Kok P. M. van Kessel

Chemotaxis Inhibitory Protein of Staphylococcus aureus, a Bacterial Antiinflammatory Agent

Leukocyte migration is a key event both in host defense against invading pathogens as well as in inflammation. Bacteria generate chemoattractants primarily by excretion (formylated peptides), complement activation (C5a), and subsequently through activation of leukocytes (e.g., leukotriene B4, platelet-activating factor, and interleukin 8). Here we describe a new protein secreted by Staphylococcus aureus that specifically impairs the response of neutrophils and monocytes to formylated peptides and C5a. This chemotaxis inhibitory protein of S. aureus (CHIPS) is a 14.1-kD protein encoded on a bacteriophage and is found in >60% of clinical isolates. CHIPS reduces the neutrophil recruitment toward C5a in a mouse peritonitis model, even though its activity is much more potent on human than on mouse cells. These findings suggest a new immune escape mechanism of S. aureus and put forward CHIPS as a potential new antiinflammatory therapeutic compound.

Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases

The complement system is pivotal in host defense but also contributes to tissue injury in several diseases. The assembly of C3 convertases (C4b2a and C3bBb) is a prerequisite for complement activation. The convertases catalyze C3b deposition on activator surfaces. Here we describe the identification of staphylococcal complement inhibitor, an excreted 9.8-kilodalton protein that blocks human complement by specific interaction with C4b2a and C3bBb. Staphylococcal complement inhibitor bound and stabilized C3 convertases, interfering with additional C3b deposition through the classical, lectin and alternative complement pathways. This led to a substantial decrease in phagocytosis and killing of Staphylococcus aureus by human neutrophils. As a highly active and small soluble protein that acts exclusively on surfaces, staphylococcal complement inhibitor may represent a promising anti-inflammatory molecule.

Lipoprotein metabolism in patients with severe sepsis

ObjectiveLipoproteins have been implicated to play a role in innate immunity. Changes in lipoprotein levels have been reported in a variety of inflammatory disorders. Not much is known about lipoprotein metabolism in patients with severe sepsis. We conducted an ancillary study in a multiple-center phase III sepsis trial to investigate the dynamics of plasma lipoproteins in patients with severe sepsis. DesignProspective analysis in patients meeting criteria for severe sepsis as part of a multiple-center sepsis study (KyberSept) with antithrombin III (Kybernin P). SettingUniversity hospital intensive care unit. PatientsSeventeen patients were included in the study. InterventionsRandomized patients received a loading dose of 6000 IU of antithrombin III (Kybernin P) or placebo followed by a 96-hr continuous infusion of 250 IU/hr antithrombin III (Kybernin P) or placebo. In each patient, serial blood samples for total cholesterol, lipoprotein cholesterol, triglycerides, apolipoprotein A-1, apolipoprotein B, and C-reactive protein determination as well as clinical data were collected over 28 days. Measurements and Main ResultsPlasma cholesterol levels rapidly decreased from 2.67 ± 2.02 mmol/L on day 0 to a nadir of 1.41 ± 0.70 mmol/L on day 3, followed by a slow increase to 4.18 ± 1.94 mmol/L on day 28. High-density lipoprotein (HDL) cholesterol concentrations decreased rapidly from 0.84 ± 0.92 mmol/L to a nadir of 0.42 ± 0.35 mmol/L on day 3, to show a slow increase during the following 4 wks to 0.84 ± 0.42 mmol/L. The low-density lipoprotein (LDL) cholesterol concentrations were already low (0.94 ± 0.81 mmol/L) at study entry, to show a progressive increase to subnormal values (2.01 ± 0.94 mmol/L) at 4 wks. Nadir and recovery lipoprotein concentrations were significantly different (paired Student’s t-test, p < .05). A significant correlation was found between HDL cholesterol and apolipoprotein A-1 (r = .714, p < .05) and between LDL cholesterol and apolipoprotein B (r = .733, p < .05). There was no statistical difference in lipoprotein concentrations either between survivors and nonsurvivors or between patients receiving antithrombin III or placebo.Serum amyloid A was a major apoprotein (45%) in HDL at the start of the sepsis and was slowly replaced by apolipoprotein A-1 during recovery. A positive correlation was found between plasma C-reactive protein concentrations and serum amyloid A concentrations in HDL (r = .684, p < .05). No other relevant correlations were found between inflammatory and lipoprotein parameters. ConclusionsIn patients with severe sepsis, lipoprotein concentrations rapidly change and can be reduced to 50% of recovery concentrations. The pattern of early rapid decline is found primarily in the HDL and a slow recovery in both HDL and LDL fractions. The correlation between apolipoprotein and lipoprotein cholesterol concentrations suggests a decline in lipoprotein particles. During severe sepsis, HDL is shifted to acute phase HDL, which is enriched in serum amyloid A and depleted of cholesterol and apolipoprotein A-1. Lipoprotein concentrations are unable to discriminate between survivors and nonsurvivors.

Staphylococcus aureus Strains Lacking d-Alanine Modifications of Teichoic Acids Are Highly Susceptible to Human Neutrophil Killing and Are Virulence Attenuated in Mice

Staphylococcus aureus is resistant to alpha-defensins, antimicrobial peptides that play an important role in oxygen-independent killing of human neutrophils. The dlt operon mediates d-alanine incorporation into teichoic acids in the staphylococcal cell envelope and is a determinant of defensin resistance. By using S. aureus wild-type (WT) and Dlt- bacteria, the relative contributions of oxygen-dependent and -independent antimicrobial phagocyte components were analyzed. The Dlt- strain was efficiently killed by human neutrophils even in the absence of a functional respiratory burst, whereas the killing of the WT organism was strongly diminished when the respiratory burst was inhibited. Human monocytes, which do not produce defensins, inactivated the WT and Dlt- bacteria with similar efficiencies. In addition, mice injected with the Dlt- strain had significantly lower rates of sepsis and septic arthritis and fewer bacteria in the kidneys, compared with mice infected with the WT strain.

Chemotaxis Inhibitory Protein of Staphylococcus aureus Binds Specifically to the C5a and Formylated Peptide Receptor

Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is an exoprotein produced by several strains of S. aureus, and a potent inhibitor of neutrophil and monocyte chemotaxis toward C5a and formylated peptides like fMLP. These chemoattractants act on their target cells by binding and activating the C5aR and formylated peptide receptor (FPR), respectively. In the present report, we examined the mechanism by which CHIPS affects both of these receptors. We showed that CHIPS blocked binding of anti-C5aR mAb and formylated peptide to human neutrophils as efficiently at temperatures of 0 and 37°C, implying that it is independent of signal transducing systems. This was confirmed by showing that CHIPS acts completely independently of ATP. Additionally, CHIPS was not internalized upon binding to neutrophils. Furthermore, we showed that CHIPS binds specifically to the C5aR and FPR expressed on U937 cells. This binding was functional in blocking C5a- and fMLP-induced calcium mobilization in these cell lines. These results suggest that CHIPS binds directly to the C5aR and FPR, thereby preventing the natural ligands from activating these receptors. The apparent Kd values of CHIPS for the C5aR and FPR were 1.1 ± 0.2 nM and 35.4 ± 7.7 nM, respectively. Moreover, after screening a wide variety of other G protein-coupled receptors, CHIPS was found to affect exclusively the C5aR and FPR. This selectivity and high-affinity binding with potent antagonistic effects makes CHIPS a promising lead for the development of new anti-inflammatory compounds for diseases in which damage by neutrophils plays a key role.

The Staphylococcal Toxin Panton-Valentine Leukocidin Targets Human C5a Receptors

Panton-Valentine Leukocidin (PVL) is a staphylococcal bicomponent pore-forming toxin linked to severe invasive infections. Target-cell and species specificity of PVL are poorly understood, and the mechanism of action of this toxin in Staphylococcus aureus virulence is controversial. Here, we identify the human complement receptors C5aR and C5L2 as host targets of PVL, mediating both toxin binding and cytotoxicity. Expression and interspecies variations of the C5aR determine cell and species specificity of PVL. The C5aR binding PVL component, LukS-PV, is a potent inhibitor of C5a-induced immune cell activation. These findings provide insight into leukocidin function and staphylococcal virulence and offer directions for future investigations into individual susceptibility to severe staphylococcal disease.

Staphylococcal superantigen-like 5 binds PSGL-1 and inhibits P-selectin-mediated neutrophil rolling.

Staphylococcus aureus secretes several virulence factors interfering with host-cell functions. Staphylococcal superantigen-like (SSL) proteins are a family of 11 exotoxins with structural homology to superantigens but with generally unknown functions. Recently, we described that chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS(31-121)), a potent inhibitor of C5a-induced responses, is structurally homologous to the C-terminal domain of SSL5. Here, we identify P-selectin glycoprotein ligand-1 (PSGL-1), involved in the initial rolling of neutrophils along the endothelium, as a target for SSL5. SSL5 specifically bound to Chinese hamster ovary cells stably expressing PSGL-1 (CHO-PSGL-1), which was dependent of sulfation and sialylation. Furthermore, SSL5 bound to PSGL-1/Ig fusion protein immobilized on a biosensor chip. SSL5 affected binding of soluble P-selectin/Fc chimera, the principle ligand of PSGL-1, to CHO-PSGL-1 cells and inhibited adhesion of neutrophils to immobilized P-selectin under static conditions. Under flow conditions SSL5 strongly decreased neutrophil rolling on immobilized P-selectin/Fc and activated human endothelial cells. In conclusion, SSL5 interferes with the interaction between PSGL-1 and P-selectin, suggesting that S aureus uses SSL5 to prevent neutrophil extravasation toward the site of infection. This makes SSL5 a potential lead for the development of new anti-inflammatory compounds for disorders characterized by excessive recruitment of leukocytes.

Quantitation of surface CD14 on human monocytes and neutrophils.

The absolute number of membrane‐expressed CD14, the most important endotoxin receptor, on human monocytes and neutrophils shows remarkable variation in the literature. To quantify these numbers two fluorescence methods using fluorescein isothiocyanate (FITC)‐labeled monoclonal antibodies (mAb) were applied. A commercially available set of standard beads was used in flow cytometry to quantitate CD14 with eight different mAbs. Independent from their isotype the various mAbs showed minor differences and indicated that peripheral blood monocytes expressed 99,500–134,600 (115,400 ± 10,600) and neutrophils 1,900–4,400 (3,300 ± 800) CD14 receptors. There was no significant difference in CD14 expression on leukocytes in unprocessed freshly obtained whole blood and after a Ficoll isolation procedure. However, a short temperature shift resulted in a 1.3‐ to 1.6‐fold upregulation of CD14. The results obtained with the reference beads were verified with fluorescence Scatchard analysis and spectrofluorometry using mAb 26ic‐FITC and showed 109,500 CD14 per monocyte and 6,700 CD14 per neutrophil. For comparison the number of CD14 on the monocytic THP‐1 cells and Fcγ‐receptors on human leukocytes were determined using the reference beads and flow cytometry and gave results comparable to published data. Our data indicate that resting human monocytes express roughly 110,000 CD14 molecules on their surface using a simple fluorometric assay. Correct determination of the number of CD14 and other cell surface receptors is of importance in the monitoring of septic patients. J. Leukoc. Biol. 61: 721–728; 1997.

Plasma vascular endothelial growth factor in severe sepsis.

Vascular endothelial growth factor (VEGF) is a potent vascular permeability factor. The development of capillary leak is common in septic patients, and several sepsis-associated mediators may induce VEGF production. The potential role of VEGF during sepsis has not been studied to date. The aim of the study was first to assess whether circulating VEGF levels increase during sepsis, and second, to examine whether plasma VEGF levels are associated with disease severity. VEGF levels were measured in serial plasma samples of 18 patients with severe sepsis and in 40 healthy controls. VEGF levels were correlated to clinical signs and symptoms. VEGF levels were significantly elevated in sepsis patients compared with healthy controls (134 vs. 55 pg/mL; P < 0.001). Serum albumin levels used as an indirect measure of vascular leak were decreased in septic patients. Increased plasma VEGF levels at study entry were correlated to severity of multiple organ dysfunction during the course of disease (Pearson correlation coefficient r = 0.75; P = 0.001). Moreover, maximum VEGF levels in nonsurvivors were significantly higher than those in survivors (P = 0.018). These data show that plasma VEGF levels are elevated during severe sepsis. Furthermore, our data indicate that plasma VEGF levels are associated with disease severity and mortality. Further study of the potential role of VEGF in the development of sepsis-associated capillary leak is indicated.

A New Staphylococcal Anti-Inflammatory Protein That Antagonizes the Formyl Peptide Receptor-Like 1

Bacteria have developed mechanisms to escape the first line of host defense, which is constituted by the recruitment of phagocytes to the sites of bacterial invasion. We previously described the chemotaxis inhibitory protein of Staphylococcus aureus, a protein that blocks the activation of neutrophils via the formyl peptide receptor (FPR) and C5aR. We now describe a new protein from S. aureus that impaired the neutrophil responses to FPR-like1 (FPRL1) agonists. FPRL1 inhibitory protein (FLIPr) inhibited the calcium mobilization in neutrophils stimulated with MMK-1, WKYMVM, prion-protein fragment PrP106–126, and amyloid β1–42. Stimulation with low concentrations of fMLP was partly inhibited. Directed migration was also completely prevented toward MMK-1 and partly toward fMLP. Fluorescence-labeled FLIPr efficiently bound to neutrophils, monocytes, B cells, and NK cells. HEK293 cells transfected with human C5aR, FPR, FPRL1, and FPRL2 clearly showed that FLIPr directly bound to FPRL1 and, at higher concentrations, also to FPR but not to C5aR and FPRL2. FLIPr can reveal unknown inflammatory ligands crucial during S. aureus infections. As a novel described FPRL1 antagonist, it might lead to the development of therapeutic agents in FPRL1-mediated inflammatory components of diseases such as systemic amyloidosis, Alzheimer’s, and prion disease.