Gram-positive and Gram-negative bacteria elicit different patterns of pro-inflammatory cytokines in human monocytes

Pro-inflammatory cytokines secreted by tissue macrophages recruit polymorphonuclear leukocytes and evoke fever, cachexia and production of acute phase proteins. This study investigates whether Gram-positive and Gram-negative bacteria equally and efficiently trigger production of the pro-inflammatory cytokines IL-1 beta, IL-6, IL-8 and TNF-alpha in human monocytes. A range of aerobic and anaerobic Gram-positive and Gram-negative bacteria were killed by UV-light and added in different concentrations to human monocytes. Cytokines were measured in 24 h supernatants by ELISA. Gram-positive and Gram-negative bacteria were equally efficient inducers of IL-1 beta, but Gram-positive bacteria generated twice as much TNF-alpha as did Gram-negative bacteria (p<0.001 for 25 and 250 bacteria/cell). In contrast, Gram-negative bacteria induced at least twice as much IL-6 and IL-8 as did Gram-positive bacteria (p<0.001 for 2.5, 25 and 250 bacteria/cell). While the cytokine responses to LPS were similar to those induced by the corresponding amount of Gram-negative bacteria, the strong IL-1 beta and TNF-alpha responses to Gram-positive bacteria could not be induced by soluble peptidoglycan or lipotheicoic acid. The particular nature of the bacteria, thus seem to modify the response to Gram-positive bacterial components. The different cytokine profiles evoked by Gram-positive and Gram-negative bacteria might optimize clearance of bacteria that differ in cell wall structure.     

Beta 2 integrins are involved in cytokine responses to whole Gram-positive bacteria

Proinflammatory cytokines have an important pathophysiologic role in septic shock. CD14 is involved in cytokine responses to a number of purified bacterial products, including LPS. However, little is known of monocyte receptors involved in cytokine responses to whole bacteria. To identify these receptors, human monocytes were pretreated with different mAbs and TNF-alpha was measured in culture supernatants after stimulation with whole heat-killed bacteria. Human serum and anti-CD14 Abs significantly increased and decreased, respectively, TNF-alpha responses to the Gram-negative Escherichia coli. However, neither treatment influenced responses to any of the Gram-positive bacteria tested, including group A and B streptococci, Listeria monocytogenes, and Staphylococcus aureus. Complement receptor type III (CR3 or CD18/CD11b) Abs prevented TNF-alpha release induced by heat-killed group A or B streptococci. In contrast, the same Abs had no effects when monocytes were stimulated with L. monocytogenes or S. aureus. Using either of the latter bacteria, significant inhibition of TNF-alpha release was produced by Abs to CD11c, one of the subunits of CR4. To confirm these blocking Ab data, IL-6 release was measured in CR3-, CR4-, or CD14-transfected Chinese hamster ovary cells after bacterial stimulation. Accordingly, streptococci triggered moderate IL-6 production (p < 0.05) in CR3 but not CD14 or CR4 transfectants. In contrast, L. monocytogenes and S. aureus induced IL-6 release in CR4 but not CR3 or CD14 transfectants. Collectively our data indicate that beta 2 integrins, such as CR3 and CR4, may be involved in cytokine responses to Gram-positive bacteria. Moreover, CD14 may play a more important role in responses to whole Gram-negative bacteria relative to Gram-positive ones.     

CD14 plays no major role in shock induced by Staphylococcus aureus but down-regulates TNF-alpha production

Recent in vitro studies have suggested that CD14, a major receptor for LPS, may also be a receptor for cell wall components of Gram-positive bacteria and thus play a role in Gram-positive shock. To analyze the in vivo role of CD14 in responses to Gram-positive bacteria, CD14-deficient and control mice were injected with Staphylococcus aureus, and the effects on lethality, bacterial clearance, and production of cytokines were analyzed. Survival of CD14-deficient and control mice did not differ significantly after administration of various doses of either unencapsulated or encapsulated S. aureus; furthermore, mice in both groups displayed similar symptoms of shock. In addition, inflammatory cytokines such as TNF-alpha and IL-6 were readily detectable in the serum of CD14-deficient mice injected with live or antibiotic-killed S. aureus. Surprisingly, the serum concentration of TNF-alpha in CD14-deficient mice was at least threefold higher than in control mice after injection of either unencapsulated or encapsulated S. aureus, suggesting that CD14 down-regulates TNF-alpha. A similar increase in serum TNF-alpha occurred when CD14-deficient animals were injected with gentamicin-killed bacteria even though no symptoms of shock were observed. These studies indicate that CD14, in contrast to its key function in responses to the Gram-negative bacterium, Escherichia coli 0111, does not play a prominent role in septic shock induced by S. aureus, and that the symptoms of S. aureus shock are not due solely to TNF-alpha.     

Interleukin-6 is the main mediator of the interaction between monocytes and platelets in the killing of Schistosoma mansoni

We demonstrate the existence of a cooperation between monocytes and platelets for the killing of Schistosoma mansoni. Indeed, supernatants obtained after a 24 hr adherence of normal human monocytes were able to induce, in a dose dependent manner, the cytotoxicity of normal human platelets towards the young larvae of S.mansoni in vitro. The physicochemical analysis of the supernatants showed that a factor exhibiting a pl of 4.8-4.9 was responsible of this effect, suggesting a role of IL-6, detected in the supernatants, in this induction. This was confirmed by the neutralization of the cytotoxic effect by a polyclonal serum against IL6 whereas polyclonal sera against IL-1 beta or TNF-alpha, the other cytokines present in the supernatants, did not modify the cytotoxicity observed. Finally human recombinant IL-6 induces the platelet cytotoxic function, demonstrating a direct effect of IL6 on blood platelets.     

The involvement of CD14 in the activation of human monocytes by peptidoglycan monomers.

BACKGROUND: Cell-wall components of Gram-positive and Gram-negative bacteria induce the production of cytokines in human peripheral blood mononuclear cells. These cytokines are the main mediators of local or systemic inflammatory reaction that can contribute to the development of innate immunity. AIMS: This study was performed to analyze the involvement of CD14 molecule in the activation of human monocytes by peptidoglycan monomer (PGM) obtained by biosynthesis from culture fluid of penicillin-treated Brevibacterium divaricatum NRLL-2311. METHODS: Cytokine release of interleukin (IL)-1, IL-6 and tumor necrosis factor-alpha from human monocytes via soluble CD14 (sCD14) or membrane-associated (mCD14) receptor using anti-CD14 monoclonal antibody (MEM-18) or lipid A structure (compound 406) was measured in bioassays. RESULTS: The results demonstrated that PGM in the presence of human serum might induce the monokine release in a dose-dependent manner. The addition of sCD14 at physiologic concentrations enhanced the PGM-induced monokine release, while the monokine inducing capacity of PGM in the presence of sCD14 was inhibited by MEM-18. Effects of PGM were also blocked by glycolipid, compound 406, suggesting the involvement of binding structures similar to those for lipopolysaccharide. CONCLUSION: Activation of human monocytes by PGM involves both forms of CD14 molecule, sCD14 and mCD14.     

Differences in Toll-like receptor expression and cytokine production after stimulation with heat-killed Gram-positive and Gram-negative bacteria

Innate immune surveillance in the blood is executed mostly by circulating monocytes, which recognise conserved bacterial molecules such as peptidoglycan and lipopolysaccharide. Toll-like receptors (TLR) play a central role in microbe-associated molecular pattern detection. Here, we compared the differences in TLR expression and cytokine production after stimulation of peripheral blood cells with heat-killed Gram-negative and Gram-positive human pathogens Neisseria meningitidis, Escherichia coli, Staphylococcus aureus and Streptococcus pneumoniae. We found that TLR2 expression is up-regulated on monocytes after stimulation with S. aureus, S. pneumoniae, E. coli and N. meningitidis. Moreover, TLR2 up-regulation was positively associated with increasing concentrations of Gram-positive bacteria, whereas higher concentrations of Gram-negative bacteria, especially E. coli, caused a milder TLR2 expression increase compared with low doses. Cytokines were produced in similar dose-dependent profiles regardless of the stimulatory pathogen; however, Gram-negative pathogens induced higher cytokine levels than Gram-positive ones at same concentrations. These results indicate that Gram-positive and Gram-negative bacteria differ in their dose-dependent patterns of induction of TLR2 and TLR4, but not in cytokine expression.     

In vitro inhibition activity of nisin A, nisin Z, pediocin PA-1 and antibiotics against common intestinal bacteria

AIMS: To evaluate the sensitivity of 21 common intestinal bacteria to six antibiotics and three broad-spectrum bacteriocins (nisins Z and A and pediocin PA-1). METHODS AND RESULTS: Neutralized cell-free culture supernatants containing active bacteriocins, and antibiotics were tested with the agar diffusion test and the disc-diffusion method, respectively. The tested intestinal strains showed high sensitivity to most antibiotics except for streptomycin and oxacillin. Nisins A and Z (8 mug per well) had similar activity spectra and inhibited all Gram-positive intestinal bacteria at different levels (except Streptococcus salivarius), with bifidobacteria (except Bifidobacterium breve and Bif. catenulatum), Collinsella aerofaciens and Eubacterium biforme being the most sensitive strains, but they were not active against Gram-negative bacteria. Surprisingly, none of the tested strains were inhibited by pediocin PA-1 (16 mug per well). CONCLUSION: Pediocin PA-1 which is very active against Listeria spp. and other food pathogens did not inhibit major intestinal species in the human intestine in contrast to both nisins A and Z. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that pediocin PA-1 has potential to inhibit Listeria within the intestinal microbiota without altering commensal bacteria.     

Aggregated immunoglobulin and Fc fragment of IgG induce IL-6 release from human monocytes

The Fc fragment of immunoglobulin (Ig) has been shown to play an important role in the regulation of humoral immunity, cellular immunity, lymphocyte and monocyte activation, and immune mediator secretion. We wished to determine if Ig or Fc fragments would induce IL-6 production from monocytes. Incubation of monocytes purified from human peripheral blood mononuclear cells with aggregated Ig or Fc fragments of Ig induced interleukin-6 (IL-6) activity in the supernatants. Monomeric Ig taken from an intravenous preparation of Ig, from which all aggregated Ig are removed, would not induce IL-6 production from monocytes whereas as a heat-treated aliquot, presumably containing aggregates, did induce IL-6. The supernatants were assayed according to their ability to induce growth in a murine hybridoma cell line B9, or enhance Ig secretion of B cells stimulated with Staphylococcus aureus Cowan 1 (SAC). The IL-6 activity in the supernatants could be neutralized by a polyclonal rabbit anti-human IL-6 antiserum in both assays of IL-6 activity. Exposure of T-enriched or B-enriched lymphocyte subpopulations to Fc fragments did not induce the release of any IL-6 after 12 hr of incubation, but small amounts of IL-6 were produced by B-enriched cells after 60 hr of exposure to Fc fragments. Hence Fc fragments and aggregated Ig induce peripheral blood monocytes to rapidly secrete large quantities of interleukin-6.     

Utilization of exogenous siderophores by enterococci]

The study was undertaken to investigate the ability of enterococci to assimilate iron via siderophores of bacteria living in the same habitats in the human organism. The potential recipients of exogenous siderophores were six Enterococcus faecalis and six Enterococcus faecium strains, isolated from clinical materials of human origin. The donors of siderophores were Gram-negative rods (various species of the Enterobacteriaceae, Pseudomonas and Acinetobacter) and Gram-positive cocci (various species of Staphylococcus and Streptococcus). All of the investigated E. faecium and only two E. faecalis strains demonstrated the ability to utilize the siderophores of the aforementioned bacterial groups, predominantly the chelators of Gram-negative rods, those of Gram-positive cocci were utilized to a smaller extent. Four recipient strains from E. faecalis species did not demonstrate the ability to utilize siderophores synthesized by all of 40 investigated donor strains.     

Cytokine secretion by stimulated monocytes depends on the growth phase and heat treatment of bacteria: a comparative study between lactic acid bacteria and invasive pathogens

The consumption of food containing lactic acid bacteria (LAB) has been shown to exert immunomodulatory effects in humans. The specific cellular interaction of these bacteria with immuno-competent cells has not yet been fully understood. Since the TNF-alpha secretion of stimulated monocytes is an important initial response to a bacterial challenge, we investigated the potential of LAB originating from the human intestine or fermented food in comparison to the effect of invasive pathogens. The challenge of monocytes with three LAB strains, Listeria monocytogenes or enterohaemorrhagic Escherichia coli (EHEC) elicited a strain specific, dose-dependent biphasic TNF-alpha secretion. The concentration (EDmax) of bacteria or bacterial cell wall components necessary to induce maximal TNF-alpha secretion (TNFmax) by monocytes was mathematically approximated. It was shown for exponentially growing LAB strains that the maximal TNF-alpha secretion (TNFmax) was stronger (57 to 78%) upon stimulation with living bacteria than with heat killed cells. In contrast to log-phase bacteria, the maximal TNF-alpha secretion of monocytes (TNFmax) was higher (15 to 55%) after the stimulation with heat killed, stationary-phase bacteria when compared to that of live LAB. Thus, monocyte stimulation was clearly affected by the growth phase of bacteria. Purified cell walls of LAB strains revealed only a limited potential for monocyte stimulation. LPS exhibited a higher capacity to stimulate monocytes than purified gram positive cell walls or muramyldipeptide. In comparison to pathogenic bacteria, the maximal secretory TNF-alpha response (TNFmax) was up to 2 fold higher with LAB strains. In general, the amount of bacteria (EDmax) necessary to induce maximal TNF-alpha secretion (TNFmax) was approximately 1 to 3 log higher for heat killed bacteria when compared to live bacterial cells illustrating the significant lower potential of heat killed bacteria to activate monocytes.     

Suppressive activity of a macrolide antibiotic, roxithromycin, on pro-inflammatory cytokine production in vitro and in vivo

This study was designed to examine the influence of a macrolide antibiotic, roxithromycin (RXM), on the production of pro-inflammatory cytokines, interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. In the first experiments, we examined the effect of RXM on in vitro cytokine production from lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes. The monocytes were cultured in the presence of various doses of the agent. After 24 h, the culture supernatants were obtained and assayed for IL-1beta and TNF-alpha contents by enzyme-linked immunosorbent assay. RXM suppressed the in vitro production of IL-1beta and TNF-alpha in response to LPS stimulation. This was dose dependent and first noted at a concentration of as little as 0.05 microg/ml, which is much lower than therapeutic blood levels. In the second part of the experiments, we examined the influence of RXM on the appearance of IL-1beta and TNF-alpha in mouse lung extract induced by LPS inhalation. RXM was administered orally into BALB/c mice at a single dose of 2.5 mg/kg once a day for 5-12 weeks. These mice were then instilled with LPS into the trachea and examined for the presence of cytokines in aqueous lung extracts. Pretreatment of mice with RXM for 5 weeks did not influence of the appearance of both IL-1beta and TNF-alpha in aqueous lung extracts. However, pretreatment for more than 7 weeks dramatically suppressed the cytokine appearance in the extracts.     

Selective activation of antigen-specific human B cells in recently immunized individuals by nonspecific factors in the absence of antigen

The in vitro effect of nonspecific factors (derived from mixed lymphocyte culture [MLC] supernatants) on human B cell responses was studied in individuals recently immunized in vivo to keyhole limpet hemocyanin, tetanus toxoid, and/or diphtheria toxin. In T cell-depleted fractions of peripheral blood mononuclear cells, nonspecific factors alone, without antigen, selectively induced a specific antibody response to the antigen to which the individual had been recently immunized, at dilutions that did not generate a significant polyclonal response in the remainder of the B cell repertoire. The source of these factors, with respect to MLC donors, did not affect the antibody response. Supernatants of MLC from nonimmunized individuals induced a specific antibody response as effectively as supernatants of MLC from immunized individuals, when added to B cells plus monocytes from recently immunized individuals. Studies in which the same individuals were followed over time showed that these factor-sensitive B cells are seen in the peripheral blood of recently immunized individuals for only a finite period of time. Thus, in vivo immunization with a specific antigen results in the transient appearance in the peripheral blood of B cells that are specific for the antigen in question. These B cells are probably preactivated in that nonspecific factors selectively induce in vitro their further differentiation into antibody-secreting cells, in the absence of added antigen or mitogen. These studies may add further insight into our understanding of the sequential steps involved in the activation and differentiation of human B lymphocytes and provide a model for the combined in vivo and in vitro study of human B cell physiology.     

The role of encapsulated anaerobic bacteria in synergistic infections

Abstract: The effect of encapsulation on the virulence, survival, and protection of anaerobic bacteria from phagocytosis is reviewed. Support for the importance of encapsulated Gram-negative anaerobic rods ( Bacteroides sp., Prevotella sp. and Porphyromonas sp.), anaerobic and facultative Gram-positive cocci (AFGPC) was provided by their higher recovery rate in oropharyngeal infections, abscesses and blood, compared to their number in the normal flora. The pathogenicity of Bacteroides, Fusobacterium, Clostridium , and AFGPC was studied by inoculating them into mice and observing their ability to induce subcutaneous abscesses. Encapsulated Bacteroides, Fusobacteria , and AFGPC generally induced abscesses, whereas non-encapsulated organisms did not. However, many of the strains that had only a minimal number of encapsulated organisms (< 1%) survived in the abscesses, and they became heavily encapsulated when inoculated with other viable or non-viable encapsulated bacteria. Thereafter, these strains were able to induce abscesses when injected alone. Encapsulated Gram-negative anaerobic rods and AFGPC-induced bacteraemia and translocation, and increased the mortality of the infected animals more often than did the non-encapsulated form of the same strains. As determined by using selective antimicrobial therapy and quantitative cultures of abscesses induced in mice, possession of a capsule generally made Gram-negative anaerobic rods more important than their aerobic counterparts. Synergistic potentials were seen between encapsulated Gram-negative anaerobic rods and all tested aerobic bacteria and most AFGPC, and also between most AFGPC and Pseudomonas aeruginosa or Staphylococcus aureus . These studies demonstrated the importance of encapsulated anaerobes in mixed infections.     

Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2

NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-alpha and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-alpha. Jak2 tyrosine kinase inhibition blocked LTA-induced production of NO but not TNF-alpha. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-beta expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-alpha production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-beta, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production.     

A novel receptor tyrosine kinase, Mer, inhibits TNF-alpha production and lipopolysaccharide-induced endotoxic shock

The regulation of monocyte function and the inhibition of TNF-alpha production during bacterial sepsis are critical in attenuating adverse host responses to endotoxemia. To study the function of a novel receptor tyrosine kinase, mer, that is expressed in monocytes, we generated mice (merkd) that lack the signaling tyrosine kinase domain. Upon LPS challenge, merkd animals died of endotoxic shock (15/17, 88.2%), whereas control wild-type mice survived (1/15, 6.7% died). Susceptible merkd mice exhibited edema, leukocyte infiltration, and signs of endotoxic shock that correlated with higher levels of TNF-alpha found in the serum of merkd mice as compared with wild-type control animals. Death due to LPS-induced endotoxic shock in merkd mice was blocked by administration of anti-TNF-alpha Ab, suggesting that overproduction of this cytokine was principally responsible for the heightened suseptibility. The increase in TNF-alpha production appeared to be the result of a substantial increase in the LPS-dependent activation of NF-kappa B nuclear translocation resulting in greater TNF-alpha production by macrophages from merkd mice. Thus, Mer receptor tyrosine kinase signaling participates in a novel inhibitory pathway in macrophages important for regulating TNF-alpha secretion and attenuating endotoxic shock.     

Phenotypic and functional characteristics of macrophage-like cells differentiated in pro-inflammatory cytokine-containing cultures

Previous studies have demonstrated an infiltration of monocytes and increased levels of IL-1beta and TNF-alpha in some chronic inflammatory tissues. Interleukin-1beta and TNF-alpha are capable of protecting monocytes from spontaneous apoptosis and thus maintain their viability in vitro. To study the possible effects of these cytokines on the differentiation and function of recruited monocytes, a model has been developed in which monocytes isolated from human peripheral blood were differentiated into macrophages in serum in the presence or absence of IL-1beta or TNF-alpha. Monocytes cultured with IL-1beta and TNF-alpha underwent substantial changes in morphology, similar to those observed in monocytes undergoing differentiation into macrophages. The cultured cells increased in size and vacuolization and their content of acid phosphates increased 10-fold. Although they exhibited the morphological characteristics of macrophages, monocytes matured in the cytokines differed functionally from those cultured in serum in a lower expression of HLA-DR, lower ability for triggering the proliferation of allogeneic lymphocytes, higher expression of mannose receptor and greater production of superoxide and TNF-alpha. This data suggests that IL-1beta and TNF-alpha direct monocyte differentiation into macrophages with a reduced antigen-presenting and an increased pro-inflammatory factor-releasing phenotype. Elevated levels of IL-1beta and TNF-alpha in the inflammatory tissues may therefore not only prolong the survival of recruited monocytes, but maintain them in an inflammatory state.     

Dual mechanism for Mycobacterium tuberculosis cytotoxicity on lung epithelial cells

Mycobacterium tuberculosis strains CDC1551 and Erdman were used to assess cytotoxicity in infected A549 human alveolar epithelial cell monolayers. Strain CDC1551 was found to induce qualitatively greater disruption of A549 monolayers than was strain Erdman, although total intracellular and cell-associated bacterial growth rates over the course of the infections were not significantly different. Cell-free culture supernatants from human monocytic cells infected with either of the 2 M. tuberculosis strains produced a cytotoxic effect on A549 cells, correlating with the amount of tumor necrosis factor alpha (TNF-α) released by the infected monocytes. The addition of TNF-α-neutralizing antibodies to the supernatants from infected monocyte cultures did prevent the induction of a cytotoxic effect on A549 cells overlaid with this mixture but did not prevent the death of epithelial cells when added prior to infection with M. tuberculosis bacilli. Thus, these data agree with previous observations that lung epithelial cells infected with M. tuberculosis bacilli are rapidly killed in vitro. In addition, the data indicate that some of the observed epithelial cell killing may be collateral damage; the result of TNF-α released from M. tuberculosis-infected monocytes.     

Human recombinant migration inhibitory factor activates human macrophages to kill tumor cells.

A recombinant form of human migration inhibitory factor (rMIF) obtained from COS-1 cells transfected with MIF-specific cDNA is able to activate cultured human peripheral blood monocytes and monocyte-derived macrophages, in a dose-dependent manner to become cytotoxic for tumor cells in vitro. The cytotoxicity exhibited by macrophages treated with rMIF is > or = 30% above that of cells incubated with control supernatants or with media and peaks 72 hr after the addition of tumor targets. rMIF also induces macrophages to produce tumor necrosis factor (TNF-alpha) and interleukin-1 beta (IL-1 beta). These results demonstrate that rMIF is able to modulate macrophage functions and plays a role in cell-mediated immune response.