Killing Of Gram-negative Bacteria Research Articles

Selective inhibitory effects of the anticoagulant activated protein C on the responses of human mononuclear phagocytes to LPS, IFN-gamma, or phorbol ester.

Recent studies have shown that infusion of the anticoagulant protein, activated protein C (APC), can ameliorate many of the systemic effects of endotoxemia in experimental animals, although the mechanisms in this action are unknown. We investigated the effects of APC on the responses of blood monocytes, alveolar macrophages, and cells of the monocyte line, THP-1, to stimulation in vitro by LPS, IFN-gamma, or PMA. Mononuclear phagocyte (MO) activation was associated with rapid production of TNF-alpha, down-regulation of the glycophosphatidylinositol-linked protein CD14 (the key MO receptor for complexes of LPS and LPS-binding protein responsible for intracellular signaling), and down-regulation of the related LPS-binding proteins CD11b and CD18. Addition of APC, but not the zymogen, PC, or active site-blocked APC, inhibited selected MO responses involving the CD14-dependent LPS-induced pathway of MO activation, or activation induced by IFN-gamma or PMA. Thus, APC inhibited the production of TNF-alpha and prevented down-regulation of membrane CD11b, CD14, and CD18, but had no effect on up-regulation of MHC class II, ICAM-1, or IL-2R, down-regulation of MO expression of another glycophosphatidylinositol-linked protein, CD59, or production of reactive oxygen intermediates. These data show that APC inhibits host cytokine production but maintains MO responses associated with adhesion, phagocytosis, and killing of Gram-negative bacteria, such that use of APC may be a logical and potent adjunctive therapy in select inflammatory diseases involving MO activation and damaging host cytokine overproduction.

A recombinant amino terminal fragment of bactericidal/permeability-increasing protein inhibits the induction of leukocyte responses by LPS

Bactericidal/permeability-increasing protein (BPI) is a major component of the granules of polymorphonuclear neutrophils (PMNs) and is involved in the killing of gram-negative bacteria. A 23-kd recombinant protein, corresponding to the NH2-terminal fragment of human BPI (rBPI23), has been shown to bind lipid A and antagonize some lipopolysaccharide (LPS)-mediated effects. In this study the ability of rBPI23 to prevent a wide range of cellular responses to LPS was investigated. In vitro assays were carried out using human blood to more closely approximate in vivo conditions. The release of proinflammatory cytokines [tumor necrosis factor (TNF), interleukin-1 beta (IL-1 beta), IL-6, IL-8], induced by E. coli O113 LPS, was markedly reduced by rBPI23 in a concentration-dependent fashion. The production of the anti-inflammatory protein IL-1ra (IL-1 receptor antagonist) was triggered by lower LPS concentrations than those necessary for the other cytokines. Furthermore, prevention of IL-1ra release required higher rBPI23 concentrations than for other cytokines. The LPS-induced production of oxygen-derived free radicals by phagocytic cells (resulting in chemiluminescence) was also prevented by rBPI23. The inhibition was specific for LPS because the activation of leukocytes by phorbol myristate acetate, zymosan, or TNF was unaffected by BPI. The ability of rBPI23 to antagonize specifically the effects of endotoxin in the complex environment of human blood along with its bactericidal activity suggests that rBPI23 may be a novel therapeutic agent in the treatment of gram-negative infections.

Molecular mechanism of hemolymph clotting system in Limulus

Limulus (horseshoe crab) hemolymph is known to be very sensitive to bacterial endotoxin (LPS), which causes a rapid coagulation response. Hemolymph contains a single type of hemocyte that undergoes aggregation, adhesion, and degranulation in response to LPS. The granule contents are released into the hemolymph, where they form an insoluble gel. We have characterized four components involved in this coagulation response that comprise a cascade of three serine protease zymogens (factor C, factor B, and proclotting enzyme) and one clottable protein (coagulogen). Of these components, factor C sensitive to LPS is a protein composed of five complement-related domains (“Sushi” or SCR), an EGF-like domain, and a C-type lectinlike domain as well as a putative amino-terminal LPS-binding domain. This domain structure is very similar to that of selectin family of cell adhesion molecules, suggesting that it might also function as a cell adhesion molecule after the release into the hemolymph. Factor B and the proclotting enzyme share a common Cys-rich motif (“cliplike” domain) in the amino-terminal portions. This domain is also found in a putative serine protease zymogen (“easter”) in Drosophila, which is essential for normal embryonic development. All four of the components of the cascade and an antibacterial protein (anti-LPS factor) are localized to a specific type of the hemocyte granule. Another antibacterial peptide (tachyplesins I and II) is localized in a distinct granule population. The contents of both granule populations are released into the hemolymph in response to LPS, where they cooperate in immobilization and killing of Gram-negative bacteria.

Killing of gram-negative bacteria by lactoferrin and lysozyme.

Although lactoferrin has antimicrobial activity, its mechanism of action is not full defined. Recently we have shown that the protein alters the Gram-negative outer membrane. As this membrane protects Gram-negative cells from lysozyme, we have studied whether lactoferrin's membrane effect could enhance the antibacterial activity of lysozyme. We have found that while each protein alone is bacteriostatic, together they can be bactericidal for strains of V. cholerae, S. typhimurium, and E. coli. The bactericidal effect is dose dependent, blocked by iron saturation of lactoferrin, and inhibited by high calcium levels, although lactoferrin does not chelate calcium. Using differing media, the effect of lactoferrin and lysozyme can be partially or completely inhibited; the degree of inhibition correlating with media osmolarity. Transmission electron microscopy shows that E. coli cells exposed to lactoferrin and lysozyme at 40 mOsm become enlarged and hypodense, suggesting killing through osmotic damage. Dialysis chamber studies indicate that bacterial killing requires direct contact with lactoferrin, and work with purified LPS suggests that this relates to direct LPS-binding by the protein. As lactoferrin and lysozyme are present together in high levels in mucosal secretions and neutrophil granules, it is probable that their interaction contributes to host defense.

Antibody‐Targeted Photolysisa

Antibody-targeted photolysis is a technique for damaging or killing cells using light and an antibody-bound photosensitizer. In the present study, immunoconjugates were constructed to selectively kill Pseudomonas aeruginosa bacteria using tin (IV) chlorin e6 which was linked to dextran and then bound to the carbohydrate moiety of a monoclonal antibody specific for Pseudomonas aeruginosa Fisher type I polysaccharide antigen. Killing of Pseudomonas during mid-log phase growth was shown to be dependent upon light dose with complete bacterial cell killing observed at an irradiation dose of 80 J/cm2. Individual components of the immunoconjugates (e.g., antibody or chlorin alone) showed no bacterial cytotoxicity and immunoconjugates constructed with nonbinding antibodies were also ineffective as cytotoxic agents. These studies demonstrate that killing of gram negative bacteria using photoradiation is feasible and suggest that this methodology may be applicable in treatment of infections in man.

Killing of gram-negative bacteria by neutrophils: role of O2-independent system in intracellular killing and evidence of O2-dependent extracellular killing.

For many strains of these two bacterial species, ingested bacteria are efficiently killed by O2-independent mechanisms. Intracellular killing depends not on 2de novo generation of toxic products of the respiratory burst but rather on intracellular delivery to pre-existing cytotoxic proteins. The principal O2- independent bactericidal system toward these bacteria appears to be BPI which rapidly binds to ingested bacteria and whose discrete action closely resembles the initial lesions produced by the intact neutrophil. In addition, at least for one strain of E. coli (S15), extracellular bacteria can be killed in an O2- dependent fashion.

Killing of gram-negative bacteria by polymorphonuclear leukocytes: role of an O2-independent bactericidal system.

Previous studies have suggested that a cationic bactericidal/permeability-increasing protein (BPI) present in both rabbit and human polymorphonuclear leukocytes is the principal O2-independent bactericidal agent of these cells toward several strains of Escherichia coli and Salmonella typhimurium (1978. J. Biol. Chem. 253: 2664--2672; 1979. J. Biol. Chem. 254: 11000--11009). To further evaluate the possible role of this protein in the killing of gram-negative bacteria by polymorphonuclear leukocytes, we have measured the bactericidal activity of intact rabbit peritoneal exudate leukocytes under aerobic or anaerobic conditions and of intact human leukocytes from a patient with chronic granulomatous disease. Anaerobic conditions were created by flushing the cells under a nitrogen stream. Effective removal of oxygen was demonstrated by the inability of nitrogen-flushed leukocytes to mount a respiratory burst (measured as increased conversion of 1-[14C]glucose leads to 14CO2 or by superoxide production) during bacterial ingestion. At a bacteria/leukocyte ratio of 10:1, killing of gram-positive, BPI-resistant, Staphylococcus epidermidis is markedly impaired in the absence of oxygen (76.4 +/- 3.3% killing in room air, 29.2 +/- 8.2% killing in nitrogen). Essentially all increased bacterial survival is intracellular. In contrast, both a nonopsonized rough strain (MR-10) and an opsonized smooth strain (MS) of S. typhimurium 395 are killed equally well in room air and nitrogen. A maximum of 70--80 MR-10 and 30--40 MS are killed per leukocyte either in the presence or absence of oxygen. There is no intracellular bacterial survival in either condition indicating that intracellular O2-independent bactericidal system(s) of rabbit polymorphonuclear leukocytes can at least match the leukocyte's ingestive capacity. Whole homogenates and crude acid extracts manifest similar bactericidal capacity toward S. typhimurium 395. This activity can be accounted for by the BPI content of these cell fractions and is virtually eliminated by immune (anti-BPI), but not by preimmune goat IgG-rich fractions. Opsonization of smooth MS, required for bacterial killing by intact leukocytes, does not alter bacterial sensitivity to BPI in crude or purified form. Leukocytes of a patient with chronic granulomatous disease killed ingested S. typhimurium 396 MS nearly as well as did normal leukocytes. The bactericidal activity toward E. coli (J5) of crude acid extracts of the CGD and normal human leukocytes was virtually the same and was nearly completely inhibited by anti-BPI IgG-rich fractions, but not by preimmune IgG-rich fractions. These findings suggest that the killing of gram-negative bacteria such as S. typhimurium by intact polymorphonuclear leukocytes may also be attributed to the action of BPI.

O2-independent killing of gram-negative bacteria by intact granulocytes. The role of a potent bactericidal membrane-perturbing protein.

During the past two decades many students of the microbicidal activity of polymorphonuclear leukocytes (PMN) have become increasingly convinced that the important role of the PMN in host defense against bacterial infection depends predominantly on the cell’s ability to generate cytotoxic systems that utilize oxygen1,2. This view has been strongly supported by the observations on patients with chronic granulomatous disease, whose leukocytes cannot generate these systemsl,2.