Expression of lysostaphin in HeLa cells protects from host cell killing by intracellular Staphylococcus aureus

The Staphylococcus aureus-specific cell wall endopeptidase lysostaphin was used as a model for an intracellular acting bactericidal antibiotic. HeLa cells were transfected with an expression vector directing the heterologous expression of lysostaphin in the cytoplasm. Expression, subcellular localization and enzymatic activity of lysostaphin were investigated by immunoblotting, fluorescent microscopy and agar diffusion assays. Both transiently and stably transfected HeLa cells showed a strong expression of active lysostaphin. After infection with S. aureus, the intracellular number of S. aureus and the host cell viability were determined. This staphylolytic activity resulted in a strong reduction of intracellular S. aureus in a time- and dose-dependent manner. Furthermore, host cells expressing lysostaphin became protected from S. aureus-induced cell death. Our data demonstrate the potential of intracellularly acting cell-wall active drugs or antibiotics that kill S. aureus without causing harm to the infected host cells.     

Fcγ receptor-mediated phagocytosis requires tyrosine kinase activity and is ligand independent

Receptors for the invariant chain of immunoglobulins (FcR) define the cellular response to specific antigens. FcγR recognize IgG and so elicit a variety of effector functions including phagocytosis. We are interested in the structural determinants for FcγR-mediated phagocytosis, specifically FcγRI(p135) and FcγRIIa isoforms. The low-affinity receptor, FcγRIIa, is found on macrophages and its cytoplasmic domain contains a tyrosine activation motif which has previously been shown to regulate endocytosis. In contrast, FcγRI has no known signaling motifs, though a functional interaction has recently been demonstrated with the γ chain of the high-affinity receptor for IgE, FcεRI. This accessory molecule has a cytoplasmic tyrosine activation motif implicated in signal transduction. Here we demonstrate that although FcγRI transiently expressed on COS-7 cells is able to rosette opsonized SRBC, it cannot phagocytose them. If the cytoplasmic domain of either γ chain or FcγRIIa replaces that of FcγRI in a chimeric receptor, efficient phagocytosis occurs. This particle ingestion is sensitive to the tyrosine kinase inhibitor genistein. Chimeric receptors where the extracellular domain of either FcγRI or FcγRIIa is replaced with that of CD2, a T cell antigen, indicate that FcγR-mediated phagocytosis is ligand independent. We conclude that phagocytosis is dependent upon close particle apposition, tyrosine kinase activity, and that the process is ligand independent.     

FcεRI β‐chain ITAM amplifies PI3K‐signaling to ensure synergistic degranulation response via FcεRI and adenosine receptors

Simultaneous stimulation with antigen and adenosine in mast cells induces a synergistic degranulation response at a low antigen dose that is insufficient to cause secretion by itself. This kind of stimulation is thought to be relevant to the immediate asthmatic response upon bronchial challenge with low‐dose allergen. In this context, FcεRI‐ and adenosine receptor‐mediated signalings cooperate to increase degranulation in mast cells. In the present study, we prepared mast cells that have mutations (Y219F/Y225F/Y229F) in three tyrosine residues of the FcεRI β‐chain (FcRβ)‐ITAM in order to elucidate the molecular mechanisms of degranulation response synergistically elicited by costimulation with low‐dose antigen and adenosine. Introduction of mutations in the FcRβ‐ITAM abolished the synergistic degranulation response. Upon costimulation with low‐dose antigen and adenosine, tyrosine phosphorylation of Grb2‐associated binder 2, which is located upstream of PI3K‐signaling, was significantly increased, but severely diminished in FcRβ‐ITAM mutant cells. These findings indicate that FcRβ acts as a critical element in mast cell synergistic degranulation response through FcεRI and adenosine receptors, and that PI3K‐signaling through FcRβ‐ITAM is a crucial participant in augmentation of FcεRI‐mediated degranulation by adenosine.     

Association of tissue factor with a γ chain homodimer of the IgE receptor type I in cultured human monocytes

Tissue factor (TF) is a high-affinity receptor for coagulation factors VII (F VII) and VIIa. The F VII/VIIa/TF complex is the major cellular initiator of the extrinsic coagulation cascade. We found that the occupancy of TF by its ligand, F VIIa, is involved with signal transduction and that TF is associated with the γ chain homodimer identified as a component of IgE receptor type I (FcϵRI). When 4-day cultured human monocytes were incubated with F VIIa, several polypeptides, especially a 70-kDa polypeptide, were transiently phosphorylated on tyrosine, residues. These phosphorylation events were inhibited by prior binding of anti-TF monoclonal antibody (mAb) HTF-K14, but not anti-TF mAb HTF-K180 to intact cultured monocytes. HTF-K14 blocked the binding of FVII/Vila to cell surface TF, whereas HTF-K180 did not. Anti-TF immunoprecipitates prepared from 1 % digitonin lysates of cultured human monocytes incorporated phosphate in a γ chain homodimer when incubated with [γ-³²P] ATP. The identity of the TF-associated structures as γ chains was established by immunoblot analysis of anti-TF mAb immunoprecipitates with anti-γ chain mAb. In addition, anti-TF immunoblot analysis showed that TF co-precipitated with anti-γ chain mAb. Our data suggest that γ chains may play an important role in signaling via TF in human monocytes/macrophages.     

Direct interaction of Syk and Lyn protein tyrosine kinases in rat basophilic leukemia cells activated via type I Fcε receptors

Activation of rat mast cells through the receptor with high affinity for IgE (FcεRI) requires a complex set of interactions involving transmembrane subunits of the FcεRI and two classes of nonreceptor protein tyrosine kinase (PTK), the Src family PTK p53/p56^lyn (Lyn) and the Syk/ZAP-family PTK p72^syk (Syk). Early activation events involve increased activity of Lyn and Syk kinases and their translocation into membrane domains containing aggregated FcεRI, but the molecular mechanisms responsible for these changes have remained largely unclear. To determine the role of FcεRI subunits in this process, we have analyzed Syk- and Lyn-associated proteins in activated rat basophilic leukemia (RBL) cells and their variants deficient in the expression of FcεRI β or γ subunits. Sepharose 4B gel chromatography of postnuclear supernatants from Nonidet-P40-solubilized antigen (Ag)- or pervanadate-activated RBL cells revealed extensive changes in the size of complexes formed by Lyn and Syk kinases and other cellular components. A fusion protein containing Src homology 2 (SH2) and SH3 domains of Lyn bound Syk from lysates of nonactivated RBL cells; an increased binding was observed when lysates from Ag- or pervanadate-activated cells were used. A similar amount of Syk was bound when lysates from pervanadate-activated variant cells deficient in the expression of FcεRI β or γ subunits were used, suggesting that FcεRI does not function as the only intermediate in the formation of the Syk-Lyn complexes. Further experiments have indicated that Syk-Lyn interactions occur in Ag-activated RBL cells under in vivo conditions and that these interactions could involve direct binding of the Lyn SH2 domain with phosphorylated tyrosine of Syk. The physical association of Lyn and Syk during mast-like cell activation supports the recently proposed functional cooperation of these two tyrosine kinases in FcεRI signaling.     

FcγRIII (CD16)‐mediated ADCC by NK cells is regulated by monocytes and FcγRII (CD32)

Monocytes are known to engage in reciprocal crosstalk with NK cells but their influence on NK‐cell‐associated antibody‐dependent cellular cytotoxicity (ADCC) is not well understood. We demonstrate that in humans FcγRIII (CD16)‐dependent ADCC by NK cells is considerably enhanced by monocytes, and that this effect is regulated by FcγRII (CD32) crosslinking in healthy individuals. It is known that during HIV‐1 infection, NK cells are known to express low levels of CD16 and exhibit reduced ADCC. We show that immune regulation of CD16‐mediated NK‐cell cytotoxicity by monocytes through CD32 engagement is substantially disturbed in chronic progressive HIV‐1 infection. Expression of activating isoform of CD32 represented a compensatory mechanism for reduced expression of CD16 on NK cells during HIV‐1 infection. As a result, the regulation of NK‐cell‐associated ADCC by monocytes is skewed and eventually constitutes a novel factor that contributes to HIV‐1‐associated immune deficiency, dysregulation and pathogenesis. Our data therefore provide evidence, for the first time, that in humans monocytes act as a rheostat for FcγRIII‐mediated NK‐cell functions maintaining a well‐balanced immune response.     

Induction of intracellular cytokine production in human monocytes/macrophages stimulated with ligands of pattern recognition receptors

Objective: This study addressed the role of the pattern recognition receptors (PRR), which recognize different molecular structures present on microorganisms, apoptotic, senescent and tumor cells, in the stimulation of human monocyte and monocyte-derived macrophages (MDM) for the production of intracellular cytokines.Materials and methods: Monocytes and MDM were stimulated with different ligands of scavenger receptors (SR) and mannose receptor (MR). Production of intracellular cytokines: tumor necrosis factor alpha (TNF), interleukin 10 and 12 (IL-10, IL-12) was determined by flow cytometry following staining with anti-cytokine monoclonal antibodies (mAbs).Results: The ligands of SR type A: fucoidan, polyguanylic acid (polyG), chemically modified low density lipoproteins (LDL), ligands of SR-B: native and chemically modified LDL, and ligand of mannose receptor (MR) – mannan induced strong expression of intracellular TNF and weaker IL-10 in monocytes, while phosphatidylserine (PdS) was without effect. IL-12 was stimulated only by fucoidan and polyG. The induction of cytokine m-RNA generally followed the pattern and the magnitude of intracellular cytokine production. In MDM, intracellular TNF and IL-12 expression was induced by mannan, native and modified LDL, but not other ligands. Expression of IL-10 was less pronounced and occurred following stimulation with fucoidan, polyG and modified, but not native, LDL.Conclusions: These results suggest that some PRR ligands may be involved in activation of monocytes/MDM for the production of mainly proinflammatory cytokines (TNF, IL-12) implicating their role in the response to microbial and tumor invasion.Received 3 August 2003; returned for revision 11 September 2003; accepted by A. Falus 3 November 2003.     

An Fcγ receptor I (CD64)-negative subpopulation of human peripheral blood monocytes is resistant to killing by antigen-activated CD4-positive cytotoxic T cells

It has been demonstrated that in monocyte/T cell co-cultures activated with recall antigens, cytotoxic T cells were generated which are able to reduce the number of antigen-presenting monocytes. In previous studies we could show that a minor subset of monocytes, the Fcγ receptor I-negative (CD64^?) monocytes, exhibits significantly higher antigen-presenting capacity than the main population of monocytes (< 90%) which are Fcγ receptor I-positive (CD64⁺). Therefore, we addressed the question whether they are also differentially susceptible to T cell-mediated killing. In the present study we demonstrate that the CD64^? monocyte subset is more resistant to killing by antigen-activated T cells than CD64⁺ monocytes, as indicated by a higher viability and recovery of CD64^? monocytes. This mechanism involves CD95 (Fas) antigen, since monocyte death in co-cultures with antigen-activated T cells could be partially reduced by blocking anti-Fas monoclonal antibodies (mAb). In agreement with this finding, although CD95 antigen was expressed on CD64⁺ and CD64^? monocytes at comparable levels, killing of CD64^? monocytes by activating anti-Fas mAb was lower than of CD64⁺ monocytes.     

On the interaction between agalactosyl IgG and Fcγ receptors

One of the serum abnormalities observed in autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) is the occurrence of IgG that lacks the terminal galactose on asparagine-linked biantennary complex type oligosaccharides [Gal(0)-IgG] located in the CH2 domain. Additionally, IgG without glycosylation is known to be defective in several effector functions due to a reduced ability to bind to its specific receptors (FcγR). It has thus been speculated that, by analogy with unglycosylated IgG, Gal(0)-IgG may also be functionally impaired or exert altered effector mechanisms. If this were true, Gal(0)-IgG could contribute to the phenotype of above-mentioned autoimmune diseases, like impaired immune complex clearance and defective down-regulation of activated B cells. Here, we show by three different methods that the interaction of Gal(0)-IgG and normally glycosylated IgG with the low-affinity FcγRII (CD32) is indistinguishable with respect both to binding and receptor-mediated signalling. These data argue against a prominent role for FcγR-dependent Gal(0)-IgG interactions in the etiology or pathogenesis of autoimmune diseases.     

Cytosolic free calcium is essential for immunoglobulin G-stimulated intracellular killing of Staphylococcus aureus by human monocytes.

Earlier studies have shown that the intracellular killing of Staphylococcus aureus by human monocytes requires continuous stimulation by serum factors, e.g., immunoglobulin G (IgG). In the present study, we demonstrate that IgG, at concentrations that stimulate the intracellular killing of S. aureus, induces a transient increase in the intracellular free calcium concentration ([Ca2+]i) in monocytes. The Ca2+ ionophores A23187 and ionomycin stimulate the killing process as efficiently as IgG does and initiate O2- production in resting monocytes but not in monocytes containing bacteria. The Ca2+ ionophore-stimulated killing process was markedly inhibited by the NADPH oxidase inhibitor diphenyleneiodonium bisulfate, which indicates that these ionophores stimulate oxygen-dependent bactericidal mechanisms. Reduction of the [Ca2+]i to values below 1 nM, obtained by loading monocytes with MAPT/AM (1,2-bis-5-methyl-aminophenoxylethane-N,N,N',N'-tetraacetoxymet hyl acetate) in the absence of extracellular Ca2+, rendered the cells unresponsive to IgG or Ca2+ ionophore stimulation of the intracellular killing of S. aureus, but the response could be restored by reincubating these cells in the presence of extracellular Ca2+. It is concluded that cytosolic free Ca2+ is essential for the IgG-stimulated intracellular killing of S. aureus by human monocytes.     

Substitutions at the Glu62 residue of human interleukin-2 differentially affect its binding to the α chain and the βγ complex of the interleukin-2 receptor

Human interleukin-2 (IL-2) α helix B is more conserved than the whole molecule, but has been less studied than other α helices of IL-2. Using site-directed mutagenesis, several IL-2 mutants in this helix were obtained. We found that the IL-2 mutant containing Leu at position 62 (Leu⁶²-IL-2) loses its ability to bind IL-2 receptor subunit α (IL-2Rα), but retains binding affinity to IL-2R subunit βγ as well as some bioactivity; nevertheless, another substitution at the same residue, Arg⁶² IL-2, loses its binding ability to both IL-2Rα and IL-2Rβγ, and can no longer stimulate IL-2-dependent cell growth, showing that Glu⁶² not only takes part in IL-2Rα binding, but can also affect IL-2 binding to IL-2Rβγ. In this regard, Glu⁶² may be a key site in the IL-2/IL-2Rα interaction, and can facilitate IL-2R ternary-complex formation, leading to IL-2Rα-mediated, IL-2-stimulated signal transduction.     

Effect of probenecid on phagocytosis and intracellular killing of Staphylococcus aureus and Escherichia coli by human monocytes and granulocytes.

The present study concerns the effects of probenecid on the phagocytosis and intracellular killing of Staphylococcus aureus and Escherichia coli by human monocytes and granulocytes. In both monocytes and granulocytes the inhibitory effect on phagocytosis was very small. Inhibition of intracellular killing of S. aureus by monocytes and granulocytes by probenecid was concentration dependent, being half-maximal at about 2 mM probenecid, and near-maximal at about 5 mM probenecid. The intracellular killing could also be inhibited when probenecid was added when this process was already started. Probenecid also inhibited the intracellular killing of E. coli by granulocytes, but not by monocytes. In the concentration range used, probenecid had no toxic effect on phagocytes or bacteria during the 2 hr of the experiments.     

Kinetics of intracellular killing of Staphylococcus aureus and Escherichia coli by human granulocytes

The intracellular killing of Staphylococcus aureus and Escherichia coli by human granulocytes was investigated independently of the ingestion of these bacteria. Granulocytes were allowed to phagocytose preopsonized bacteria for only 3 min, after which the noningested bacteria were removed by differential centrifugation and 2 washes. With this technique, the number of viable cell-associated bacteria at the start of the assay, determined after lysis of the granulocytes, includes about 80% intracellular bacteria. Intracellular killing depends on (a) the temperature (no killing occurring at 4°C, maximal killing at 33–39 °C, and a decrease in the capacity of the granulocytes to kill ingested bacteria at temperatures above 42 °C), and (b) the number of bacteria ingested (after phagocytosis at bacteria-to-cell ratios of 100 : 1 and 1000 : 1, not all of the ingested bacteria are killed, whereas after phagocytosis at lower bacteria-to-cell ratios, almost all ingested bacteria are killed). To determine the maximum number of bacteria that can be killed by granulocytes, intracellular killing was measured after phagocytosis of bacteria at various bacteria-to-granulocyte ratios in the presence of phenylbutazone, a drug which inhibits killing during the ingestion period. Phenyl-butazone proved to be a useful tool in the study of intracellular killing, since this drug provides a reversible inhibition of the killing when granulocytes are incubated in its presence for up to 3 min, whereas after longer incubation, the inhibitory effect is irreversible. Calculation based on the data obtained in this study gave maximum rates of intracellular killing amounting to 3.7 × 10⁵ bacteria/5 × 10⁶ granulocytes/min for Staph. aureus and 8.5 × 10⁵ bacteria/5 × 10⁶ granulocytes/min for E. coli. Using the rate of intracellular killing after phagocytosis at a bacteria-to-granulocyte ratio of 1 : 1 and the rate of ingestion obtained in an earlier study, we were able to compute the theoretical numbers of viable extracellular not (yet) ingested, viable intracellular, killed intracellular and total intracellular bacteria. The theoretical curves fit well with the experimental data.     

TLR8‐mediated activation of human monocytes inhibits TL1A expression

TLR play important roles in inflammation and innate immune response to pathogens. TLR8 recognizes ssRNA and induces NF‐κB via MyD88 signaling. TL1A is a member of the TNF superfamily that markedly enhances IFN‐γ production by IL‐12/IL‐18‐stimulated peripheral and mucosal CD4⁺ T cells. TL1A expression is increased in the mucosa of patients with inflammatory bowel disease and is considered a key mediator of Crohn's disease (CD). We have previously shown that TL1A is strongly induced by immune complexes (IC) but not TLR ligands in antigen‐presenting cells. However, a potential interaction between these pro‐inflammatory signaling pathways has not been investigated. IC‐induced TL1A expression of monocytes was potently inhibited by a TLR8 or TLR7/8 ligand (R848) in a dose‐dependent manner. Furthermore, when co‐cultured with CD4⁺ T cells, TLR8 ligands inhibited TL1A production, resulting in almost complete inhibition of IFN‐γ production by the CD4⁺ T cells. Furthermore, we demonstrate that IFN‐α is not required for this suppressive effect by TLR8 signaling. Our data demonstrate for the first time a direct interaction between TLR and TL1A signaling pathways. TLR8 activation may be an important, novel pathway for targeted treatment of Th1‐mediated diseases, such as CD.     

Protein tyrosine kinase activity is essential for Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes.

Our previous study revealed that the intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma receptor I (Fc gamma RI) or Fc gamma RII is a phospholipase C (PLC)-dependent process. The aim of the present study was to investigate whether protein tyrosine kinase (PTK) activity plays a role in the Fc gamma R-mediated intracellular killing of bacteria and activation of PLC in these cells. The results showed that phagocytosis of bacteria by monocytes was not affected by the PTK inhibitors genistein and tyrphostin-47. The intracellular killing of S. aureus by monocytes after cross-linking Fc gamma RII or Fc gamma RII with anti-Fc gamma R monoclonal antibody and a bridging antibody or with human immunoglobulin G (IgG) was inhibited by these compounds in a dose-dependent fashion. The production of O2- by monocytes after stimulation with IgG or IgG-opsonized S. aureus was almost completely blocked by the PTK inhibitor. These results indicate that inhibition of PTK impairs the oxygen-dependent bactericidal mechanisms of monocytes. Genistein and tyrphostin-47, which do not affect the enzymatic activity of purified PLC, prevented activation of PLC after cross-linking Fc gamma RI or Fc gamma RII, measured as an increase in the intracellular inositol 1,4,5-trisphosphate concentration. Cross-linking Fc gamma RI or Fc gamma RII induced rapid tyrosine phosphorylation of several proteins in monocytes, one of which was identified as PLC-gamma 1, and the phosphorylation could be completely blocked by PTK inhibitors, leading to the conclusion that activation of PLC after cross-linking Fc gamma R in monocytes is regulated by PTK activity. Together, these results demonstrate that PTK activity is essential for the activation of PLC which is involved in the Fc gamma R-mediated intracellular killing of S. aureus by human monocytes.     

Activated monocytes augment TRAIL‐mediated cytotoxicity by human NK cells through release of IFN‐γ

Natural killer (NK) cells are innate lymphocytes that are able to directly kill tumor cells through different mechanisms including ligation of TNF‐related apoptosis‐inducing ligand (TRAIL) receptors. Zoledronic acid (ZA) is a bisphosphonate known to upregulate the expression of TRAIL on human γδ T cells. Here, we investigated whether exposure to ZA would upregulate TRAIL expression on human NK cells and augment their cytotoxicity against tumor cells. When cocultured with monocytes, treatment with ZA and IL‐2 resulted in a significant upregulation of TRAIL expression on human NK cells (p = 0.002). Consequently, ZA‐primed NK cells were significantly more cytotoxic against TRAIL sensitive tumor cells (p < 0.0001). In the presence of ZA and IL‐2, monocytes produced high levels of IFN‐γ; when cultured in the presence of neutralizing antibodies to IFN‐γ, TRAIL expression and TRAIL‐mediated cytotoxicity of NK cells were significantly reduced. Furthermore, in tumor‐bearing SCID/Beige mice, a significant delayed tumor progression and prolonged survival was observed after infusion of ZA‐primed NK cells compared with that observed in mice infused with unprimed NK cells. These findings represent a novel approach to potentiate TRAIL‐mediated apoptosis by adoptively infused NK cells that could improve the outcome in patients with cancer.     

The internalization of the IgG2a antigen receptor does not require the association with Ig-α and Ig-β but the activation of protein tyrosine kinases does

The B cell antigen receptor of class IgM is a multimeric protein complex containing the membrane-bound immunoglobulin molecule and a heterodimer of the two B cell-specific transmembrane proteins Ig-α and Ig-β. The B cell antigen receptor fulfills a dual role on the surface of B cells. First, it is a signal transduction complex which can activate protein tyrosine kinases and induce the release of Ca²⁺ ions from intracellular stores. Second, its internalization mediates the specific uptake of bound antigens, which are processed intracellularly and presented as major histocompatibility complex-bound peptides on the cell surface. In case of the IgM antigen receptor, the association with the heterodimer is necessary for expression of large amounts of IgM on the surface. We show here that the IgG2a antigen receptor can be expressed on the surface of myeloma cells in two structurally different forms: either with or without the Ig-α/Ig-β heterodimer. A functional comparison of the two forms of antigen receptors demonstrates that the Ig-α and Ig-β molecules are required for the activation of protein tyrosine kinases after cross-linking of the B cell antigen receptor. In contrast, both forms of IgG2a are equally well internalized. This suggests that Ig-α and Ig-β are essential for signal transduction through the IgG2a antigen receptor, whereas internalization can occur independently of the heterodimer.     

Arachidonic acid, but not its metabolites, is essential for FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes

Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcgamma receptor (FcgammaR)-mediated respiratory burst and phagocytosis of opsonized erythrocytes by monocytes and macrophages, we focused in this study on the role of AA and its metabolites in the FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes. The results revealed that the PLA2 inhibitors, but not inhibitors of cyclo-oxygenase and lipoxygenase, markedly suppressed the FcgammaR-mediated killing process. The production of O-2 by monocytes upon FcgammaR cross-linking was inhibited by 4-bromophenacyl bromide in a dose-dependent fashion, indicating that inhibition of PLA2 activity impairs the oxygen-dependent bactericidal mechanisms of monocytes, which could be partially restored by addition of exogenous AA and docosahexaenoic acid, but not myristic acid. These polyunsaturated fatty acids, but not myristic acid, stimulated the intracellular killing of S. aureus by monocytes, although not as effectively as FcgammaR cross-linking. Furthermore, FcgammaR cross-linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcgammaR-mediated activation of PLA2 is dependent on Ca2+ and tyrosine kinase activity. Together these results indicate a key role for PLA2/AA, but not its major metabolites, in mediating the FcgammaR-stimulated intracellular killing of S. aureus by monocytes.