Human lipopolysaccharide-binding protein potentiates bactericidal activity of human bactericidal/permeability-increasing protein.

Human bactericidal/permeability-increasing protein (BPI) from neutrophils and a recombinant amino-terminal fragment, rBPI23, bind to and are cytotoxic for gram-negative bacteria both in vitro and ex vivo in plasma or whole blood. To function in vivo as an extracellular bactericidal agent, rBPI23 must act in the presence of the lipopolysaccharide-binding protein (LBP), which also binds to but has no reported cytotoxicity for gram-negative bacteria. LBP, which is present at 5 to 10 micrograms/ml in healthy humans and at much higher levels in septic patients, mediates proinflammatory host responses to gram-negative infection. On the basis of these previous observations, we have examined the effect of recombinant LBP (rLBP) on the bactericidal activity of rBPI23 against Escherichia coli J5 in vitro. Physiological concentrations of rLBP (5 to 20 micrograms/ml) had little or no bactericidal activity but reduced by up to approximately 10,000-fold the concentration of BPI required for bactericidal or related activities in assays which measure (i) cell viability as CFUs on solid media or growth in broth culture and (ii) protein synthesis following treatment with BPI. LBP also potentiated BPI-mediated permeabilization of the E. coli outer membrane to actinomycin D by about 100-fold but had no permeabilizing activity of its own. Under optimal conditions for potentiation, fewer than 100 BPI molecules were required to kill a single E. coli J5 bacterium.     

Sensitivity of K1-Encapsulated Escherichia coli to Killing by the Bactericidal/Permeability-Increasing Protein of Rabbit and Human Neutrophils

The presence of K1 capsular polysaccharides increases the resistance of Escherichia coli to killing by serum and phagocytosis by polymorphonuclear leukocytes (PMNs). To determine whether K1 capsule impedes the action of intracellular bactericidal systems of PMNs, we compared the sensitivity of several K1-encapsulated and non-encapsulated strains of E. coli to killing by the bactericidal/permeability-increasing protein (BPI) isolated from rabbit and human PMNs. BPI appears to be the principal bactericidal agent of PMNs toward E. coli and other gram-negative bacteria (Weiss et al., J. Clin. Invest. 69:959-970, 1982). The presence of K1 capsule was monitored by sensitivity to K1-specific bacteriophages. The non-encapsulated strains used represent both random bacteremic isolates and non-encapsulated derivatives of K1-encapsulated strains obtained by selection for resistance to K1-specific phages. We found little or no difference in the sensitivity of K1-encapsulated and non-encapsulated E. coli to killing by neutralized acid extracts of rabbit PMNs. Bacterial killing by these crude fractions can be attributed to the action of BPI because: (i) bacterial killing was blocked by immune (anti-BPI) immunoglobulin but not by preimmune immunoglobulin and (ii) comparison of the dose-response curves of bacterial killing by crude extracts and by purified BPI showed that the bactericidal activity of crude fractions corresponded closely to the BPI content. Human and rabbit BPIs exhibited similar bactericidal potency toward K1-encapsulated E. coli; i.e., <5 μg of either protein killed >90% of 2.5 × 10⁷ bacteria. Thus, the potent bactericidal action of BPI toward E. coli is not impeded by K1 capsule, suggesting that the virulence of K1-encapsulated E. coli is a consequence of extracellular survival but not of resistance to intracellular killing.     

Potent CD14-mediated signalling of human leukocytes by Escherichia coli can be mediated by interaction of whole bacteria and host cells without extensive prior release of endotoxin.

How invading microorganisms are detected by the host has not been well defined. We have compared the abilities of Escherichia coli and lipopolysaccharides (LPS) purified from these bacteria to prime isolated neutrophils for phorbol myristate acetate-stimulated arachidonate release, to trigger respiratory burst in 1% blood, and to increase steady-state levels of tumor necrosis factor alpha mRNA in whole blood. In all three assays, bacteria were > or = 10-fold more potent than equivalent amounts of LPS and could trigger maximal cellular responses at ratios as low as one bacterium per 20 to 200 leukocytes. Both E. coli and LPS-triggered responses were enhanced by LPS-binding protein and inhibited by an anti-CD14 monoclonal antibody and the bactericidal/permeability-increasing protein (BPI). However, whereas O polysaccharide did not affect the potency of isolated LPS, intact E. coli carrying long-chain LPS (O111:B4) was less potent than rough E. coli (J5). Furthermore, material collected by filtration or centrifugation of bacteria incubated under conditions used to trigger arachidonate release or chemiluminescence was 5- or 30-fold less active, respectively, than whole bacterial suspensions. Extracellular BPI (not bound to bacteria) inhibited bacterial signalling, but BPI bound to bacteria was much more potent. Taken together, these findings indicate that E. coli cells can strongly signal their presence to human leukocytes not only by shedding LPS into surrounding fluids but also by exposing endotoxin at or near their surface during direct interaction with host cells.     

From infection to autoimmunity: a new model for induction of ANCA against the bactericidal/permeability increasing protein (BPI)

Antineutrophil cytoplasmic autoantibodies against the neutrophil granule bactericidal/permeability increasing protein (BPI-ANCA) have been found in diseases of different etiologies, such as cystic fibrosis, TAP deficiency or inflammatory bowel diseases. A common feature of these conditions is the chronic or profuse exposure of the host to Gram-negative bacteria and their endotoxin. BPI plays an important role in killing Gram-negative bacteria as well as neutralization and disposal of their endotoxin. During this interaction BPI can direct the delivery of complexes which contain endotoxin and bacterial outer membrane proteins to antigen presenting cells. Based on recent findings on how complexes of endotoxin and protein antigens need to be processed by dendritic cells in order to become presented on MHC class II molecules, a model can be proposed how Gram-negative bacterial infections can be linked to the generation of autoantibodies against BPI.     

The endotoxin-binding bactericidal/permeability-increasing protein (BPI): a target antigen of autoantibodies

The bactericidal/permeability-increasing protein (BPI) is an endotoxin-binding neutrophil leukocyte-granule protein with antibacterial and anti-endotoxin properties. A recombinant form of BPI (rBPI21) has been developed and is being tested as a therapeutic agent to treat gram-negative bacterial infections and exposure to gram-negative bacterial endotoxin. BPI is also a target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA). BPI-ANCA are present in cystic fibrosis, inflammatory bowel disease, vasculitis, and primary sclerosing cholangitis; presence of BPI-ANCA appears associated with a higher inflammatory disease activity and greater organ damage. BPI-ANCA as well as ANCA directed at other neutrophil-granule proteins may exacerbate inflammation by nonspecific effects of extracellular and cell-associated immune complexes. BPI-ANCA may further worsen inflammation by reducing the ability of BPI to promote clearance of gram-negative bacteria and bacterial-associated endotoxin.     

Competition between rBPI23, a recombinant fragment of bactericidal/permeability-increasing protein, and lipopolysaccharide (LPS)-binding protein for binding to LPS and gram-negative bacteria.

Lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) are two structurally related lipid A-binding proteins with divergent functional activities. LBP mediates activation of macrophage and other proinflammatory cells. In contrast, BPI has potent bactericidal and LPS-neutralizing activities. A recombinant fragment of BPI (rBPI23) retains the potent biological activities of the holo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis, and endotoxemia. For therapeutic effectiveness in many clinical situations, rBPI23 will have to successfully compete with high serum levels of LBP for binding to endotoxin and gram-negative bacteria. The relative binding affinities of rBPI23 and human recombinant LBP (rLBP) for lipid A and gram-negative bacteria were evaluated. The binding of both proteins to lipid A was specific and saturable with apparent Kds of 2.6 nM for rBPI23 and 58 nM for rLBP. rBPI23 was approximately 75-fold more potent than rLBP in inhibiting the binding of 125I-rLBP to lipid A. The binding affinity of rBPI23 (Kd = 70 nM) for Escherichia coli J5 bacteria was also significantly higher than that of rLBP (Kd = 1,050 nM). In addition, rBPI23 at 0.2 micrograms/ml was able to inhibit LPS-induced tumor necrosis factor release from monocytes in the presence of 20 micrograms of rLBP per ml. These results demonstrate that rBPI23 binds more avidly to endotoxin than does rLBP and that, even in the presence of a 100-fold weight excess of rLBP, rBPI23 effectively blocks the proinflammatory response of peripheral blood mononuclear cells to endotoxin.     

Potency of bactericidal proteins purified from the large granules of bovine neutrophils.

The novel population of large granules of bovine neutrophils, which is the cell store of bactericidal activity independent of O2 derivatives, was extracted with an acid medium. Several fractions were resolved from the extract by ion-exchange chromatography (with carboxymethyl-cellulose) and gel filtration (with Sephadex G-50). Some of these fractions contained only a very limited number of major components, as detected by polyacrylamide gel electrophoresis. The purified bactericidal proteins exhibited their activity at 0.1 to 10 micrograms/0.3 ml of assay mixture containing 1 X 10(6) to 2 X 10(6) CFU of Staphylococcus aureus or Escherichia coli in media with physiological concentrations of Na+, K+, Mg2+, and Ca2+. Two fractions, containing polypeptides with apparent molecular weights ranging from 28,000 to less than 12,000, caused rather selective and rapid (5 to 20 min) killing of S. aureus. Their action was accompanied by significant binding to the gram-positive bacteria of some low (less than 12,000)-molecular-weight components. Other Sephadex G-50 fractions, containing the first emerging proteins with relatively high molecular weights, were more active on E. coli than on S. aureus. With the gram-negative bacteria there was a 10-min delay in the onset of bactericidal activity, which thereafter developed very fast. On the basis of the in vitro potency of the large-granule bactericidal proteins, we suggest that even partial discharge of granule content into the phagosomes may supply the phagocytic vacuoles of bovine neutrophils with a very efficient nonoxidative bactericidal system acting on both gram-positive and gram-negative microorganisms.     

OmpA of a septicemic Escherichia coli O78--secretion and convergent evolution

OmpA is an important constituent of the outer membrane of Gram-negative bacteria. OmpA is involved in a variety of host-bacteria interactions, including crossing of the blood-brain barrier by E. coli strains causing newborn meningitis, and elicits a significant response by the immune system of the host. The bactericidal effect of neutrophil elastase (NE) is also attributed to degradation of the bacterial OmpA. Here we examined the OmpA of septicemic E. coli 078 strains and show that two surface-exposed loops are conserved among invasive strains of E. coli and other pathogenic Enterobacteriaceae. In addition, there is evidence for convergent evolution, implying the existence of selective pressure. Our results also indicate that large quantities of OmpA are secreted into the medium during all phases of growth, where it is present both in secreted vesicles and as a soluble secreted protein. We assume that secreted OmpA can play a role in protection of bacteria from NE by competitive inhibition. Support for this assumption was obtained from experiments indicating that addition of exogenous, purified OmpA reduces killing of bacteria by NE.     

Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment.

Although the antimicrobial activity of lactoferrin has been well described, its mechanism of action has been poorly characterized. Recent work has indicated that in addition to binding iron, human lactoferrin damages the outer membrane of gram-negative bacteria. In this study, we determined whether bovine lactoferrin and a pepsin-derived bovine lactoferrin peptide (lactoferricin) fragment have similar activities. We found that both 20 microM bovine lactoferrin and 20 microM lactoferricin release intrinsically labeled [3H]lipopolysaccharide ([3H]LPS) from three bacterial strains, Escherichia coli CL99 1-2, Salmonella typhimurium SL696, and Salmonella montevideo SL5222. Under most conditions, more LPS is released by the peptide fragment than by whole bovine lactoferrin. In the presence of either lactoferrin or lactoferricin there is increased killing of E. coli CL99 1-2 by lysozyme. Like human lactoferrin, bovine lactoferrin and lactoferricin have the ability to bind to free intrinsically labeled [3H]LPS molecules. In addition to these effects, whereas bovine lactoferrin was at most bacteriostatic, lactoferricin demonstrated consistent bactericidal activity against gram-negative bacteria. This bactericidal effect is modulated by the cations Ca2+, Mg2+, and Fe3+ but is independent of the osmolarity of the medium. Transmission electron microscopy of bacterial cells exposed to lactoferricin show the immediate development of electron-dense "membrane blisters." These experiments offer evidence that bovine lactoferrin and lactoferricin damage the outer membrane of gram-negative bacteria. Moreover, the peptide fragment lactoferricin has direct bactericidal activity. As lactoferrin is exposed to proteolytic factors in vivo which could cleave the lactoferricin fragment, the effects of this peptide are of both mechanistic and physiologic relevance.     

Enhancement of neonatal innate defense: effects of adding an N-terminal recombinant fragment of bactericidal/permeability-increasing protein on growth and tumor necrosis factor-inducing activity of gram-negative bacteria tested in neonatal cord blood ex vivo

Innate defense against microbial infection requires the action of neutrophils, which have cytoplasmic granules replete with antibiotic proteins and peptides. Bactericidal/permeability-increasing protein (BPI) is found in the primary granules of adult neutrophils, has a high affinity for lipopolysaccharides (or "endotoxins"), and exerts selective cytotoxic, antiendotoxic, and opsonic activity against gram-negative bacteria. We have previously reported that neutrophils derived from newborn cord blood are deficient in BPI (O. Levy et al., Pediatrics 104:1327-1333, 1999). The relative deficiency in BPI of newborns raised the possibility that supplementing the levels of BPI in plasma might enhance newborn antibacterial defense. Here we determined the effects of addition of recombinant 21-kDa N-terminal BPI fragment (rBPI(21)) on the growth and tumor necrosis factor (TNF)-inducing activity of representative gram-negative clinical isolates. Bacteria were tested in citrated newborn cord blood or adult peripheral blood. Bacterial viability was assessed by plating assay, and TNF-alpha release was measured by enzyme-linked immunosorbent assay. Whereas adult blood limited the growth of all isolates except Klebsiella pneumoniae, cord blood also allowed logarithmic growth of Escherichia coli K1/r and Citrobacter koseri. Bacteria varied in their susceptibility to rBPI(21)'s bactericidal action: E. coli K1/r was relatively susceptible (50% inhibitory concentration [IC(50)], approximately 10 nM), C. koseri was intermediate (IC(50), approximately 1,000 nM), Klebsiella pneumoniae was resistant (IC(50), approximately 10,000 nM), and Enterobacter cloacae and Serratia marcescens were highly resistant (IC(50), >10,000 nM). All isolates were potent inducers of TNF-alpha activity in both adult and newborn cord blood. In contrast to its variable antibacterial activity, rBPI(21) consistently inhibited the TNF-inducing activity of all strains tested (IC(50), 1 to 1,000 nM). The antibacterial effects of rBPI(21) were additive with those of a combination of conventional antibiotics typically used to treat bacteremic newborns (ampicillin and gentamicin). Whereas ampicillin and gentamicin demonstrated little inhibition of bacterially induced TNF release, addition of rBPI(21) either alone or together with ampicillin and gentamicin profoundly inhibited release of this cytokine. Thus, supplementing newborn cord blood with rBPI(21) potently inhibited the TNF-inducing activity of a variety of gram-negative bacterial clinical pathogens and, in some cases, enhanced bactericidal activity. These results suggest that administration of rBPI(21) may be of clinical benefit to neonates suffering from gram-negative bacterial infection and/or endotoxemia.     

Antibiotics enhance binding by human lipid A-reactive monoclonal antibody HA-1A to smooth gram-negative bacteria.

The effect of antibiotic exposure of phenotypically smooth gram-negative bacteria on binding by the human lipid A-reactive monoclonal antibody HA-1A (trademark of Centocor, Inc.) was examined by liquid-phase immunoassay and by dual-parameter flow cytometry (fluorescence-activated cell sorter [FACS]) analysis. HA-1A exhibited dose-dependent binding to untreated rough gram-negative bacteria such as the Escherichia coli D21F2 Re chemotype strain but little binding to untreated smooth strains such as E. coli O111:B4, or to gram-positive bacteria. However, overnight incubation of E. coli O111:B4 with inhibitory concentrations of ceftazidime produced dose-dependent enhancement of HA-1A binding. Similar augmentation of HA-1A binding was observed when other smooth strains were exposed to cell wall-active agents. Dual-parameter FACS analysis of E. coli O111:B4 exposed overnight to two times the MIC of ceftazidime revealed a decrease in forward light scatter, indicating a reduction in average cell size or bacterial fragmentation, accompanied by a striking increase in lipid A-inhibitable HA-1A binding. Moreover, ceftriaxone, but not gentamicin, produced a marked increase in propidium iodide uptake, indicating an increase in bacterial cell permeability, and a corresponding enhancement of HA-1A binding. Antibiotic-induced enhancement of HA-1A binding to smooth strains of gram-negative bacteria thus appears related to specific alterations in bacterial cell morphology resulting in exposure of the epitope recognized by HA-1A.     

Role of endotoxin in acute inflammation induced by gram-negative bacteria: specific inhibition of lipopolysaccharide-mediated responses with an amino-terminal fragment of bactericidal/permeability-increasing protein.

A recombinant 23-kDa amino-terminal fragment of human bactericidal/permeability-increasing protein (rBPI23), a potent lipopolysaccharide (LPS)-binding/neutralizing protein, was used as a probe to assess the role of endotoxin in the acute inflammatory responses elicited by gram-negative bacteria in rat subcutaneous air pouches. In initial experiments, rBPI23 prevented the Escherichia coli O111:B4 LPS-induced accumulation of polymorphonuclear leukocytes (PMN), tumor necrosis factor alpha (TNF-alpha), and nitrite (a stable end product of nitric oxide formation) in exudate fluids. Significant inhibition of TNF-alpha production was still evident when rBPI23 treatment was delayed for 30 min after LPS instillation. In subsequent experiments, rBPI23 also prevented the nitrite and early (2-h) TNF-alpha accumulation induced by three different strains of formaldehyde-killed gram-negative bacteria (E. coli O7:K1, E. coli O111:B4, and Pseudomonas aeruginosa 12.4.4) but did not inhibit the PMN or late (6-h) TNF-alpha accumulation induced by these bacteria. As with LPS challenge, a significant inhibition of early TNF-alpha production was still evident when rBPI23 treatment was delayed for 30 to 60 min after instillation of killed bacteria. The results indicate that in this experimental model the NO and early TNF-alpha responses to gram-negative bacterial challenge are mediated predominantly by endotoxin, whereas the PMN and late TNF-alpha responses may be mediated by other bacterial components. Moreover, the results indicate that rBPI23 can inhibit the bacterially induced production of certain potentially harmful mediators (TNF-alpha and NO) without entirely blocking the host defense, i.e., PMN response, against the bacteria.     

High-affinity binding of the bactericidal/permeability-increasing protein and a recombinant amino-terminal fragment to the lipid A region of lipopolysaccharide.

Bactericidal/permeability-increasing protein (BPI) is a 55-kDa cationic protein (nBPI55) elaborated by polymorphonuclear neutrophils (PMN). BPI has potent bactericidal activity against a wide variety of gram-negative organisms and neutralizes endotoxin activities. An N-terminal fragment of nBPI55 exhibits the bactericidal and antiendotoxin properties of the holoprotein. To further characterize the biological activities of the N-terminal fragment, a recombinant protein (rBPI23) corresponding to the first 199 amino acids of human BPI was produced and purified. rBPI23 had antibacterial activity equivalent to that of nBPI55 against Escherichia coli J5. Furthermore, both rBPI23 and nBPI55 bound identically to a broad range of R- and S-form lipopolysaccharides (LPS) and to natural and synthetic lipid A. Binding of radiolabeled nBPI55 to LPS was inhibited in an identical fashion by either nBPI55 or rBPI23. The binding of both proteins to immobilized E. coli J5 lipid A was inhibited in a comparable fashion by long- or short-chain LPS or lipid A. The binding of both rBPI23 and nBPI55 was specific, saturable, and of high affinity, with an apparent Kd of approximately 2 to 5 nM for all ligands tested. These results demonstrate that BPI recognizes the highly conserved lipid A region of bacterial LPS via residues contained within the amino-terminal portion of the BPI molecule.     

Killing of an encapsulated strain of Escherichia coli by human serum.

Changes in cell viability and in factors affecting metabolic integrity were examined after exposure of Escherichia coli LP1092 to human serum. Antibody-dependent classical pathway activity accounted for the rapid killing of strain LP1092 by complement. Removal of serum lysozyme by bentonite absorption or by neutralization with anti-human lysozyme immunoglobulin G resulted in a reduction in the rate of killing; optimal activity could be restored by the addition of physiological amounts of egg-white lysozyme. The pattern of 86Rb+ and alkaline phosphatase release obtained after serum treatment did not support the view that complement simultaneously disrupts cytoplasmic and outer membrane integrity. Macromolecular synthesis was affected late in the reaction sequence; complete inhibition of precursor incorporation into RNA, DNA, and protein occurred only after almost total loss of bacterial colony-forming ability. Addition of chloramphenicol, an inhibitor of protein synthesis, to the bactericidal system resulted in a marked reduction in the rate of serum killing. Killing was completely inhibited by an inhibitor (KCN) and an uncoupler (2,4-dinitrophenol) of oxidative phosphorylation. Exposure of LP1092 cells to serum was followed by a rapid and large increase in intracellular ATP levels; ATP synthesis did not occur when bacteria were exposed to dialyzed serum, which killed LP1092 cells at a much reduced rate. Addition of glucose or serum ultrafiltrate to dialyzed serum restored optimal bactericidal activity. We suggest that optimal killing of gram-negative bacteria is an energy-dependent process requiring an input of bacterially generated ATP.     

Bactericidal/permeability-increasing protein promotes complement activation for neutrophil-mediated phagocytosis on bacterial surface

The neutrophil bactericidal/permeability-increasing protein (BPI) has both bactericidal and lipopolysaccharide-neutralizing activities. The present study suggests that BPI also plays an important role in phagocytosis of Escherichia coli by neutrophils through promotion of complement activation on the bacterial surface. Flow cytometric analysis indicated that fluorescein-labelled E. coli treated with BPI were phagocytosed in the presence of serum at two- to five-fold higher levels than phagocytosis of the bacteria without the treatment. In contrast, phagocytosis of the fluoresceined bacteria with or without treatment by BPI did not occur at all in the absence of serum. The phagocytosis stimulated by BPI and serum was dose-dependent. The effect of BPI on phagocytosis in the presence of serum was not observed on Gram-positive bacteria (Staphylococcus aureus). Interestingly, the complement C3b/iC3b fragments were deposited onto the bacterial surface also as a function of the BPI concentration under conditions similar to those for phagocytosis. Furthermore, the BPI-promoted phagocytosis was blocked completely by anti-C3 F(ab')(2) and partially by anti-complement receptor (CR) type 1 and/or anti-CR type 3. These findings suggest that BPI accelerates complement activation to opsonize bacteria with complement-derived fragments, leading to stimulation of phagocytosis by neutrophils via CR(s).     

Determinants of activation by complement of group II phospholipase A2 acting against Escherichia coli.

Prompt killing of many strains of Escherichia coli during phagocytosis in vitro by isolated polymorphonuclear leukocytes (PMN) requires the presence of nonlethal doses of nonimmune serum (B. A. Mannion, J. Weiss, and P. Elsbach, J. Clin. Invest. 86:631-641, 1990). Because this requirement is bypassed in a phospholipase A (PLA)-rich mutant (pldA ) of E. coli, we have examined the effect of serum on bacteria] phospholipid (PL) degradation during phagocytosis of wild-type (pldA+) and PLA-deficient (pldA) E. coli. In parallel with increased killing, nonlethal doses of serum increased the degradation of prelabeled bacterial PL during phagocytosis by two- to fivefold, to nearly the same levels (ca. 50 to 60%) as those produced during phagocytosis of E. coli pldA in the absence of serum. The effects on the E. coli pldA mutant imply that there is a serum-mediated enhancement of granule-associated group II PMN PLA2 activity. At the same doses, serum promoted action against E. coli in the presence of purified rabbit and human group II PLA2 but did not activate bacterial PLA. Related PLA2s that lack specific structural determinants needed for optimal activity against E. coli treated with the bactericidal/permeability-increasing protein (BPI) of PMN are also less active than wild-type group II PLA2 against serum-treated E. coli. Treatment of E. coli with C7- or C9-depleted serum did not enhance bacterial killing or PL degradation during phagocytosis or the action of purified PLA2. In summary, these findings suggest that (i) nonlethal assemblies of the membrane attack complex promote intracellular killing and destruction of E. coli ingested by PMN, in part by promoting the action of granule-associated PLA2 against ingested bacteria, and (ii) structural determinants first implicated in PLA2 action against BPI-treated E. coli are also important in PLA2 action in concert with other host defense systems, such as complement.     

Bactericidal activity of human eosinophilic granulocytes against Escherichia coli

Eosinophils participate in allergic inflammation and may have roles in the body's defense against helminthic infestation. Even under noninflammatory conditions, eosinophils are present in the mucosa of the large intestine, where large numbers of gram-negative bacteria reside. Therefore, roles for eosinophils in host defenses against bacterial invasion are possible. In a system for bacterial viable counts, the bactericidal activity of eosinophils and the contribution of different cellular antibacterial systems against Escherichia coli were investigated. Eosinophils showed a rapid and efficient killing of E. coli under aerobic conditions, whereas under anaerobic conditions bacterial killing decreased dramatically. In addition, diphenylene iodonium chloride (DPI), an inhibitor of the NADPH oxidase and thereby of superoxide production, also significantly inhibited bacterial killing. The inhibitor of nitric oxide (NO) production L-N(5)-(1-iminoethyl)-ornithine dihydrochloride did not affect the killing efficiency, suggesting that NO or derivatives thereof are of minor importance under the experimental conditions used. To investigate the involvement of superoxide and eosinophil peroxidase (EPO) in bacterial killing, EPO was blocked by azide. The rate of E. coli killing decreased significantly in the presence of azide, whereas addition of DPI did not further decrease the killing, suggesting that superoxide acts in conjunction with EPO. Bactericidal activity was seen in eosinophil extracts containing granule proteins, indicating that oxygen-independent killing may be of importance as well. The findings suggest that eosinophils can participate in host defense against gram-negative bacterial invasion and that oxygen-dependent killing, i.e., superoxide acting in conjunction with EPO, may be the most important bactericidal effector function of these cells.     

Outer membranes are competitive inhibitors of Escherichia coli O157:H7 adherence to epithelial cells.

Escherichia coli of serotype O157:H7 are Vero cytotoxin-producing enteric pathogens that have been associated recently with sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic-uremic syndrome. Adherence of many enteropathogenic bacteria to mucosal surfaces is a critical step in the pathogenesis of diarrheal disease. We showed previously that adherence of E. coli O157:H7 strain CL-56 to epithelial cells in vitro is inhibited by outer membranes. In this study we examined whether outer membranes from a series of E. coli O157:H7 strains mediated competitive inhibition of bacterial binding to epithelial cells grown in tissue culture. We also determined which constituents of the outer membrane mediated inhibition of CL-56 adherence. Binding of six O157:H7 strains to HEp-2 cells was determined by quantitating the number of adherent bacteria in the presence and absence of outer membranes which were extracted from each strain with N-lauroyl sarcosinate (1.7%, wt/vol). After separation of outer membranes by gel electrophoresis, four bands (94, 40, 36, and 30 kDa) were collected by electroelution. Immune sera were raised in rabbits to each of the four eluted bands. Outer membrane extracts from each of the six O157:H7 strains inhibited binding of homologous organisms to the HEp-2 cells. At dilutions which did not cause bacterial agglutination, antiserum raised against the 94-kDa outer membrane protein showed maximal inhibition of bacterial adherence (17.0 +/- 7.3% adherence of control levels). Growth of bacteria in iron-depleted broth did not affect their binding to HEp-2 cells, suggesting that iron-regulated outer membranes were not involved. Fluid accumulation in ileal ligated loops of rabbits in response to E. coli O157:H7 challenge was diminished following both parenteral immunization with outer membranes extracted from the homologous strain and coincubation of organisms with immune serum which contained antibodies to outer membrane extracts. These data indicate that outer membranes are competitive inhibitors of E. coli O157:H7 adherence. Specific constituents of the outer membrane may function as bacterial attachment factors (i.e., adhesins) for E. coli O157:H7 adherence to epithelial cell surfaces.     

Molecular characterization of the acute inflammatory response to infections with gram-negative versus gram-positive bacteria

Sepsis caused by gram-negative bacteria and that caused by gram-positive bacteria often manifest similar clinical features. We investigated plasma proinflammatory cytokine profiles in patients with sepsis due to gram-positive and gram-negative bacteria and studied the cytokine production and differential gene regulation of leukocytes stimulated ex vivo with Escherichia coli lipopolysaccharide or heat-killed Staphylococcus aureus. Concentrations of tumor necrosis factor alpha, interleukin 1 receptor antagonist (IL-1Ra), IL-8, IL-10, IL-18 binding protein, procalcitonin, and protein C in plasma did not differ between patients with sepsis due to gram-negative and gram-positive bacteria. However, plasma IL-1beta, IL-6, and IL-18 concentrations were significantly higher in patients with sepsis due to gram-positive bacteria. Ex vivo stimulation of whole blood with heat-killed S. aureus markedly increased IL-1beta and IL-18 levels more than E. coli lipopolysaccharide stimulation. Microarray analysis revealed at least 359 cross-validated probe sets (genes) significant at the P < 0.001 level whose expression discriminated among gram-negative-organism-stimulated, gram-positive-organism-stimulated, and unstimulated whole-blood leukocytes. The host inflammatory responses to gram-negative and gram-positive stimuli share some common response elements but also exhibit distinct patterns of cytokine appearance and leukocyte gene expression.     

Bacterial killing in vitro by abscess-derived neutrophils

In the absence of antimicrobial therapy, bacteria such as Bacteriodes fragilis, Escherichia coli and Proteus mirabilis may persist within an intra-abdominal abscess in the presence of large numbers of neutrophils which, under optimal conditions in vitro, can readily phagocytose and kill the same bacterial strains. Neutrophils taken from abscesses induced by gram-negative bacteria such as those above contain viable organisms. On incubation in vitro in the presence of serum, these neutrophils kill the bacteria phagocytosed in the abscess poorly, if at all, yet can readily kill organisms added in vitro. To determine possible mechanisms that might explain this, we examined the bactericidal activity in vitro of neutrophils from a range of abscesses induced by one or two species of bacteria plus an abscess-potentiating agent, bran. The organisms studied were B. fragilis, E. coli, P. mirabilis and Staphylococcus aureus. The killing in vitro of E. coli and P. mirabilis, engulfed within an abscess, was significantly less than that of the same organisms when they were added to the in-vitro assay. In contrast, the killing of S. aureus was similar, whether engulfed in vivo or in vitro. However, S. aureus was less susceptible to phagocytosis and killing in vitro than P. mirabilis or E. coli, and the killing of S. aureus during in-vitro incubation of neutrophils that had engulfed the organism with in the abscess was similar to that of the gram-negative bacteria engulfed within the abscess.(ABSTRACT TRUNCATED AT 250 WORDS)