Toxic oxygen metabolite production by circulating phagocytic cells in inflammatory bowel disease.

To investigate the possibility that the oxidative capacity of phagocytic cells may be defective in inflammatory bowel disease, toxic oxygen metabolite production by circulating neutrophils and monocytes has been measured by luminol dependent chemiluminescence. Neutrophils from patients with Crohn's disease and ulcerative colitis produced significantly lower chemiluminescent responses after chemotactic stimulation with formylmethionylleucylphenylalanine (fMLP) than neutrophils from control patients, p = 0.018 and 0.043 respectively. Chemiluminescent responses of neutrophils from patients with inflammatory bowel disease, however, were similar to control responses when cells were stimulated with latex beads or phorbol myristate acetate. Monocytes from patients with Crohn's disease produced significantly greater levels of chemiluminescence than control monocytes when stimulated with either fMLP (p less than 0.002), phorbol myristate acetate (p less than 0.0005) or latex beads (p less than 0.002). Monocytes from patients with ulcerative colitis also produced significantly greater levels of chemiluminescence than controls when stimulated with latex beads (p less than 0.5) or phorbol myristate acetate (p less than 0.0005), although there was no difference in the level of chemiluminescence in response to fMLP. These results exclude a generalised defect in phagocytic cell oxidase activity in inflammatory bowel disease and suggest that circulating monocytes are 'activated'.     

Cytoprotection against neutrophil derived hypochlorous acid: a potential mechanism for the therapeutic action of 5-aminosalicylic acid in ulcerative colitis.

The aim of the present study was to investigate the effects of 5-aminosalicyclic acid (5-ASA) on the cell injury mediated by activated neutrophils. We used a system constituted of neutrophils, triggered with phorbol myristate acetate, and 51Cr-labelled Daudi cells as targets. The results show that 5-ASA is capable of efficiently preventing neutrophil-mediated lysis. 5-ASA was up to 10-fold more effective than taurine, which acts as an hypochlorous acid scavenger. Moreover, 5-ASA was found to compete with taurine for the neutrophil derived hypochlorous acid. The results are consistent with the conclusion that 5-ASA is capable of limiting the neutrophil mediated cell damage by scavenging the generated hypochlorous acid. This may represent a potential mechanism for the therapeutic action of 5-ASA in ulcerative colitis.     

Phagocytic and oxidative burst activity of neutrophils in the end stage of liver cirrhosis

AIM: To evaluate the phagocytic activity and neutrophil oxidative burst in liver cirrhosis. METHODS: In 45 patients with advanced postalcoholic liver cirrhosis (aged 45±14 years) and in 25 healthy volunteers (aged 38±5 years), the percentage of phagocytizing cells after in vitro incubation with E. coli (Phagotest Kit), phagocytic activity (mean intensity of fluorescence, MIF) and the percentage of neutrophil oxidative burst (Bursttest Kit), and the level of free oxygen radical production (MIF of Rodamine 123) were analyzed by flow cytometry. The levels of soluble sICAM-1, sVCAM-1, sP-selectin, sE-selectin, sL-selectin, and TNF-α were determined in blood serum. RESULTS: The percentage of E. coli phagocytizing neu-trophils in liver cirrhosis patients was comparable to that in healthy subjects. MIF of neutrophil - ingested E. coli was higher in patients with liver cirrhosis. The oxidative burst in E. coll phagocytizing neutrophils generated less amount of active oxygen compounds in liver cirrhosis patients (MIF of R123: 24.7±7.1 and 29.7±6.6 in healthy, P<0.01). Phorbol myristate acetate (PMA) - stimulated neutrophilsproduced less reactive oxidants in liver cirrhosis patients than in healthy subjects (MIF of R123: 42.7±14.6 vs 50.2±13.3, P<0.01). A negative correlation was observed between oxidative burst MIF of PMA-stimulated neutrophils and ALT and AST levels (r -0.35, P<0.05; r-0.4, P<0.03). sVCAM-1, sICAM-1, sE-selectin concentrations correlated negatively with the oxygen free radical production (MIF of R123) in neutrophils after PMA stimulation in liver cirrhosis patients (r-0.45, P<0.05; r-0.41, P<0.05; r-0.39, P<0.05, respectively). CONCLUSION: Neutrophil metabolic activity diminishes together with the intensification of liver failure. The metabolic potential of phagocytizing neutrophils is significantly lower in liver cirrhosis patients, which can be one of the causes of immune mechanism damage. The evaluation of oxygen metabolism of E. coli-stimulated neutrophils reveals that the amount of released oxygen metabolites is smaller in liver cirrhosis patients than in healthy subjects.     

An oxygen-dependent mechanism of neutrophil-mediated cytotoxicity

Human neutophils stimulated with phorbol myristate acetate were able to rapidly destroy autologous red blood cell targets. Neutrophil-mediated cytotoxicity was related to phorbol myristate acetate concentration and neutrophil number. The ability of stimulated neutrophils to lyse red blood cell targets was markedly impaired by catalase or superoxide dismutase but not by heat-inactivated enzymes or albumin. Despite a simultaneous requirement for O2.- and H2O2 in the cytotoxic event, a variety of OH. and 1O2 did not effect cytolysis. The myeloperoxidase inhibitor cyanide did not reduce red blood destruction, while azide consistently impaired cytolysis. The inability of cyanide to reduce cytotoxicity coupled with the protective effect of superoxide dismutase suggests that cytotoxicity is independent of the classic myeloperoxidase-H2O2-halide system. We propose that neutrophils, stimulated with phorbol myristate acetate, generate O2.- and H2O2, which play an integral role in a novel cytotoxic mechanism.     

Oxidative mechanisms of monocyte-mediated cytotoxicity.

Human monocytes stimulated with phorbol myristate acetate were able to rapidly destroy autologous erythrocyte targets. Monocyte-mediated cytotoxicity was related to phorbol myristate acetate concentration and monocyte number. Purified preparations of lymphocytes were incapable of mediating erythrocyte lysis in this system. The ability of phorbol myristate acetate-stimulated monocytes to lyse erythrocyte targets was markedly impaired by catalase or superoxide dismutase but not by heat-inactivated enzymes or albumin. Despite a simultaneous requirement for superoxide anion and hydrogen peroxide in the cytotoxic event, a variety of hydroxyl radical and singlet oxygen scavengers did not effect cytolysis. However, tryptophan significantly inhibited cytotoxicity. The myeloperoxidase inhibitor cyanide enhanced erythrocyte destruction, whereas azide reduced it modestly. The inability of cyanide to reduce cytotoxicity coupled with the protective effect of superoxide dismutase suggests that cytotoxicity is independent of the classic myeloperoxidase system. We conclude that monocytes, stimulated with phorbol myristate acetate, generate superoxide anion and hydrogen peroxide, which together play an integral role in this cytotoxic mechanism.     

Flow cytometric assay of phagocytic activity of human neutrophils and monocytes in whole blood by neutral red uptake

In a new, simple, and fast flow-cytometric method for the simultaneous measurement of phagocytic activity of human neutrophils and monocytes in whole blood, the fluorescence capability of the well-known vital stain, neutral red was used. The incubation of 0.5 ml heparinized human blood with the phagocytic stimulus of zymosan dose- and time-dependently increased the percentage and the red fluorescence intensity of both neutrophils (4.3±1.2 times) and monocytes (2.7±0.7 times) measured cytofluorimetrically. Decreased uptake of neutral red was observed in a patient with phagocytic disorder, based upon impaired engulfment of particles and production of reactive oxygen species. In a patient with chronic granulomatosis, however, no decrease of neutral red uptake was measured. Platelet activating factor and phorbol myristate acetate were also able to increase the uptake of neutral red by both monocytes and neutrophils, but to a lesser extent than zymosan. The advantage of this method is the possibility for the simultaneous measurement of phagocytic activities of monocytes and neutrophils stimulated by either particles or soluble activators. This method is suitable for the selective measurement of activation processes not related to the production of free radicals in the phagocytes.     

Effect of sulphasalazine and its active metabolite, 5-amino-salicylic acid, on toxic oxygen metabolite production by neutrophils.

The possibility that the mode of action of sulphasalazine and its active metabolite 5-amino-salicylic acid (5ASA) involves modification of toxic oxygen metabolite production by neutrophils has been investigated by measuring the effect of these drugs on luminol-dependent chemiluminescence, superoxide release and oxygen consumption by stimulated neutrophils in vitro. 5ASA, and to a lesser extent sulphasalazine, had profound inhibitory effects on the luminol dependent chemiluminescent response of neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine (1 microM) + cytochalasin B (5 micrograms/ml). A concentration of 50 microM 5ASA or sulphasalazine produced 93.8 (2.3)% and 65.7 (3.7)% inhibition of control responses respectively. The concentration of 5ASA and sulphasalazine producing 50% inhibition of chemiluminescence were 3.6 (1.8) microM and 16.5 (6) microM respectively. Both drugs had little effect on the chemiluminescent response of neutrophils stimulated with phorbol myristate acetate (1 microgram/ml), producing only 11.4 (3.9)% and 34 (7)% inhibition respectively, at a concentration of 50 microM. Superoxide release from fMLP + CB stimulated neutrophils was also inhibited slightly by 5ASA (50 microM) by 35.6% and by sulphasalazine (50 microM) by 7.9%. Similarly, there was little inhibition in the rate of oxygen consumption by fMLP + CB stimulated neutrophils by either 5ASA or sulphasalazine at concentrations which produced near total abolition of luminol dependent chemiluminescence. These results show that sulphasalazine and 5ASA inhibit the reaction of toxic metabolites produced by stimulated neutrophils with luminol, without inhibition of the oxidase system producing these metabolites. The site of action of these drugs on neutrophils in vitro is thus extracellular, by scavenging a released metabolite, probably hypochlorite. This has important implications for their mode of action in vivo in inflammatory bowel disease.     

Aclacinomycin, an anti-leukemic anthracycline, impairs human neutrophil functions.

The effects of aclacinomycin, an anti-leukemic anthracycline, on human neutrophil functions were investigated. The release of superoxide (O2-) in neutrophils stimulated by opsonized zymosan, myristate, or phorbol myristate acetate was inhibited by aclacinomycin in a dose- and time-dependent manner. The phagocytosis of yeast particles and oil droplets, and membrane potential changes stimulated by phorbol myristate acetate were also inhibited by aclacinomycin. On the other hand, the O2(-)-producing enzyme (NADPH oxidase) in the particulate fraction prepared from myristate-stimulated neutrophils was not affected by aclacinomycin. When high concentrations of aclacinomycin (10-100 micrograms/ml) were employed, significant inhibition of O2- release, phagocytosis, and membrane potential changes was observed within 5 min. Phagocytic activity was also inhibited when neutrophils were preincubated for 13 h at 37 degrees C with a low concentration (40 ng/ml) of aclacinomycin, which could be obtained by intravenous administration of 20 mg aclacinomycin. Myristate-induced O2- release was not impaired by cytosine arabinoside (2-800 micrograms/ml), vincristine (0.1-100 micrograms/ml), adriamycin (25-100 micrograms/ml), or daunomycin (5-75 micrograms/ml) when the cells were preincubated with these drugs for 5 min at 37 degrees C. These findings suggest that aclacinomycin inhibits the respiratory burst by impairing the activating system of NADPH oxidase and phagocytic activity.     

Bactericidal effect of polymorphonuclear neutrophils on antibiotic-induced filaments of gram-negative bacilli

Exposure of strains of Escherichia coli to ampicillin and mezlocillin and of strains of Pseudomonas aeruginosa to azlocillin and cefsulodin caused the bacilli to elongate into filaments. The bacilli and their filaments were incubated with fresh human polymorphonuclear neutrophils (PMNs), and the phagocytic process was recorded by means of phase-contrast microscopy. The bactericidal effect of PMNs on both filaments and bacilli was quantitated by counts of colony-forming units. A single PMN phagocytized one or more filaments, some of which were as long as 90 micron and contained as many as 20 genomes. Two predominant patterns of phagocytosis of filaments were observed. When the ratio of bacteria to PMNs was low (0.2-1.8), the rate of killing was 62%-81%. When the ratio was higher (5-12), the rate of killing of both filaments and bacilli was lower. As an alternative to colony-forming units, cell mass was used as a gauge of phagocytic activity. The relative mass of killed filaments was considerably greater than that of killed bacilli; this finding indicated that filaments were much more susceptible than were bacilli to the bactericidal activity of PMNs.     

Defective oxidative metabolic responses in vitro of alveolar macrophages in chronic granulomatous disease.

After stimulation with bacteria, alveolar macrophages (AM) from uninfected normal subjects or persons with pneumonia approximately doubled their rates of O2 consumption, superoxide anion generation, and glucose (1(-14)C) oxidation. In contrast, bacteria-stimulated AM from a patient with chronic granulomatous disease (CGD) failed to consume more O2, make superoxide anion, or oxidize glucose. In addition, AM from the patient with CGD did not respond to stimulation by a chemical agent, phorbol myristate acetate, which increased the metabolic activities of AM from control subjects. The appearance, esterase and Gomori acid phosphatase staining, phagocytic ability, unstimulated O2 consumption, and response to methylene blue of AM from the CGD patient were normal. The results extend the biochemical defect in patients with CGD beyond abnormalities in their circulating neutrophils and monocytes, to their tissue-associated lung macrophages. The results also indicate that AM from patients with CGD may have an additional abnormality in metabolism, which is a lack of enhanced mitochondrial respiration during phagocytosis. The studies also document the selective action of phorbol myristate acetate, which stimulated the metabolic activities of normal AM, but not of those from the patient with CGD.     

Rapid deactivation of NADPH oxidase in neutrophils: continuous replacement by newly activated enzyme sustains the respiratory burst

The cell-free system for activation of the neutrophil NADPH oxidase allowed us to examine activation of the oxidase in the absence of its NADPH-dependent turnover. The covalent sulfhydryl-modifying reagent N- ethylmaleimide completely inhibited the activation step (Ki = 40 mumol/L) in the cell-free system but had no effect on turnover of the preactivated particulate NADPH oxidase (up to 1 mmol/L). When N- ethylmaleimide was added to intact neutrophils during the period of maximal O2 generation in response to stimuli that activate the respiratory burst (phorbol myristate acetate, f-Met-Leu-Phe, opsonized zymosan, arachidonic acid), O2- generation ceased within seconds. Study of components of the cell-free activation system indicated that the cytosolic cofactor was irreversibly inhibited by N-ethylmaleimide whereas the N-ethylmaleimide-treated, membrane-associated oxidase could be activated by arachidonate and control cytosolic cofactor. Likewise, the cell-free system prepared from intact neutrophils that had been briefly exposed to N-ethylmaleimide and then washed reflected the effects of N-ethylmaleimide on the isolated cell-free components: cytosolic cofactor activity was absent, but the membrane oxidase remained fully activatable. Thus inhibition of oxidase activation by N- ethylamaleimide unmasked a rapid deactivation step that was operative in intact neutrophils but not in isolated particulate NADPH oxidase preparations. The demonstrated specificity of N-ethylmaleimide for oxidase activation and lack of effect on turnover of the NADPH oxidase suggested that sustained O2- generation by intact neutrophils was a result of continued replenishment of a small pool of active oxidase. The existence of an inactive pool of NADPH oxidase molecules in particulate preparations from stimulated neutrophils was supported more directly by activating these preparations again in the cell-free system.     

Serum amyloid A is an innate immune opsonin for Gram-negative bacteria

Serum amyloid A (SAA) is the major acute-phase protein in man and most mammals. Recently we demonstrated that SAA binds to many Gram-negative bacteria including Escherichia coli and Pseudomonas aeruginosa through outer membrane protein A (OmpA) family members. Therefore we investigated whether SAA altered the response of innate phagocytic cells to bacteria. Both the percentage of neutrophils containing E coli and the number of bacteria per neutrophil were greatly increased by SAA opsonization, equivalent to the increase seen for serum opsonization. In contrast, no change was seen for Streptococcus pneumoniae, a bacteria that did not bind SAA. Neutrophil reactive oxygen intermediate production in response to bacteria was also increased by opsonization with SAA. SAA opsonization also increased phagocytosis of E coli by peripheral blood mononuclear cell-derived macrophages. These macrophages showed strong enhancement of TNF-alpha and IL-10 production in response to SAA-opsonized E coli and P aeruginosa. SAA did not enhance responses in the presence of bacteria to which it did not bind. These effects of SAA occur at normal concentrations consistent with SAA binding properties and a role in innate recognition. SAA therefore represents a novel innate recognition protein for Gram-negative bacteria.     

The chlorinating potential of the human monocyte

Human monocytes incubated with phorbol myristate acetate (PMA) or opsonized zymosan particles can chlorinate the beta-amino acid taurine to its monochloramine derivative. Taurine monochloramine can then be quantitated by its ability to oxidize 5-thio-2-nitrobenzoic acid to its disulfide or by its characteristic absorption peak at 252 nm. Stimulated, but not resting, monocytes chlorinated taurine by a process dependent on time, cell concentration, and pH. The formation of taurine chloramine by stimulated monocytes could be inhibited by catalase, azide, or cyanide, was unaffected by superoxide dismutase, and was stimulated by exogenous myeloperoxidase. Thus, taurine chloramine generation by human monocytes appeared dependent on both H2O2 and myeloperoxidase. Compared to human neutrophils, the monocyte could generate similar amounts of chloramine when stimulated with phorbol myristate acetate, but far less if opsonized zymosan particles were used as the trigger. Based on the known ability of the H2O2- myeloperoxidase-Cl- system to generate free HOCl, it would seem that this oxidant is the most likely species responsible for the monocyte- mediated chlorination reactions. Thus, we have used a simple quantitative assay to demonstrate the ability of the human monocyte to generate large quantities of a highly reactive and toxic oxygen metabolite.     

Effect of fish oil on neutrophil chemiluminescence induced by different stimuli in patients with rheumatoid arthritis.

Lipid composition plays an important part in the structural and metabolic functions of cell membranes. In particular the production of inflammatory mediators such as prostaglandins and leukotrienes is dependent on polyunsaturated fatty acid precursors. Neutrophil leucocytes participate in inflammatory processes by their phagocytic and killing activities which can be monitored by measuring the photon emission (chemiluminescence). Chemiluminescence was measured in a luminol dependent system after stimulation by either particulate (zymosan) or soluble (phorbol myristate acetate) stimulus in a group of 10 patients with rheumatoid arthritis before and 21 and 45 days after treatment with a diet supplemented with eicosapentaenoic and docosahexaenoic acids. Ten patients with rheumatoid arthritis continuing their usual diet were used as control subjects. A progressive reduction of chemiluminescence stimulated by zymosan and phorbol myristate acetate was found in the patients treated with fish oil supplementation. This result correlated well with the reduction in erythrocyte sedimentation rate and an improvement of clinical parameters. The effects of fish oil derived lipids on neutrophil chemiluminescence are probably due to a change of the lipid composition of the cell membrane which is dependent on the esterification of eicosapentaenoic acid and docosahexaenoic acid in cellular membrane phospholipids. The modification of membrane lipid composition seems to interact in a non-specific way with the metabolic activation of neutrophils during phagocytosis.     

Quantitative assessment of bactericidal ability of leukemic cells in childhood acute leukemia

Bactericidal ability of leukemic cells was evaluated in four children with acute myeloblastic leukemia (AML) and in two with acute monocytic leukemia (AMoL) by means of quantitative assay of intracellular viable bacteria and by electron microscopy. The electron microscopy confirmed phagocytosis of Staphylococcus aureus by the leukemic cells. The leukemic cells killed a significantly smaller proportion of phagocytized bacteria than normal neutrophils. In some patients the leukemic cells virtually lacked bactericidal ability, despite presence of phagocytic ability. The defect of the cells in killing bacteria was more prominent in AML than in AMoL. The clinical importance of the defective bactericidal ability of leukemic cells is discussed.     

Phagocytic cells metabolize 25-hydroxyvitamin D3 in vitro.

Phagocytic cells are widely distributed in tissues known to be important in the metabolism of vitamin D. Incubation of human polymorphonuclear leukocytes and monocytes and resident rat peritoneal macrophages with 3H-labeled 25-hydroxyvitamin D3 leads to the formation of three radioactive peaks. Peak I is most consistent with a lactone derivative of 25-hydroxyvitamin D3, and peak II has been identified as putative 24,25-dihydroxyvitamin D3. Peak III is a novel metabolite of 25-hydroxyvitamin D3 unlike any of the synthetic standards available in our laboratories. Human neutrophils converted more substrate than did the other phagocytes examined. The stimulation of neutrophils by opsonized zymosan or phorbol myristate acetate led to a 4-fold increase in synthesis of the metabolites. These results suggest that vitamin D metabolism by phagocytic cells may play a role in the microenvironmental events that surround bony metabolism and calcium homeostasis.     

Selective depletion of neutrophils by a monoclonal antibody, RP-3, suppresses dextran sulphate sodium-induced colitis in rats

Administration of dextran sulphate sodium to animals induces acute colitis characterized by infiltration of large numbers of neutrophils into the colonic mucosa, which histologically resembles human active ulcerative colitis. It has been reported that neutrophils and the reactive oxygen metabolites produced by them are involved in the progress of ulcerative colitis. This study was intended to clarify their roles by using this animal model. First, possible sources and species of reactive oxygen metabolites were determined using luminol-dependent chemiluminescence with addition of enzyme inhibitors and reactive oxygen metabolite scavengers. Next, to examine whether neutrophils and hypochlorous acid derived from them contribute to tissue injury, we administered RP-3, a monoclonal antibody capable of selectively depleting neutrophils, and taurine, a hypochlorous acid scavenger, to rats treated with dextran sulphate sodium. Addition of azide, taurine, catalase, superoxide dismutase and dimethyl sulphoxide into colonic mucosal scrapings significantly inhibited chemiluminescence production, but allopurinol and indomethacin had no effects. These results suggest that excessive hypochlorous acid, hydrogen peroxide, superoxide anion and hydroxyl radical are generated by the inflamed colonic mucosa. Intraperitoneal injections of RP-3 significantly suppressed bleeding, tissue myeloperoxidase activity, chemiluminescence production and erosion formation. On the other hand, administration of taurine tended to inhibit bleeding and erosion formation to some extent, although it could not significantly suppress them. These data suggest that neutrophils play an important role in the development of this colitis and that hypochlorous acid might be one of the causes of tissue injury induced by neutrophils.     

Studies on defense effects of recombinant human granulocyte colony-stimulating factor (G-CSF) to infections. II. Priming effect for superoxide production by human neutrophil]

In present study, we have investigated superoxide (O2-) production from human neutrophils by recombinant human granulocyte colony-stimulating factor (G-CSF) using the microtiter plate for the purpose of being close to the inflammatory site. G-CSF by itself did not induce the release of O2- in human neutrophil on either Fetal Bovine Serum (FBS)-coated plate or plate uncoated with FBS, even if neutrophils were exposed for maximum 3 hr. However, the optimal concentration of G-CSF (50 ng/ml) was able to prime human neutrophils with enhance of O2- release stimulated by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) from 10(-6) to 10(-8) M, but not by the non chemoattractant such as phorbol myristate acetate (PMA), concanavalin A, and ionomycin. These findings indicate that G-CSF might enhance bactericidal activity of neutrophils by priming them penetrating into the inflammatory site.     

Peritoneal macrophages and opsonins: antibacterial defense in patients undergoing chronic peritoneal dialysis

The antibacterial activity of phagocytic cells and opsonins in peritoneal dialysis effluents from 21 patients undergoing chronic peritoneal dialysis (CPD) was studied. Effluents contained an average of 12 x 10(6) cells per liter that were predominantly macrophages. Macrophages phagocytized and killed opsonized Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli as efficiently as did polymorphonuclear neutrophils (PMNs) from healthy donors. Macrophage chemiluminescence was one-third of that observed with donor PMNs. In the absence of opsonins, macrophages efficiently phagocytized and killed S. aureus by binding S. aureus cell wall protein A to macrophage surface IgG. Nine (43%) of 21 effluents failed to opsonize S. epidermidis, and none opsonized E. coli. When present, titers of S. epidermidis opsonins were 50- to 100-fold lower than that of normal serum. IgG and C3 concentrations in effluent reflected its opsonic capacity. Macrophages from patients undergoing CPD thus have intact phagocytic and bactericidal functions. However, the low level of opsonic molecules and inadequate numbers of macrophages in the peritoneal cavity may predispose patients undergoing CPD to peritonitis.     

Inefficient bacteriolysis of Escherichia coli by serum from human neonates.

To assess bacteriolysis in human neonates, Escherichia coli O7w:K1:NM were incubated with sera from eight healthy neonates, serum pooled from the eight neonates, and serum pooled from healthy adults. The adult serum killed E. coli. In contrast, the bacteria were not killed during incubation with sera from the eight neonates, the pooled neonatal serum, or with heat-inactivated adult serum. However, the combination of pooled neonatal serum and heat-inactivated adult serum killed the bacteria. Supplemental IgG-containing antibodies that bound to E. coli did not enhance the bactericidal activity of the neonatal serum. Ten of 12 blood isolates of E. coli from septic neonates but only 8 of 15 isolates from septic adults were serum-sensitive (killed during incubation with adult serum) (P less than .05). Therefore, neonatal serum killed E. coli inefficiently and was deficient in non-IgG heat-stabile component(s) required for bacterial killing. Compared with adults, neonates were more frequently septic with serum-sensitive strains of E. coli.