Leukotriene B4 triggers the in vitro and in vivo release of potent antimicrobial agents

Leukotriene B(4) (LTB(4)) is a bioactive lipid derived from the metabolism of arachidonic acid. Mainly produced by polymorphonuclear leukocytes (PMN) and macrophages, LTB(4) triggers several functional responses important in host defense, including the secretion of lysosomal enzymes, the activation of NADPH oxidase activity, NO formation, and phagocytosis. We report that LTB(4), but not structural analogs thereof, stimulates primed human PMN to release molecules having potent antimicrobial activities. Exposure of bacteria (Escherichia coli and Staphylococcus aureus) or viruses (herpes simplex virus type 1 and HIV type 1) to supernatants of LTB(4)-activated PMN led to > or =90% reduction in infectivity. ELISA and mass spectroscopy analysis of proteins released from LTB(4)-activated PMN have identified several antimicrobial proteins, including alpha-defensins, cathepsin G, elastase, lysozyme C, and LL-37, that are likely to participate in the killing of microorganisms. In addition to these in vitro observations, i.v. injections of LTB(4) (50 microg/kg) to monkeys led to an increase in alpha-defensin plasmatic levels and enhanced ex vivo antimicrobial activities of plasma. These results demonstrate the ability of LTB(4) to cause the release of potent antimicrobial agents from PMN in vitro as well as in vivo and add further support to the important role of LTB(4) in host defense.     

Differential antibacterial activity of nitric oxide from the immunological isozyme of nitric oxide synthase transduced into endothelial cells.

Primary cultures of endothelial cells, grown on the three-dimensional matrix Gelfoam where they take on the morphology of these cells in vivo, were found to phagocytose Staphylococcus aureus and two strains of Escherichia coli. The phagocytosis was independent of opsonization, although once opsonized, these bacteria were phagocytosed by endothelial cells. As cytochalsin D inhibited the internationalization of S. aureus and E. coli, the phagocytosis by endothelial cells appears to be actin-dependent. Transducing the gene for nitric oxide synthase (NOS) II into endothelial cells allowed us to determine the importance of NO(*) in host immunity against these bacteria. While the growth of S. aureus was impeded by NOS II endothelial cells, two strains of E. coli were killed by an NO(*)-dependent pathway. We conclude that endothelial cells have microbicidal mechanisms that are selective for the type of pathogen encountered.     

The effect of cytokines on bactericidal activity of murine neutrophils

Abstract A range of recombinant cytokines have now been shown to modify aspects of the phenotype and function of human and murine neutrophils. However, few reports describe modification of the bactericidal activity of neutrophils. We therefore examined the recombinant murine cytokines tumor necrosis factor-α (TNF-α, 10–1000 ng ml⁻¹) and granulocyte macrophage-colony stimulating factor (GM-CSF, 10–1000 U ml⁻¹) for their ability to increase the bacterial killing capacity of murine neutrophils. Neutrophils from either bone marrow (fresh or cultured), or peritoneal exudates, or abscesses, were pre-incubated with either cytokine for 30–60 min and the killing of Proteus mirabilis, Escherichia coli , or Bacteriodes fragilis was examined in the presence or absence of serum over a 90 min period. Only for one combination was a small but significantly enhanced level of bacterial killing observed, the phagocytic killing of P. mirabilis by peritoneal exudate neutrophils in the presence of GM-CSF and serum. With this exception there was no enhancement of bacterial killing for the range of combinations of neutrophils and bacterial species tested. In contrast, at the concentrations tested for effect on bactericidal activity, TNF-α and GM-CSF were able to significantly upregulate CR3(but not FcγRII) expression on mouse neutrophils. There results indicate that upregulation of CR3 as an index of neutrophil activation does not necessarily correlate with increased bactericidal activity.     

Phagocytosis and killing of bacteria and yeast by human milk cells after opsonisation in aqueous phase of milk.

Macrophages and neutrophils from human milk phagocytose and kill Staphylococcus aureus and Escherichia coli in vitro after opsonisation by the aqueous phase of milk as effectively as blood leucocytes in serum. They also phagocytose Candida albicans. The overgrowth of E coli resulting from the addition of iron to cultures of the organism in the aqueous phase of milk is not influenced by the presence of cells. We conclude that the phagocytosis and killing of bacteria by milk cells may contribute to the lower incidence of infection among breast-fed than artificially fed babies.     

Polymorphonuclear leukocyte migration across model intestinal epithelia enhances Salmonella typhimurium killing via the epithelial derived cytokine,IL-6

The host response to Salmonella typhimurium involves movement of polymorphonuclear leukocytes (PMN) across the epithelium and into the intestinal lumen. Following their arrival in the lumen, the PMN attempt to combat bacterial infection by activating antimicrobial defenses such as granule release, oxidative burst, phagocytosis, and cell signaling. We sought to examine PMN-S. typhimurium interaction following PMN arrival in the lumenal compartment. Here, for the first time, we demonstrate that PMN that have transmigrated across model intestinal epithelia have an enhanced ability to kill S. typhimurium. Our data provide evidence to indicate that the extracellular release of the primary and secondary granules of PMN, myeloperoxidase and lactoferrin, respectively, is correlated with enhanced bacterial killing. Furthermore, epithelial cells, during PMN transmigration, release the cytokine IL-6. IL-6 is known to increase intracellular stores of Ca(2+), and we have determined that this epithelial released cytokine is not only responsible for priming the PMN to release their granules, but also stimulating the PMN to kill S. typhimurium. These results substantiate the pathway in which PMN transmigration activates the epithelial release of IL-6, which in turn increases intracellular Ca(2+) storage. Our results, herein, extend this pathway to include an enhanced PMN granule release and an enhanced killing of S. typhimurium.     

Selective regulation of IL-10 signaling and function by zymosan

Balanced activity of pro- and anti-inflammatory cytokines during innate immune responses is required to allow effective host defense while avoiding tissue damage and autoimmunity. Induction of cytokine production after recognition of pathogen-associated molecular patterns (PAMPs) by innate immune cells has been well demonstrated, but modulation of cytokine function by PAMPs is not well understood. In this study we show that stimulation of macrophages with zymosan, which contains PAMPs derived from yeast, rapidly extinguished macrophage responses to IL-10, a suppressive cytokine that limits inflammatory tissue damage but also compromises host defense. The mechanism of inhibition involved protein kinase Cbeta and internalization of IL-10R, and was independent of TLR2 and phagocytosis. Inhibition of IL-10 signaling and function required direct contact with zymosan, and cells in an inflammatory environment that had not contacted zymosan remained responsive to the paracrine activity of zymosan-induced IL-10. These results reveal a mechanism that regulates IL-10 function such that antimicrobial functions of infected macrophages are not suppressed, but the activation of surrounding noninfected cells and subsequent tissue damage are limited. The fate of individual cells in an inflammatory microenvironment is thus specified by dynamic interactions among host cells, microbes, and cytokines that determine the balance between protection and pathology.     

Role of leukotrienes in killing of Mycobacterium bovis by neutrophils

The neutrophil (PMN) plays an important role in the phagocytosis and killing of microorganisms. Pro-inflammatory leukotrienes (LT) play an important role in various disease states. However LT elaborated by PMN have also been shown to be important in host defense, specifically phagocytosis and killing of microorganisms. Defective LT synthesis by phagocytes correlates with their reduced anti-microbial activity. Therefore, we determined if LT played an important role in the killing of Mycobacteria bovis (M. bovis) by PMN. Endogenous LT play a role in the killing of mycobacteria since the LT synthesis inhibitor MK-886 reversed the killing of M. bovis by PMN. Increased synthesis of LT occurred following incubation of PMN with M. bovis. Treatment with granulocyte-colony stimulating factor, which augments PMN LT synthesis, also boosted anti-microbial activity. Furthermore, exogenous LTB4 augmented dose-dependent killing of M. bovis by PMN. In conclusion, LT play a vital role in promoting mycobactericidal actions of PMN.     

Critical role of prostaglandin E2 overproduction in impaired pulmonary host response following bone marrow transplantation

The success of bone marrow transplantation (BMT) as a therapy for malignant and inherited disorders is limited by infectious complications. We previously demonstrated syngeneic BMT mice are more susceptible to Pseudomonas aeruginosa pneumonia due to defects in the ability of donor-derived alveolar macrophages (AMs), but not polymorphonuclear leukocytes (PMNs), to phagocytose bacteria. We now demonstrate that both donor-derived AMs and PMNs display bacterial killing defects post-BMT. PGE2 is a lipid mediator with potent immunosuppressive effects against antimicrobial functions. We hypothesize that enhanced PGE2 production post-BMT impairs host defense. We demonstrate that lung homogenates from BMT mice contain 2.8-fold more PGE2 than control mice, and alveolar epithelial cells (2.7-fold), AMs (125-fold), and PMNs (10-fold) from BMT animals all overproduce PGE2. AMs also produce increased prostacyclin (PGI2) post-BMT. Interestingly, the E prostanoid (EP) receptors EP2 and EP4 are elevated on donor-derived phagocytes post-BMT. Blocking PGE2 synthesis with indomethacin overcame the phagocytic and killing defects of BMT AMs and the killing defects of BMT PMNs in vitro. The effect of indomethacin on AM phagocytosis could be mimicked by an EP2 antagonist, AH-6809, and exogenous addition of PGE2 reversed the beneficial effects of indomethacin in vitro. Importantly, in vivo treatment with indomethacin reduced PGE2 levels in lung homogenates and restored in vivo bacterial clearance from the lung and blood in BMT mice. Genetic reduction of cyclooxygenase-2 in BMT mice also had similar effects. These data clearly demonstrate that overproduction of PGE2 post-BMT is a critical factor determining impaired host defense against pathogens.     

Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte adhesion and phagocytosis.

Several structural homologues of the chemotactic peptide neutrophil-activating peptide 1/IL-8 (NAP-1/IL-8) were tested for their ability to influence the expression and function of adhesion-promoting receptors on human polymorphonuclear leukocytes (PMN). NAP-2, melanoma growth stimulatory activity, and two forms of NAP-1/IL-8 (ser-NAP-1/IL-8 and ala-NAP-1/IL-8, consisting of 72 and 77 amino acids, respectively), each caused an increase in the expression of CD11b/CD18 (CR3) and CR1, which was accompanied by a decrease in the expression of leukocyte adhesion molecule-1 (LAM-1, LECAM-1). The binding activity of CD11b/CD18 was also enhanced 3- to 10-fold by these peptides, but enhanced function was transient: binding of erythrocytes coated with C3bi reached a maximum by 30 min and declined thereafter. Ser-NAP-1/IL-8, ala-NAP-1/IL-8, NAP-2, and melanoma growth stimulatory activity also caused a two- to threefold enhancement of the phagocytosis of IgG-coated erythrocytes (EIgG) by PMN without causing a large increase in the expression of Fc gamma receptors. Enhanced phagocytosis of EIgG appeared to be mediated through CD11b/CD18, because F(ab')2 fragments of an antibody directed against CD18 inhibited NAP-1/IL-8-stimulated ingestion of EIgG. The four active peptides caused a rapid, transient increase in the amount of F-actin within PMN, indicating that they are capable of influencing the structure of the microfilamentous cytoskeleton, which participates in phagocytosis. Two other NAP-1/IL-8-related peptides, platelet factor 4 and connective tissue-activating peptide III, were without effect on expression of CD11b/CD18, CR1, and LAM-1, binding activity of CD11b/CD18, or Fc-mediated phagocytosis, and increased actin polymerization only slightly. Our observations indicate that several members of the NAP-1/IL-8 family of peptides were capable of promoting integrin-mediated adhesion and Fc-mediated phagocytosis, processes important in the recruitment of PMN to sites of inflammation and antimicrobial responses of PMN.     

Local IL-13 gene transfer prior to immune-complex arthritis inhibits chondrocyte death and matrix-metalloproteinase-mediated cartilage matrix degradation despite enhanced joint inflammation

During immune-complex-mediated arthritis (ICA), severe cartilage destruction is mediated by Fcγ receptors (FcγRs) (mainly FcγRI), cytokines (e.g. IL-1), and enzymes (matrix metalloproteinases (MMPs)). IL-13, a T helper 2 (Th2) cytokine abundantly found in synovial fluid of patients with rheumatoid arthritis, has been shown to reduce joint inflammation and bone destruction during experimental arthritis. However, the effect on severe cartilage destruction has not been studied in detail. We have now investigated the role of IL-13 in chondrocyte death and MMP-mediated cartilage damage during ICA. IL-13 was locally overexpressed in knee joints after injection of an adenovirus encoding IL-13 (AxCAhIL-13), 1 day before the onset of arthritis; injection of AxCANI (an empty adenoviral construct) was used as a control. IL-13 significantly increased the amount of inflammatory cells in the synovial lining and the joint cavity, by 30% to 60% at day 3 after the onset of ICA. Despite the enhanced inflammatory response, chondrocyte death was diminished by two-thirds at days 3 and 7. The mRNA level of FcγRI, a receptor shown to be crucial in the induction of chondrocyte death, was significantly down-regulated in synovium. Furthermore, MMP-mediated cartilage damage, measured as neoepitope (VDIPEN) expression using immunolocalization, was halved. In contrast, mRNA levels of MMP-3, -9, -12, and -13 were significantly higher and IL-1 protein, which induces production of latent MMPs, was increased fivefold by IL-13. This study demonstrates that IL-13 overexpression during ICA diminished both chondrocyte death and MMP-mediated VDIPEN expression, even though joint inflammation was enhanced.     

Bactericidal/permeability-increasing protein has endotoxin-neutralizing activity

Neutrophil granules contain proteins important in host defense against bacterial pathogens. Granule proteins released from activated neutrophils facilitate opsonization, phagocytosis, tissue digestion, and antimicrobial activity. Three similar, if not identical, neutrophil proteins, bactericidal/permeability-increasing protein (BPI), 57,000 m.w. cationic antimicrobial protein, and bactericidal protein have been described that specifically kill gram negative bacteria. Since LPS is a structure common to all gram-negative bacteria, we investigated whether the microbicidal protein BPI affects biologic activity of LPS in vitro. Human neutrophils can be activated both in vitro and in vivo by LPS. Upon stimulation, surface expression of CR1 and CR3 increases markedly. Using flow microfluorimetry, we analyzed surface expression of CR1 and CR3 as a measure of neutrophil stimulation in response to LPS. CR up-regulation on neutrophils was TNF independent, suggesting direct LPS stimulation of neutrophils in this system. Purified BPI completely inhibited CR up-regulation on neutrophils stimulated with both rough and smooth LPS chemotypes at 1.8 to 3.6 nM (100 to 200 ng/ml). By comparison, the polypeptide antibiotic polymyxin B completely inhibited the same dose of LPS at 0.4 nM. The inhibitory activity of BPI appeared to be specific for LPS because neutrophil stimulation by formylated peptide or TNF was unaffected. The specificity of BPI for LPS was further demonstrated by inhibition of LPS activity in the limulus amebocyte lysate assay. Therefore, the role of BPI in infection may not be limited to its microbicidal activity, but it may also regulate the neutrophil response to LPS.     

Surfactant protein A enhances apoptotic cell uptake and TGF-beta1 release by inflammatory alveolar macrophages

The phagocytosis of apoptotic inflammatory cells by alveolar macrophages (AMs) is a key component of inflammation resolution within the air space. Surfactant protein A (SP-A) has been shown to stimulate the phagocytosis of apoptotic neutrophils (PMNs) by normal AMs. We hypothesized that SP-A promotes the resolution of alveolar inflammation by enhancing apoptotic PMN phagocytosis and anti-inflammatory cytokine release by inflammatory AMs. Using an LPS lung inflammation model, we determined that SP-A stimulates the phagocytosis of apoptotic PMNs threefold by normal AMs and AMs isolated after LPS injury. Furthermore, SP-A enhances transforming growth factor-beta1 (TGF-beta1) release from both AM populations. Inflammatory AMs release twofold more TGF-beta1 in culture than do normal AMs. SP-A and apoptotic PMNs together stimulate TGF-beta1 release equivalently from normal and inflammatory cultured AMs (330% of unstimulated release by normal AMs). In summary, SP-A enhances apoptotic PMN uptake, stimulates AM TGF-beta1 release, and modulates the amount of TGF-beta1 released when AMs phagocytose apoptotic PMNs. These findings support the hypothesis that SP-A promotes the resolution of alveolar inflammation.     

Cutting edge: neutrophil granulocyte serves as a vector for Leishmania entry into macrophages

Macrophages (MF) are the final host cells for multiplication of the intracellular parasite Leishmania major (L. major). However, polymorphonuclear neutrophil granulocytes (PMN), not MF, are the first leukocytes that migrate to the site of infection and encounter the parasites. Our previous studies indicated that PMN phagocytose but do not kill L. major. Upon infection with Leishmania, apoptosis of human PMN is delayed and takes 2 days to occur. Infected PMN were found to secrete high levels of the chemokine MIP-1beta, which attracts MF. In this study, we investigated whether MF can ingest parasite-infected PMN. We observed that MF readily phagocytosed infected apoptotic PMN. Leishmania internalized by this indirect way survived and multiplied in MF. Moreover, ingestion of apoptotic infected PMN resulted in release of the anti-inflammatory cytokine TGF-beta by MF. These data indicate that Leishmania can misuse granulocytes as a "Trojan horse" to enter their final host cells "silently" and unrecognized.     

The IκB family member Bcl-3 coordinates the pulmonary defense against Klebsiella pneumoniae infection

Bcl-3 is an atypical member of the IκB family that has the potential to positively or negatively modulate nuclear NF-κB activity in a context-dependent manner. Bcl-3's biologic impact is complex and includes roles in tumorigenesis and diverse immune responses, including innate immunity. Bcl-3 may mediate LPS tolerance, suppressing cytokine production, but it also seems to contribute to defense against select systemic bacterial challenges. However, the potential role of Bcl-3 in organ-specific host defense against bacteria has not been addressed. In this study, we investigated the relevance of Bcl-3 in a lung challenge with the Gram-negative pathogen Klebsiella pneumoniae. In contrast to wild-type mice, Bcl-3-deficient mice exhibited significantly increased susceptibility toward K. pneumoniae pneumonia. The mutant mice showed increased lung damage marked by neutrophilic alveolar consolidation, and they failed to clear bacteria in lungs, which correlated with increased bacteremic dissemination. Loss of Bcl-3 incurred a dramatic cytokine imbalance in the lungs, which was characterized by higher levels of IL-10 and a near total absence of IFN-γ. Moreover, Bcl-3-deficient mice displayed increased lung production of the neutrophil-attracting chemokines CXCL-1 and CXCL-2. Alveolar macrophages and neutrophils are important to antibacterial lung defense. In vitro stimulation of Bcl-3-deficient alveolar macrophages with LPS or heat-killed K. pneumoniae recapitulated the increase in IL-10 production, and Bcl-3-deficient neutrophils were impaired in intracellular bacterial killing. These findings suggest that Bcl-3 is critically involved in lung defense against Gram-negative bacteria, modulating functions of several cells to facilitate efficient clearance of bacteria.     

Immunomodulatory effects of glycine on LPS-treated monocytes: reduced TNF-alpha production and accelerated IL-10 expression.

Cytokines play a pivotal role in the pathogenesis of septic shock. Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) stimulate the progression of septic shock whereas the anti-inflammatory cytokine IL-10 has counterregulative potency. The amino acid glycine (GLY) has been shown to protect against endotoxin shock in the rat by inhibiting TNF-alpha production. In the current study we investigated the role of GLY on lipopolysaccharide (LPS) -induced cell surface marker expression, phagocytosis, and cytokine production on purified monocytes from healthy donors. GLY did not modulate the expression of HLA-DR and CD64 on monocytes, whereas CD11b/CD18 expression (P<0.05) and E. coli phagocytosis (P<0.05) decreased significantly. GLY decreased LPS-induced TNF-alpha production (P<0.01) and increased IL-10 expression of purified monocytes. Similarly, in a whole blood assay, GLY reduced TNF-alpha (P<0.0001) and IL-1beta (P<0.0001) synthesis and increased IL-10 expression (P<0.05) in a dose-dependent manner. The inhibitory effects of GLY were neutralized by strychnine, and the production of IL-10 and TNF-alpha was augmented by anti-IL-10 antibodies. Furthermore, GLY decreased the amount of IL-1beta and TNF-alpha-specific mRNA. Our data indicate that GLY has a potential to be used as an additional immunomodulatory tool in the early phase of sepsis and in different pathophysiological situations related to hypoxia and reperfusion.     

A role for triggering receptor expressed on myeloid cells-1 in host defense during the early-induced and adaptive phases of the immune response

Triggering receptor expressed on myeloid cells (TREM)-1 is a cell surface molecule expressed on neutrophils and monocytes implicated in the propagation of the inflammatory response. To further characterize the function of this molecule in different phases of the immune response, we examined TREM-1 in the context of host defense against microbial pathogens. In primary human monocytes TREM-1 activation did not trigger innate antimicrobial pathways directed against intracellular Mycobacterium tuberculosis, and only minimally improved phagocytosis. However, activation of TREM-1 on monocytes did drive robust production of proinflammatory chemokines such as macrophage inflammatory protein-1alpha and IL-8. Engagement of TREM-1 in combination with microbial ligands that activate Toll-like receptors also synergistically increased production of the proinflammatory cytokines TNF-alpha and GM-CSF, while inhibiting production of IL-10, an anti-inflammatory cytokine. Expression of TREM-1 was up-regulated in response to TLR activation, an effect further enhanced by GM-CSF and TNF-alpha but inhibited by IL-10. Functionally, primary monocytes differentiated into immature dendritic cells following activation through TREM-1, evidenced by higher expression of CD1a, CD86, and MHC class II molecules. These cells had an improved ability to elicit T cell proliferation and production of IFN-gamma. Our data suggest that activation of TREM-1 on monocytes participates during the early-induced and adaptive immune responses involved in host defense against microbial challenges.     

IL-12, but not IL-18, is critical to neutrophil activation and resistance to polymicrobial sepsis induced by cecal ligation and puncture

Sepsis is a systemic inflammatory response resulting from local infection due, at least in part, to impaired neutrophil migration. IL-12 and IL-18 play an important role in neutrophil migration. We have investigated the mechanism and relative role of IL-12 and IL-18 in polymicrobial sepsis induced by cecal ligation and puncture (CLP) in mice. Wild-type (WT) and IL-18(-/-) mice were resistant to sublethal CLP (SL-CLP) sepsis. In contrast, IL-12(-/-) mice were susceptible to SL-CLP sepsis with high bacteria load in peritoneal cavity and systemic inflammation (serum TNF-alpha and lung neutrophil infiltration). The magnitude of these events was similar to those observed in WT mice with lethal CLP sepsis. The inability of IL-12(-/-) mice to restrict the infection was not due to impairment of neutrophil migration, but correlated with decrease of phagocytosis, NO production, and microbicidal activities of their neutrophils, and with reduction of systemic IFN-gamma synthesis. Consistent with this observation, IFN-gamma(-/-) mice were as susceptible to SL-CLP as IL-12(-/-) mice. Moreover, addition of IFN-gamma to cultures of neutrophils from IL-12(-/-) mice restored their phagocytic, microbicidal activities and NO production. Mortality of IL-12(-/-) mice to SL-CLP was prevented by treatment with IFN-gamma. Thus we show that IL-12, but not IL-18, is critical to an efficient host defense in polymicrobial sepsis. IL-12 acts through induction of IFN-gamma and stimulation of phagocytic and microbicidal activities of neutrophils, rather than neutrophil migration per se. Our data therefore provide further insight into the defense mechanism against this critical area of infectious disease.