfunction observed. A significant correlation was observed between the degree of PMN locomotory abnormality or injury severity score and the infection rate. Eighteen infections (six suspected and 12 definite) were observed in 11 of the 24 patients. Twelve (67%) infections involved the lungs. Nine patients (82%) showed evidence of infection by day 6. PMN dysfunction in trauma is associated with increased infection rate and is not due solely to increased numbers of immature forms of PMNs.

Leukotactic dystunction in sarcoidosis.

The leukotactic function of patients with sarcoidosis was studied. A defect was found in 19 of the 20 patients tested and was due to moderately elevated serum levels of the chemotactic factor inactivator. The chemotactic factor inactivator levels were not as high as those previously reported in patients with Hodgkin's disease or cirrhosis of the liver. The effect of the inactivator was irreversible and was directed toward all three of the chemotactic factors tested. The physicochemical characteristics of chemotactic factor inactivator in serum from sarcoid patients resembled in most respects the features of chemotactic factor inactivator in normal serum. As expected, the generation of chemotactic activity in some sarcoid serums by zymosan was impaired. The results of this study may relate to some of the reported defects in expression of immunity in sarcoid patients.

Inappropriate activation, deactivation, and probable autooxidative damage as a mechanism of neutrophil locomotory defect in trauma.

We studied 46 patients who suffered from serious blunt trauma to examine the possible mechanism of their acquired neutrophil (PMN) locomotory dysfunction. Concentrations of plasma C3adesArg were higher in patients than in controls (310 +/- 190 ng/ml vs. 90 +/- 28 ng/ml, respectively; P = 3 X 10(-5)). Both resting and phagocytosing PMNs from the patients produced higher quantities of H2O2 (0.31 +/- 0.29 and 5.2 +/- 3.4 nmol/10(6) PMNs per hr, respectively). These levels resemble the H2O2 production of normal PMNs preactivated with chemotactic factor (0.85 +/- 0.03 for normal and 8.2 +/- 1.6 nmol/10(6) PMNs per hr for preactivated PMNs). Concentrations of oxidized glutathione were not significantly higher in PMNs from patients compared with PMNs from controls (0.053 +/- 0.057 vs. 0.037 +/- 0.046 nmol/10(6) PMNs, respectively; P = .5). A higher percentage of PMNs from trauma patients than from controls were capped with concanavalin A (66% +/- 11% vs. 37% +/- 14%, respectively; P = 4 X 10(-5)), a result indicating microtubular dysfunction. These findings suggest that in trauma, activation of intravascular complement results in inappropriate chemotactic stimulation and subsequent deactivation and autoxidative damage of circulating PMNs.

The effect of betamethasone on neonatal neutrophil chemotaxis.

Antenatal maternal glucocorticoid administration has been widely used to accelerate fetal lung maturation. Glucocorticoids have also been used postnatally in selected neonates as antiinflammatory agents. Numerous studies have shown that glucocorticoids inhibit multiple components of the immune system including neutrophil (PMN) function in children and adults. Since PMNs are of critical importance in host defense against bacterial infection, impaired PMN function in newborn infants is thought to be an important cause of their increased morbidity and mortality from bacterial infection. Further compromise of neonatal PMN function by exogenous factors such as glucocorticoids may therefore be of significant clinical importance. A micropore filter chemotactic assay was used to determine the in vitro effect of betamethasone on the random migration and directed migration (chemotaxis) of PMNs from 18 neonates. The addition of a concentration of betamethasone (0.01 microgram/ml) similar to that found in cord blood following a standard dose administered to the mother resulted in a significant (p less than 0.01) inhibition in mean neonatal PMN random migration (-15.0 +/- 0.8%) and chemotaxis (-23.5 +/- 3.0%). A similar inhibition was not found when PMNs from 14 adults were exposed to the same concentrations of betamethasone. Betamethasone administration to pregnant women or their newborn infants may further impair PMN motility and lead to an increased morbidity and mortality from bacterial infection in neonates.

Comparative efficacy of cimetidine, famotidine, ranitidine, and mylanta in postoperative stress ulcers. Gastric pH control and ulcer prevention in patients undergoing coronary artery bypass graft surgery.

To determine the comparative efficacy of several histamine (H2)-receptor antagonists (cimetidine, famotidine, and ranitidine) and the antacid Mylanta-II (Stuart Pharmaceuticals, Wilmington, DE) in gastric pH control and the prevention of postoperative stress ulceration, a prospective, randomized study was performed in a homogeneous population of patients with elective coronary artery bypass. None of the 57 patients in the study population had a documented history of ulcer disease. There were four treatment groups, each with similar demographics (age and sex). Cimetidine-treated group consisted of 15, famotidine-treated group of 18, ranitidine-treated group of 19, and antacid-treated group of 5 patients. There was no hemodynamically significant postoperative gastrointestinal bleeding in any of the patients. When the agents were compared for efficacy of gastric pH control, statistically better pH control was found in the famotidine- and ranitidine-treated groups (P less than 0.003) than in the cimetidine-treated group (pH less than or equal to 4.0) during the 20-hour observation period. Side effects (hematologic and neurological) were noted only in the cimetidine-treated group. The results of this study indicate that in patients in postoperative intensive care, better gastric pH control, and thus prevention of gastric stress ulcers, is achieved with either famotidine or ranitidine rather than cimetidine or antacid.