Staphylococcus aureus protects its immune-evasion proteins against degradation by neutrophil serine proteases.

Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti-staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune-evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune-escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus.

Bovine Staphylococcus aureus Secretes the Leukocidin LukMF' To Kill Migrating Neutrophils through CCR1.

UNLABELLED: Although Staphylococcus aureus is best known for infecting humans, bovine-specific strains are a major cause of mastitis in dairy cattle. The bicomponent leukocidin LukMF', exclusively harbored by S. aureus of ruminant origin, is a virulence factor associated with bovine infections. In this study, the molecular basis of the host specificity of LukMF' is elucidated by identification of chemokine receptor CCR1 as its target. Bovine neutrophils, the major effector cells in the defense against staphylococci, express significant cell surface levels of CCR1, whereas human neutrophils do not. This causes the particular susceptibility of bovine neutrophils to pore formation induced by LukMF'. Bovine S. aureus strains produce high levels of LukMF' in vitro. In culture supernatant of the mastitis field isolate S1444, LukMF' was the most important cytotoxic agent for bovine neutrophils. In a fibrin gel matrix, the effects of the in situ secreted toxins on neutrophils migrating toward S. aureus were visualized. Under these physiological ex vivo conditions, bovine S. aureus S1444 efficiently killed approaching neutrophils at a distance through secretion of LukMF'. Altogether, our findings illustrate the coevolution of pathogen and host, provide new targets for therapeutic and vaccine approaches to treat staphylococcal diseases in the cow, and emphasize the importance of staphylococcal toxins in general. IMPORTANCE: This study explains the mechanism of action of LukMF', a bicomponent toxin found in bovine lineages of S. aureus that is associated with mastitis in cattle. At a molecular level, we describe how LukMF' can specifically kill bovine neutrophils. Here, we demonstrate the contribution of toxins in the determination of host specificity and contribute to the understanding of mechanisms of coevolution of pathogen and host. Our study provides new targets that can be used in therapeutic and vaccine approaches to treat staphylococcal diseases in the cow. We also demonstrate the importance of toxins in specific elimination of immune cells, which has broader implications, especially in human infections.

Microenvironment involved in FPR1 expression by human glioblastomas.

Formyl peptide receptor 1 (FPR1) activity in U87 glioblastoma (GBM) cells contributes to tumor cell motility. The present study aimed to evaluate the FPR1 expression in human GBM, the possibility to elicit agonist induced FPR1 activation of GBM cells and inhibit this activation with chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Immunohistochemistry was used to assess FPR1 expression in GBM patient samples, which was present in all 178 samples. Also FPR1 mRNA levels measured with quantitative PCR, could be detected in all 25 GBM patient samples tested. Activation of FPR1 in U87 cells, as measured by human mitochondrial-derived agonists, increased calcium mobilization, AKT and ERK1/2 phosphorylation, and ligand-induced migration. Inhibition of all responses could be achieved with CHIPS. Eight early passage human Groningen Glioma (GG) cell lines, isolated from primary GBM tissue were screened for the presence of FPR1. FPR1 mRNA and protein expression as well as receptor activation could not be detected in any of these early passage GG cell lines. However FPR1 was present in ex vivo tumors formed by the same GG cell lines after being implanted in mouse brains. FPR1 is highly expressed in human GBM specimens, it can be activated by human mitochondrial-derived agonists in U87 and inhibited with CHIPS. FPR1 cannot be detected in early passage GG cell lines in vitro, however when engrafted in the mouse brain these cells show FPR1 expression. These results suggest a role of the brain microenvironment in FPR1 expression in GBM.

Staphylococcal alpha-phenol soluble modulins contribute to neutrophil lysis after phagocytosis.

Staphylococcus aureus community-acquired (CA) MRSA strains are highly virulent and can cause infections in otherwise healthy individuals. The most important mechanism of the host for clearing S. aureus is phagocytosis by neutrophils and subsequent killing of the pathogen. Especially CA-MRSA strains are very efficient in circumventing this neutrophil killing. Interestingly, only a relative small number of virulence factors have been associated with CA-MRSA, one of which are the phenol soluble modulins (PSMs). We have recently shown that the PSMs are functionally inhibited by serum lipoproteins, indicating that PSMs may exert their cytolytic function primarily in the intracellular environment. To further investigate the intracellular role of the PSMs we measured the effect of the α-type and ß-type PSMs on neutrophil killing after phagocytosis. Using fluorescently labelled S. aureus, we measured bacterial survival after phagocytosis in a plate reader, which was employed next to flow cytometry and time-lapse microscopy. Phagocytosis of the CA-MRSA strain MW2 by human neutrophils resulted in rapid host cell death. Using mutant strains of MW2, we demonstrated that in the presence of serum, the intracellular expression of only the psmα operon is both necessary and sufficient for both increased neutrophil cell death and increased survival of S. aureus. Our results identify PSMα peptides as prominent contributors to killing of neutrophils after phagocytosis, a finding with major implications for our understanding of S. aureus pathogenesis and strategies for S. aureus vaccine development.

Inhibition of formyl peptide receptor in high-grade astrocytoma by CHemotaxis Inhibitory Protein of S. aureus.

BACKGROUND: High-grade astrocytomas are malignant brain tumours that infiltrate the surrounding brain tissue and have a poor prognosis. Activation of formyl peptide receptor (FPR1) on the human astrocytoma cell line U87 promotes cell motility, growth and angiogenesis. We therefore investigated the FPR1 inhibitor, Chemotaxis Inhibitory Protein of S. aureus (CHIPS), as a potential anti-astrocytoma drug. METHODS AND RESULTS: FPR1 expression was studied immunohistochemically in astrocytomas WHO grades I-IV. With intracellular calcium mobilisation and migration assays, human ligands were tested for their ability to activate FPR1 on U87 cells and on a cell line derived from primary astrocytoma grade IV patient material. Thereafter, we selectively inhibited these ligand-induced responses of FPR1 with an anti-inflammatory compound called Chemotaxis Inhibitory Protein of S. aureus (CHIPS). U87 xenografts in NOD-SCID mice served to investigate the effects of CHIPS in vivo. FPR1 was expressed in 29 out of 32 (90%) of all grades of astrocytomas. Two human mitochondrial-derived formylated peptides, formyl-methionil-leucine-lysine-isoleucine-valine (fMLKLIV) and formyl-methionil-methionil-tyrosine-alanine-leucine-phenylalanine (fMMYALF), were potent activators of FPR1 on tumour cells. Ligand-induced responses of FPR1-expressing tumour cells could be inhibited with FPR1 inhibitor CHIPS. Treatment of tumour-bearing mice with CHIPS slightly reduced tumour growth and improved survival as compared to non-treated animals (P=0.0019). CONCLUSION: Targeting FPR1 with CHIPS reduces cell motility and tumour cell activation, and prolongs the survival of tumour-bearing mice. This strategy could be explored in future research to improve treatment results for astrocytoma patients.

Evasion of Toll-like receptor 2 activation by staphylococcal superantigen-like protein 3.

Toll-like receptors (TLRs) are crucial for our host defense against microbial infections. TLR2 is especially important to fight bacterial infections, as it specifically recognizes bacterial lipoproteins of both Gram-positive and Gram-negative origin. Present on a variety of immune cells, TLR2 is critical for host protection against several bacterial infections, including those caused by Staphylococcus aureus. This major human pathogen causes increasing health care problems due to its increased resistance to antibiotics. S. aureus secretes a wide variety of proteins that inhibit innate immune responses. Recently, several staphylococcal superantigen-like proteins (SSLs) have been described to mediate immune evasive properties. Here, we describe that SSL3 specifically binds and inhibits TLR2 activation on human and murine neutrophils and monocytes. Through binding of the extracellular TLR2 domain, SSL3 inhibits IL-8 production by HEK cells expressing TLR1/2 and TLR2/6 dimers, stimulated with their specific ligands. The SSL3-TLR2 interaction is partially glycan dependent as binding of SSL3 to TLR2 is affected upon removal of sialic acid residues. Moreover, the SSL3(R308A) mutant lacking glycan-binding properties shows lower TLR2 inhibition. An SSL3 mutant, lacking the N-terminal 126 amino acids, still retains full TLR2 inhibiting activity. Of other SSLs tested, only SSL4, which shares the highest homology with SSL3, blocks TLR2 activation. SSL3 is the first-described bacterial protein that blocks TLR2 activation through direct extracellular interaction with the receptor. This unique function of SSL3 adds to the arsenal of immune evasive molecules that S. aureus can employ to subvert both innate and adaptive immunity.

Staphylococcal superantigen-like 5 activates platelets and supports platelet adhesion under flow conditions, which involves glycoprotein Ibalpha and alpha IIb beta 3.

OBJECTIVES: Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus aureus that has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). METHODS AND RESULTS: When purified recombinant SSL5 was added to washed platelets in an aggregometry set-up, complete and irreversible aggregation was observed. Proteolysis of the extracellular part of GPIb alpha or the addition of dRGDW abrogated platelet aggregation. When a mixture of isolated platelets and red cells was perfused over immobilized SSL5 at a shear rate of 300 s(-1), stable platelet aggregates were observed, and platelet deposition was substantially reduced after proteolysis of GPIb or after addition of dRGDW. SSL5 was shown to interact with glycocalicin, a soluble GPIb alpha fragment, and binding of SSL5 to platelets resulted in GPIb-mediated signal transduction as evidenced by translocation of 14-3-3 zeta. In addition, SSL5 was shown to interact with endothelial cell matrix (ECM) and this interaction enhanced aggregation of platelets from whole blood to this ECM. CONCLUSIONS: SSL5 activates and aggregates platelets in a GPIb alpha-dependent manner, which could be important in colonization of the vascular bed and evasion of the immune system by S. aureus.

Anti-opsonic properties of staphylokinase.

Recently we described a novel bacteriophage-encoded pathogenicity island in Staphylococcus aureus that harbors a number of virulence factors that are all involved in the evasion of innate immunity. Here we describe a mechanism by which staphylokinase (SAK), frequently present on this pathogenicity island, interferes with innate immune defenses: SAK is anti-opsonic. By activating human plasminogen (PLG) into plasmin (PL) at the bacterial surface, it creates bacterium-bound serine protease activity that leads to degradation of two major opsonins: human immunoglobulin G (IgG) and human C3b. Incubation of opsonized bacteria with PLG and SAK resulted in removal of anti-staphylococcal IgGs and C3b from the bacterial surface. In phagocytosis assays this proved to be a very efficient mechanism to reduce the opsonic activity of human IgG and serum. The fact that SAK activates human PLG at the bacterial surface and removes IgG as well as C3b makes this protein a unique anti-opsonic molecule.

The role of tumour necrosis factor in the kinetics of lipopolysaccharide-mediated neutrophil priming in whole blood.

Neutrophils can be primed by bacterial lipopolysaccharide (LPS) for an enhanced oxidative burst, which is a key element in the pathogenesis of Gram-negative sepsis. Some serum proteins (e.g. lipopolysaccharide-binding protein) avidly bind LPS and markedly enhance receptor binding and cellular activation while other serum factors (lipoproteins, bactericidal/permeability-increasing protein) neutralize LPS and prevent neutrophil activation. In this paper we examined the kinetics of this priming reaction in whole blood. To study the balance between neutrophil activation and LPS neutralization a sensitive chemiluminescence assay was used in a whole blood system. LPS was able to prime neutrophils for enhanced oxidative burst in whole blood with an optimum incubation time of 25 min. However, LPS was neutralized very rapidly with a t(1/2) of 10 min. After 20 min a second priming factor was already generated, which was shown to be monocyte-derived tumour necrosis factor (TNF).

Novel engagement of CD14 and multiple toll-like receptors by group B streptococci.

Group B streptococcus (GBS) imposes a major health threat to newborn infants. Little is known about the molecular basis of GBS-induced sepsis. Both heat-inactivated whole GBS bacteria and a heat-labile soluble factor released by GBS during growth (GBS-F) induce nuclear translocation of NF-kappaB, the secretion of TNF-alpha, and the formation of NO in mouse macrophages. Macrophages from mice with a targeted disruption of MyD88 failed to secrete TNF-alpha in response to both heat-inactivated whole bacteria and GBS-F, suggesting that Toll-like receptors (TLRs) are involved in different aspects of GBS recognition. Immune cell activation by whole bacteria differed profoundly from that by secreted GBS-F. Whole GBS activated macrophages independently of TLR2 and TLR6, whereas a response to the secreted GBS-F was not observed in macrophages from TLR2-deficient animals. In addition to TLR2, TLR6 and CD14 expression were essential for GBS-F responses, whereas TLR1 and TLR4 or MD-2 did not appear to be involved. Heat lability distinguished GBS-F from peptidoglycan and lipoproteins. GBS mutants deficient in capsular polysaccharide or beta-hemolysin had GBS-F activity comparable to that of wild-type streptococci. We suggest that CD14 and TLR2 and TLR6 function as coreceptors for secreted microbial products derived from GBS and that cell wall components of GBS are recognized by TLRs distinct from TLR1, 2, 4, or 6.

Characteristics of surfactant protein A and D binding to lipoteichoic acid and peptidoglycan, 2 major cell wall components of gram-positive bacteria.

Infection with gram-positive bacteria is a major cause of pneumonia. Surfactant proteins A (SP-A) and D (SP-D) are thought to play an important role in the innate immunity of the lung. Both proteins can bind to gram-positive bacteria. Until now, it was not known with which surface component(s) of gram-positive bacteria SP-A and SP-D interact. Lipoteichoic acid (LTA) and peptidoglycan (PepG) are components of the cell wall of gram-positive bacteria. By use of a solid phase-based binding assay, LTA of Bacillus subtilis was shown to be bound by SP-D but not by SP-A. Unmodified PepG of Staphylococcus aureus was bound by SP-D. SP-D binding to both LTA and PepG was calcium dependent and carbohydrate inhibitable. These results indicate that SP-D interacts with gram-positive bacteria via binding to the cell wall components LTA and PepG and that the carbohydrate recognition domain is responsible for this binding.

A novel flow cytometric assay to quantify soluble CD14 concentration in human serum.

BACKGROUND: CD14, the major lipopolysaccharide (LPS)-binding protein of myeloid cells, is found as a soluble molecule in human serum. Recent data describe the presence of elevated soluble CD14 (sCD14) concentration in various disorders, confirming disease activity. A novel, easy, and rapid flow cytometric assay was developed to measure sCD14 levels in serum. METHODS: The assay is based on the competition between membrane-expressed CD14 of isolated monocytes from healthy volunteers and sCD14 in the sample sera for binding to anti-CD14 monoclonal antibodies (mAb; 26ic or 60bca). The amount of cell-associated mAb is determined with a fluorescein isothiocyanate (FITC)-labeled anti-mouse conjugate and flow cytometry. The fluorescence signal is inversely proportional with the amount of serum sCD14. Using dilutions of a standard serum, the concentration of sCD14 in the samples is calculated and compared with results obtained by a commercial sCD14 enzyme-linked immunosorbent assay (ELISA). RESULTS: After optimization, the assay showed log-log linearity of 122.1-984.7 ng/ml sCD14 using mAb 26ic and 29.5-246.2 ng/ml sCD14 using mAb 60bca. It revealed similar results as the ELISA (mAb 26ic: r = 0.88, mAb 60bca: r = 0.92) and provided significantly elevated sCD14 levels in systemic lupus erythematosus patients compared with controls (26ic: 2,213 versus 1,676 ng/ml, P < 0.002; 60bca: 2,625 versus 1,907 ng/ml, P < 0.0002). Receiver operating characteristic curve analysis suggested a reasonable diagnostic efficacy of sCD14 quantification in this autoimmune disease. CONCLUSIONS: The method is easy, rapid, sensitive, and can be used in the follow-up of patients suffering from sepsis or chronic inflammatory disorders.

Potent inhibition of neutrophil migration by cryptococcal mannoprotein-4-induced desensitization.

Cryptococcal capsular Ags induce the production of proinflammatory cytokines in patients with cryptococcal meningitis. Despite this, their cerebrospinal fluid typically contains few neutrophils. Capsular glucuronoxylomannan is generally considered to mediate the inhibition of neutrophil extravasation. In the current study, culture supernatant harvested from the nonglucuronoxylomannan-producing strain CAP67 was found to be as potent as supernatant from wild-type strains in preventing migration. We identified capsular mannoprotein (MP)-4 as the causative agent. Purified MP-4 inhibited migration of neutrophils toward platelet-activating factor, IL-8, and fMLP, probably via a mechanism involving chemoattractant receptor cross-desensitization, as suggested by its direct chemotactic activity. Supporting this hypothesis, MP-4 elicited Ca(2+) transients that were inhibited by preincubation with either fMLP, IL-8, or C5a, but not platelet-activating factor, and vice versa. Moreover, MP-4 strongly decreased the neutrophil surface expression of L-selectin and induced shedding of TNF receptors p55/p75, whereas CD11b/18 increased. Finally, MP-4 was clearly detectable in both serum and cerebrospinal fluid of patients suffering from cryptococcal meningitis. These findings identify MP-4 as a novel capsular Ag prematurely activating neutrophils and desensitizing them toward a chemoattractant challenge.

Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine.

Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance mechanism. We describe a novel staphylococcal gene, mprF, which determines resistance to several host defense peptides such as defensins and protegrins. An mprF mutant strain was killed considerably faster by human neutrophils and exhibited attenuated virulence in mice, indicating a key role for defensin resistance in the pathogenicity of S. aureus. Analysis of membrane lipids demonstrated that the mprF mutant no longer modifies phosphatidylglycerol with l-lysine. As this unusual modification leads to a reduced negative charge of the membrane surface, MprF-mediated peptide resistance is most likely based on repulsion of the cationic peptides. Accordingly, inactivation of mprF led to increased binding of antimicrobial peptides by the bacteria. MprF has no similarity with genes of known function, but related genes were identified in the genomes of several pathogens including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Enterococcus faecalis. MprF thus constitutes a novel virulence factor, which may be of general relevance for bacterial pathogens and represents a new target for attacking multidrug resistant bacteria.

Diverging pathways for lipopolysaccharide and CD14 in human monocytes.

BACKGROUND: CD14 is considered to be the major endotoxin (lipopolysaccharide [LPS]) binding molecule on human monocytes. It initiates cellular response, but its role in the clearance of LPS is not well understood. Under conditions that ensure totally CD14-dependent LPS binding on human monocytes, the internalization mechanisms of LPS and CD14 were studied. METHODS: The uptake and intracellular distribution of fluorescein isothiocyanate (FITC)-LPS and CD14 was determined by flow cytometry, trypan blue quenching, and confocal fluorescence microscopy. Incubation of surface-biotinylated cells with LPS at 37 degrees C or 4 degrees C and subsequent subfractionation was used to further characterize CD14 internalization. The amount of the intracellular CD14 was estimated by CD14 enzyme-linked immunosorbent assay (ELISA). RESULTS: The internalization rate of 10 ng/ml FITC-LPS with 1% human serum was 1% of bound endotoxin per minute, whereas CD14 expression did not decrease at the same time surface. We proved the presence of an intracellular CD14 pool (2.68 x 10(6) molecules per unstimulated monocyte) and could show that internalized FITC-LPS molecules can be found in different intracellular compartments than CD14. Subfractionation of LPS-treated biotinylated monocytes showed no change in biotinylated CD14 in the membrane fraction independently of the incubation temperature (37 degrees C or at 4 degrees C) used, indicating that these CD14 molecules were not taken up by an active process. CONCLUSIONS: These data indicate the presence of a large intracellular CD14 pool in monocytes with a yet unknown function, and suggest that LPS and CD14 molecules can be internalized independently after association on the cell surface.

Analysis of lipopolysaccharide (LPS)-binding characteristics of serum components using gel filtration of FITC-labeled LPS.

Lipopolysaccharide (LPS)-binding components in serum play an important role in modifying LPS toxicity. We analyzed the binding characteristics of LPS in the presence of serum using gel filtration of FITC-labeled LPS (FITC-LPS) with on line detection of optical density and fluorescence. FITC-LPS separately behaves as an aggregate resulting in a low, dequenched, fluorescence. Binding of single LPS molecules, segregated from the aggregate, to serum components results in an increase in the fluorescence due to dequenching, and a comigration of fluorescence and optical density signals using gel filtration. This method, in combination with the use of specific antibodies inducing additional shifts, demonstrated that in serum high-density lipoproteins (HDL), albumin and low-density lipoproteins (LDL) were able to monomerize LPS. An ELISA on collected fractions of the gel filtration revealed binding of the recently identified LPS-binding protein, serum amyloid P component (SAP), to the high molecular weight LPS aggregate. In serum, binding of soluble CD14 (sCD14) and LPS-binding protein (LBP) to LPS could not be detected. However, this was probably due to an overshadowing effect of albumin, as an extra addition of recombinant sCD14 to serum clearly monomerized FITC-LPS. Biosensor technology revealed that, of all LPS-binding components tested, only SAP clearly bound to the LPS-coated sensor chip. These results show that gel filtration of FITC-LPS is a quick and reliable method to study the binding characteristics of LPS-binding components.

Modulation of neutrophil chemokine receptors by Staphylococcus aureus supernate.

In a previous study, we showed that Staphylococcus aureus supernate (SaS) is a potent agonist for both neutrophils and mononuclear cells. To further investigate the immunomodulating effects of SaS, the effect on different neutrophil receptors was studied. Expression of various neutrophil receptors, before and after treatment with SaS, was quantified by flow cytometry. We found that SaS treatment of neutrophils resulted in a specific and total downregulation of the C5a and the fMLP receptor, both serpentine receptors, while other receptors were totally unaffected. Since these two receptors are both involved in chemotaxis, we tested the effect of SaS in calcium flux and chemotaxis assays. We showed that preincubation with SaS abrogated the rise in intracellular calcium concentration upon triggering with fMLP and C5a. We also showed that SaS is a potent inhibitor of neutrophil chemotaxis towards fMLP and C5a, but does not interfere with chemotaxis towards interleukin-8. These findings indicate that S. aureus produces a virulence factor extracellularly, which impairs chemotaxis towards the infected site.

Serum amyloid P component prevents high-density lipoprotein-mediated neutralization of lipopolysaccharide.

Lipopolysaccharide (LPS) is an amphipathic macromolecule that is highly aggregated in aqueous preparations. LPS-binding protein (LBP) catalyzes the transfer of single LPS molecules, segregated from an LPS aggregate, to high-density lipoproteins (HDL), which results in the neutralization of LPS. When fluorescein isothiocyanate-labeled LPS (FITC-LPS) is used, this transfer of LPS monomers to HDL can be measured as an increase in fluorescence due to dequenching of FITC-LPS. Recently, serum amyloid P component (SAP) was shown to neutralize LPS in vitro, although only in the presence of low concentrations of LBP. In this study, we show that SAP prevented HDL-mediated dequenching of FITC-LPS, even in the presence of high concentrations of LBP. Human bactericidal/permeability-increasing protein (BPI), a very potent LPS-binding and -neutralizing protein, also prevented HDL-mediated dequenching of FITC-LPS. Furthermore, SAP inhibited HDL-mediated neutralization of both rough and smooth LPS in a chemiluminescence assay quantifying the LPS-induced priming of neutrophils in human blood. SAP bound both isolated HDL and HDL in serum. Using HDL-coated magnetic beads prebound with SAP, we demonstrated that HDL-bound SAP prevented the binding of LPS to HDL. We suggest that SAP, by preventing LPS binding to HDL, plays a regulatory role, balancing the amount of LPS that, via HDL, is directed to the adrenal glands.

Functional human monoclonal antibodies of all isotypes constructed from phage display library-derived single-chain Fv antibody fragments.

We have constructed a series of eukaryotic expression vectors that permit the rapid conversion of single chain (sc) Fv antibody fragments, derived from semi-synthetic phage display libraries, into intact fully human monoclonal antibodies (mAb) of each isotype. As a model, a scFv fragment specific for sheep red blood cells (SRBC) was isolated from a semi-synthetic phage antibody (Ab) display library, and used to produce human mAbs of IgM, IgG1-IgG4, IgA1, IgA2m(1) and IgE isotype in vitro in stably transfected cells. N-terminal protein sequence analysis of purified immunoglobulin heavy (H) and light (L) chains revealed precise proteolytic removal of the leader peptide. Biochemical analysis of purified recombinant human mAbs demonstrated that properly glycosylated molecules of the correct molecular size were produced. The IgG and IgA mAbs retained SRBC-binding activity, interacted with different Fc receptor-transfectants, and induced complement-mediated hemolysis and Ab-dependent phagocytosis of SRBC by neutrophils in a pattern consistent with the immunoglobulin (Ig) H chain isotype. We conclude that in vitro produced recombinant human mAbs constructed from phage display library-derived scFv fragments mirror their natural counterparts and may represent a source of mAbs for use in human therapy.