Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gargir, A.
Right arrow Articles by Nissim, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gargir, A.
Right arrow Articles by Nissim, A.
(Journal of Leukocyte Biology. 2001;70:537-542.)
© 2001 by Society for Leukocyte Biology

Inhibition of antibody-dependent stimulation of lipoteichoic acid-treated human monocytes and macrophages by polyglycerolphosphate-reactive peptides

Ari Gargir*, Itzhak Ofek*, David Hasty2, Shiri Meron-Sudai*, Hayim Tsubery3, Yona Keisari* and Ahuva Nissim4,||

* Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel;
{dagger} Department of Anatomy and Neurobiology, University of Tennessee and Research Service (151) VAMC, Memphis;
{ddagger} Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel;
§ Bone and Joint Research Unit, St. Bartholomew’s and Royal London School of Medicine and Dentistry, Queen Mary, Charterhouse Square, United Kingdom; and
|| Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel Aviv University, Rabin Medical Center, Belinson Campus, Petach Tikva, Israel

Correspondence: Dr. Itzhak Ofek, Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel. E-mail: aofek{at}ccsg.tau.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
By itself, lipoteichoic acid (LTA) obtained from S. pyogenes, S. aureus, or E. hirae poorly stimulated cytokine production by macrophages, whereas in the presence of anti-polyglycerol phosphate (PGP), the cells secreted significant amounts of IL-6. Two peptides constructed from the deduced sequence of the selected anti-PGP phage-antibody’s complementary-determining region 3 of the variable heavy chain (VH-CDR3) reacted specifically with PGP. The monomeric form of the peptides markedly inhibited cytokine production by macrophages pretreated with LTA and anti-LTA. In contrast, the polyvalent form of biotinylated peptides complex with streptavidin-induced cytokine production by the LTA-treated macrophages. The data taken together support the concept that cross-linking of macrophage-bound LTA by anti-PGP is required for cytokine release by these cells. Importantly, these studies identified small, PGP-reactive peptides as potential tools in reducing this proinflammatory process.

Key Words: LTA • PGP • phage display • Streptococci • macrophage stimulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipoteichoic acids (LTA) are components of the cell membrane cell walls of virtually all Gram-positive bacteria, and they can also be secreted into the surrounding milieu [1 ]. Their structures vary among different species, but in general, they can be described as macro amphiphiles consisting of a hydrophilic backbone of approximately 30 repeating units of polyglycerol phosphate (PGP) substituted to various degrees by alanine and glycosidic substitutions and covalently linked by an ester bond to glycolipid [2 ]. LTAs have been shown to bind to many types of animal cells, and in most cases, this binding is lipid- and not PGP-dependent.

Although the central role of LTA in mediating adhesion of Gram-positive pathogens has been documented [3 ], its role in post-adhesion pathophysiology of Gram-positive infections is controversial. Its ability to bind to and subsequently lyse animal cells upon interaction with anti-PGP and complement has been suggested to contribute to tissue damage caused by group A streptococcal infection [4 ]. Normal human sera contain anti-PGP antibodies whose levels become elevated during streptococcal infection [4 ]. Almost 5% of the human population exhibits high titers of anti-LTA antibody [5 ]. Anti-LTA-dependent arthritis has been shown to occur in mice injected intra-articularly with LTA [6 ]. However, the specific roles that LTA and anti-LTA might play in the various forms of streptococcal infection and poststreptococcal sequelae are not settled.

During the last decade, many studies focused on the role of LTA in Gram-positive shock, mainly because of the remarkable, structural and functional analogies between LTA and lipopolysaccharide (LPS) of Gram-negative bacteria [1 , 7 ]. For example, with the exception of pyrogenicity, many biological effects of LTA are reminiscent of those of LPS, including immunogeneicity, mitogenicity, bone resorption, stimulation of alternative complement pathway, Schwartzmann reaction, hypersensitivity, stimulation of nonspecific immunity, nephrotoxicity, limulus assay activity, and adjuvant activity [8 ]. Furthermore, whenever it has been tested, deacylated LTA monomers have been without effect, suggesting that as for LPS, the glycolipid portion of the LTA molecule is essential in eliciting its biological effect [1 ]. Because LPS stimulates macrophages to release proinflammatory cytokines, some of which [e.g., tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin (IL)-1] are directly responsible for the symptoms of shock, most in vitro studies on the role of LTA in Gram-positive shock have also focused on the ability of the molecule to stimulate macrophages.

Although LTA alone is not pyrogenic in vivo [7 ], the pioneering studies of Yamamoto et al. [9 ] showing release of cytokines in Propionibacterium acne-primed animals following injection with LTA have been confirmed [10 , 11 ] and extended to show that the effects included release of nitric oxide [12 13 14 ]. Moreover, it was shown that Staphylococcus aureus LTA synergizes with peptidoglycan to cause multiple organ failure and shock in rats, although neither of these components alone was active [12 ]. Contradictory findings have been demonstrated regarding the ability of LTA to stimulate release of cytokines by macrophages in vitro. For example, S. aureus LTA stimulated the release of TNF-{alpha} by rat macrophage in one study [7 ], whereas another study showed that LTA did not stimulate human monocytes [15 ]. Moreover, even in tests conducted within a single laboratory, a commercial preparation of S. aureus LTA was active in monocyte stimulation, although the laboratory’s own preparation of LTA extracted from the same organism was inactive, even though the chemical composition of both LTA preparations was shown to be similar [16 ]. In other cases, however, it appears that the sources of LTA and purification procedures are important with regard to functional activity. There are several instances where the same investigator extracted LTA from two different strains of Streptococcus faecalis, which were found to differ significantly in activity [15 ]. Butanol or phenol extraction of LTA from the same strain may yield preparations with differing alanine contents and different stimulatory activities [17 ]. Recently, one group concluded that it was a mannose-rich glycolipid contaminant of their LTA preparations from S. faecalis that was responsible for the cytokine stimulation observed and that the purified LTA was inactive [18 ]. Although many of these results are contradictory, whenever tested, the observed stimulation was dependent on the lipid moiety of the molecule, suggesting that binding of LTA to macrophages is lipid-mediated and probably required for stimulation [1 ]. On their surface, the divergence of these results does not help much to clarify our understanding of LTA’s function.

Previous studies showed that LTA-treated polymorphonuclear cells (PMN) and macrophages could be stimulated to produce cytokines by adding anti-LTA [19 , 20 ]. Thus, it was suggested that cross-linking of the PGP moiety of cell-bound LTA resulted in cross-linking of LTA receptors and that this event then triggered the cells to release the proinflammatory agents. The phage display technology [21 ] has made great progress over the last decade, enabling selection of peptides and antibodies capable of reacting with a vast variety of target molecules [22 ]. We used a phage-display, semi-synthetic, human-antibody library [23 ] to select for anti-PGP antibodies to use in these studies. This technology was also critical to test the ability of small, PGP-reactive peptides to inhibit the anti-LTA-dependent stimulation of cytokine release by LTA-treated mononuclear cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of LTA, deacylated LTA, and rabbit anti-LTA antibody
LTA was extracted by the hot phenol-water procedure and further purified from Streptococcus pyogenes M-5 by sequential ion exchange and hydrophobic columns as described previously [24 25 26 ]. This LTA preparation is poor in alanine substitutions [27 ]. Highly purified LTA from Enterococcus hirae, free from other glycolipid contaminants, was a generous gift from Dr. Shoichi Kusumoto and Dr. Yasuo Suda (Osaka University, Japan) [28 ]. S. aureus LTA was purchased from Sigma Chemical Co. (St. Louis, MO). PGP was obtained by deacylating S. pyogenes LTA using alkali ammonia hydrolysis and repeated chloroform-methanol extraction [24 ]. Anti-LTA serum was prepared by immunizing rabbits with LTA-methylated albumin complexes [29 ]. Immunoglobulin G (IgG) was purified from anti-LTA serum by a protein A affinity column following the manufacturer’s instructions (Pharmacia, Uppsala, Sweden). LPS was from Escherichia coli O55:B5 (Sigma Chemical Co.).

Phage-display antibody library selection
We used a phage-display, human semi-synthetic single-chain fragment variable (scFv) library constructed from a bank of 50 human V gene segments rearranged in vitro with random nucleotide sequences encoding a variable heavy-chain, complementary-determining region (VH-CDR3) of 4–12 amino acid residues, assembled with a fixed V{lambda}3 light-chain sequence [23 , 30 , 31 ].

Selection of the M13 phage displaying PGP-specific scFv was conducted as described by panning against PGP-coated immunotubes [23 , 32 ]. Briefly, immunotubes (Nunc-Immuno Tubes, MaxiSorp, Nunc, Denmark) were coated with PGP by adding 100 µg/ml PGP in phosphate-buffered saline (PBS) and incubating overnight at room temperature. The tubes were then blocked by 2% skimmed milk in PBS (MPBS) for 2 h, washed with PBS, and exposed to 1012–1013 transforming units (t.u.) of the phage library in 2% MPBS. After 2 h incubation, the library was washed free of unbound phage, and the PGP-bound phage was eluted in 100 mM triethylamine and neutralized with 1M Tris, pH 7.4. For enrichment of the PGP-bound phage, E. coli TG-1 was infected with the eluted phages and rescued by the helper phage as described [32 ]. The panning process was repeated four times, E. coli TG-1 was infected with the final phage preparations, and individual ampicillin-resistant colonies (phage clones) were selected for further analysis.

Screening and sequencing of PGP-specific phage clones
The entire scFv DNA fragments of the selected phage clones were amplified by polymerase chain reaction (PCR) using the commonly used phage-vector primers, LMB-3 (5'-CAGGAAACAGCTATGAC) and fd-Seq (5'-GAATTTTCTGTATGAGG), and amplicons were sequenced by the dideoxy method [33 ] using DyeDeoxy chain termination (Applied Biosystems, Foster City, CA) and an Applied Biosystems sequencer. The sequences were analyzed using SeqEd (Applied Biosystems), and the heavy-chain gene family was determined by sequence alignment using the VH database and DNAPlot (URL: http://www.genetik.uni-koeln.de/dnaplot/vsearch_human.html).

Phage purification and quantification
Monoclonal phages were prepared by 10 repeated PEG (20% polyethylene glycol, MW 6000; NaCl 2.5 M) precipitations of culture supernatants from infected bacteria to eliminate bacterial contaminants. Thereafter, they were filtered with a 0.45 µm filter unit (Corning, Corning, NY). Phage titer was determined by enumerating t.u. for infected, ampicillin-resistant E. coli TG-1 [32 ]. Protein content was determined with a protein assay kit (Bio-Rad, Munchen, Germany).

Competition enzyme-linked immunosorbent assay (ELISA)
Wells of microtiter plates (Nunclon, Nunc, Denmark) were coated with LTA by adding 100 µl LTA (10 µg/ml PBS) to each well and incubating overnight at room temperature. The wells were then washed with PBS and blocked by adding 200 µl 2% MPBS for 90 min at room temperature. Phage suspension (100 µl; 10 µg/ml protein) was added to the LTA-coated wells, alone or containing various concentrations of LTA, PGP, glycerol phosphate, or trehalose (Sigma Chemical Co.). After incubation at room temperature for 90 min, the supernatants were removed, the wells washed with PBS, and the amount of bound phage was determined as follows: Peroxidase-labeled, anti-M13 antibodies (100 µl; Pharmacia) diluted to 1:5000 in 2% MPBS were added to the wells and incubated for 30 min, and the wells were washed three times with PBS containing 0.05% Tween 20, followed by three washes in PBS. The bound, peroxidase-labeled anti-phage was determined by the TMB (3, 3' 5,5' tetramethylbenzidine) reaction following the manufacturer’s instructions (Sigma Chemical Co.), and the development of color was monitored in a Spectra Max 340 ELISA reader (Molecular Devices, Sunnyville, CA) at 450 nm with a reference wavelength of 650 nm.

Preparation of monocytes and macrophages
Human monocytes and human monocyte-derived macrophages (hMoDM) were obtained from peripheral blood of healthy donors as described [34 ] with a few modifications. Adherent monocytes were used for experiments as fresh monocytes or after overnight incubation in RPMI-1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% newborn bovine serum (NBS; Biological Industries, Kibbutz Beit Ha-Emek, Israel) in a humidified incubator (37°C, 7.5% CO2) for their subsequent 10-day differentiation period to macrophages [34 ]. Briefly, monocytes incubated overnight at 37°C, 7.5% CO2, were detached by rinsing the monolayer with ice-cold Dulbecco’s PBS (DPBS) without Ca2+ and Mg2+ and were collected and counted. The cells were suspended in RPMI/10% NBS with 100 U/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Behringwerker, Marburg, Germany) at a concentration of 5 x 105 cells/ml and aliquoted in 1 ml portions in 6 ml polypropylene tubes (Falcon, Franklin Lakes, NJ), and cells were incubated for 10 days in a humidified incubator with gentle, daily resuspension to avoid cell adhesion to tubes.

Stimulation of macrophages
RPMI-1640 was the medium used to prepare all suspensions and reagent solutions as well as cell washes. Macrophages were treated with LTA by adding LTA (100 µg/ml) to a suspension of macrophages (5x105 cells) in 200 µl, and after 30 min in a humidified incubator, the cells were washed three times with medium. LTA-treated and -untreated macrophages or monocytes were stimulated with phage particles (10 µg/ml phage protein), rabbit anti-LTA IgG (300 µg/ml protein), peptide 2B-streptavidin complexes (1–1000 nM), and LPS (100 ng/ml) as a control. In some experiments, anti-LTA IgG was mixed with synthetic peptides before being added to the monocytes. Polymyxin B (25 µg/ml) was added to inhibit any possible effects of residual LPS that could be present in phage preparations [35 ]. After 30 min in a humidified incubator, the cells stimulated with phage particles were washed three times, and incubation was continued overnight. The amount of IL-6 released was determined using an ELISA kit (Endogen, Woburn, MA), according to the manufacturer’s procedures.

Peptide synthesis and characterization
Peptides were synthesized by solid-phase techniques using Fmoc/Boc chemistry, cleaved with trifluoroacetyl (TFA), and purified to homogeneity (>97%) by high-pressure liquid chromatography (HPLC) [36 ]. The peptides had the expected amino acid composition by amino acid analysis, and molecular weight and purity were verified by mass spectrometry.

Haemagglutination assay
The LTA-mediated heamagglutination assay was used as described elsewhere [24 ]. Briefly, 2% (v/v) suspension of group O, Rh-negative human red blood cells, were incubated with LTA (100 µg/ml), followed by two washes in PBS to remove nonbound LTA. Equal volumes (20 µl) of rabbit anti-LTA diluted 1:100, and PBS or PBS containing test peptide was added to 0.1 ml LTA-bound erythrocyte suspension. After incubation for 15 min at room temperature, the mixture was centrifuged for 5 min (1000 g). The erythrocyte button was gently shaken to visualize agglutination. Controls—erythrocytes alone or PBS instead of anti-sera—were always included to ensure that heamagglutination is mediated by erythrocyte-bound LTA and rabbit anti-LTA. Hemagglutination was recorded to determine the minimal peptide concentration required to inhibit rabbit anti-LTA-induced hemagglutination of LTA-treated erythrocytes.

Statistics
Unpaired Student’s t-test (two-tailed), arithmetic mean, and average deviation calculations were performed using Excel version 2000 (Microsoft Corp., Redmond, WA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence analysis of selected anti-PGP phage clone
Out of 96 clones screened by ELISA, we selected four specific, anti-PGP phage clones that showed the highest ELISA signals [optical density (OD) 450 nm>1.0] for detailed analysis. Sequence analysis revealed that all four clones were identical, and the heavy chain (VH) was determined to be DP-67, a member of the VH-4 gene family [30 ]. The sequence of the anti-PGP VH domain allows the determination of the VH-CDR3 sequence, which occupies a central position in the antigen-binding site of the antibody molecule [37 ], and was comprised of a highly basic core composed of M-A-K-R-R-K-P-F amino acids (Fig. 1 ). The light chain of the scFv is the DP{lambda}-16 gene segment, a fixed sequence in the structure of the phage-displayed, semi-synthetic scFv library used in this work [23 ]. Additional experiments were focused on one of these four clones.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 1. Amino acid sequence of PGP-specific phage clones. The entire scFv DNA fragments of the selected phage clones were amplified by PCR using the commonly used phage-vector primers, LMB-3 and fd-Seq. The specific, PGP-reactive sequence is a highly basic amino acid core of the synthetic VH-CDR3 (underlined bold letters), flanked by DP-67 and JH4 sequences, at the 5' and 3' ends, respectively. These sequence data are available from GenBank under accession number AF202544.

 
Antigenic activity of the anti-PGP phage clone
Phage preparations of the above-selected clones reacted with LTA and PGP immobilized to microtiter plates. In competition, ELISA, the binding of the anti-PGP phage, was inhibited by soluble LTA, PGP, and, to a lesser extent, glycerol-phosphate monomers with IC50 of 11, 8.6, and 100 µM, repectively. In contrast, concentration higher than 105 mM trehalose did not inhibit the binding of the phage to the immobilized LTA. This indicates that the clone is specific for PGP and binds with much lower affinity even to glycerol phosphate, which is the major backbone of PGP (IC50=100 µM; Table 1 ).


View this table:
[in this window]
[in a new window]
  		Table 1. Competition ELISA of Anti-PGP Phage

 
Stimulation of LTA-treated macrophages by anti-PGP phage
Treatment of macrophages with S. pyogenes, S. aureus, and E. hirae (S. faecalis) LTA preparations did not stimulate IL-6 release. However, addition of anti-PGP carrying-phage to macrophages pretreated with these LTA preparations resulted in a marked increase in IL-6 secretion (Fig. 2 ). The stimulation is PGP-anti-PGP-dependent because nonspecific phage neither bound to LTA nor stimulated LTA-treated macrophages to produce the cytokine.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 2. Stimulation of LTA-treated macrophages by anti-PGP phage. Macrophages pretreated with 100 µg/ml LTA from E. hirae, S. pyogenes, and S. aureus were exposed to medium alone or medium containing 10 µg/ml nonspecific or anti-PGP phage preparations. After washing nonbinding phage, the macrophages were incubated overnight in RPMI/10% NBS. IL-6 release was determined by ELISA, and its values are the mean ± SD. Macrophages stimulated by 100 ng/ml LPS released 10.2 ± 1.1 ng IL-6/ml. All experiments were performed in triplicate. The values for LTA-treated macrophages exposed to anti-PGP phage were significantly (P<0.05) higher than those obtained from macrophages exposed to LTA only or nonspecific phage.

 
Activity of VH-CDR3-derived, PGP-reactive peptides
Two peptide variants were synthesized based on the VH-CDR3 sequence of the anti-PGP phage clone: 5'-M-A-K-R-R-K-P-F-3' (Peptide I in Table 2 ) and 5'-S-G-G-G-G-M-A-K-R-R-K-P-F-G-G-G-G-S-3' (Peptide II in Table 2 ). The peptides were effective inhibitors of the hemagglutination of LTA-treated erythrocytes and rabbit serum anti-LTA (Table 2 a). Moreover, the anti-PGP Peptides I and II markedly inhibited the IL-6 release from macrophage treated with LTA and rabbit or human anti-LTA IgG serum (Table 2 b). In contrast, the peptides at these concentrations had no effect on the ability of 100 ng LPS to stimulate cytokine production (unpublished results), suggesting that they exhibit specific, anti-PGP activity. Biotinylated, peptide-strepavidin complexes, which displayed multi-valent (tetra-valent), anti-PGP reactivity, stimulated LTA-treated monocytes to release IL-6, although peptide alone was not active (Fig. 3 ). Furthermore, nonbiotinylated peptide (1 µM) inhibited the stimulation of LTA-treated monocytes by peptide-streptavidin complexes.


View this table:
[in this window]
[in a new window]
  		Table 2. Inhibition of LTA-Anti-LTA-Dependent Macrophage- Stimulation and Heamagglutination by Synthetic Anti-PGP Peptides Designed According to the VH-CDR3 Loop

 


View larger version (11K):
[in this window]
[in a new window]
  		Figure 3. Stimulation of IL-6 in LTA-treated macrophages by anti-PGP peptides. Macrophages were pretreated with 100 µg/ml LTA from S. pyogenes and were then exposed to medium containing the indicated concentrations of biotinylated, anti-PGP peptide ({blacktriangleup}) or to biotinylated peptides-streptavidin complexes (•). The biotinylated peptides-streptavidin complexes were also added to LTA-treated macrophages in the presence of 1 mM unbiotinylated peptide ({blacksquare}). After incubation and washing of nonbinding peptide, the macrophage was incubated overnight in RPMI/10% NBS. In the supernatant, IL-6 was estimated by ELISA. Values are mean ± SD of triplicate experiments. As shown, multimeric peptide formed by biotinylated peptides-streptavidin complexes stimulated IL-6 secretion of LTA-treated macrophages. This stimulation was, however, inhibited by addition of monomeric peptide ({blacksquare}).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
LTA is an amphiphile produced by most Gram-positive bacteria, which exhibits structural and functional similarity to Gram-negative LPS [1 , 7 ]. Some of these functional similarities are thought to be relevant to the remarkable similarities that can be found in the pathophysiology of shock caused by Gram-negative and Gram-positive bacteria, both of which are probably mediated in large part by TNF-{alpha} and IL-1 [1 , 9 , 38 ]. Because the role of LPS in triggering the release of these cytokines and other inflammatory mediators is well-established [39 ], it has been suggested that LTA may play a similar role. However, unlike LPS, purified LTA produced under standard procedures is unable to cause shock in vivo when administered alone [12 ]. Moreover, the ability of LTA to stimulate cytokine release in vitro is controversial. Pure, structurally defined LTA reconstituted from purified PGP and purified glycolipid does not elicit release of cytokines [40 ]. However, previous studies demonstrated that when PMN and macrophages are pretreated with LTA followed by treatment with anti-LTA serum, the cells released significant amounts of cytokines and oxygen radicals, respectively [19 , 20 ]. It was hypothesized that this effect could be a result of cross-linking the LTA on monocyte or macrophage cell surfaces.

Phage-display technology enabled us to obtain bioactive, anti-PGP polypeptides to test the foregoing hypothesis. This notion is supported by the findings showing that LTAs obtained from three different Gram-positive species alone are poor stimulators of cytokine production at best. When LTA-treated macrophages or monocytes were treated with anti-PGP phages, they secreted significant amounts of IL-6. The PGP-reactive phage mimics the effect of the anti-LTA antibodies by cross-linking the LTA-bound macrophage, because the PGP-reactive phage contains a heterogeneous mixture of wild-type pIII molecules and pIII anti-PGP fusion molecules and may carry zero to five fusion pIII anti-PGP proteins at its tip [32 ]. We observed day-to-day variation in the average number of pIII anti-PGP fusion molecules per phage particle. The average number of fusion pIII molecules per phage particle was 1.6 ± 0.13 and 0.5 ± 0.043 for two phage preparations A and B (P<0.05), respectively. This suggest that the majority of the A phages contains more than one anti-PGP fusion molecule and thus is multi-valent, and most of the B phages carry one to zero fusion molecules and thus relative to phage A, are considered monovalent. Only A phage, but not phage B, stimulated LTA-treated macrophage. After addition of anti-phage antibodies, however, A and B stimulated LTA-treated macrophage (unpublished results). This effect could be a result of cross-linking the LTA on macrophages by polyvalent anti-PGP phage B or monomeric anti-PGP phage A after addition of anti-phage antibodies.

From the foregoing discussion, we anticipated that small, PGP-reactive peptides could prevent cross-linking of LTA receptors and would, thereby, inhibit stimulation of LTA-coated macrophages by multi-valent, anti-PGP antibodies. The selection of such peptides was possible because of our ability to obtain amino acid sequence data from the reactive regions in the anti-PGP, phage-displayed antibodies. Indeed, the peptides abrogated the enhanced cytokine stimulation in LTA-coated macrophages triggered with serum-derived, anti-LTA IgG or a multi-valent, PGP-reactive peptide produced by a complex of a biotinylated, PGP-reactive peptide with streptavidin. Once again, the data suggest that the multimeric peptide mimics the anti-LTA antibodies by cross-linking a LTA-bound macrophage. However, the monomeric peptide reacts with the PGP moiety of the LTA and thus prevents the cross-linking and stimulation.

Although it is too early to speculate on the potential relevance of our findings to the development of Gram-positive shock, a number of other studies may be relevant to this discussion. LTA alone is not sufficient to elicit in vivo release of significant quantities of TNF-{alpha} or to cause shock, but it is able to do so when the animals are first primed, such as a treatment with Propionibacterium or peptidoglycan, both of which are known as potent polyclonal activators [9 , 42 ]. One mechanism through which priming acts to enable development of shock or release of TNF-{alpha} in animals injected with LTA may be by enhancing a polyclonal stimulation of the immune system to increase production of anti-LTA antibodies [42 ]. Seroepidemiological studies using LTA obtained from various Gram-positive species revealed that 1–5% of normal human sera contain elevated levels of anti-PGP antibodies [4 , 5 ]. It has also been shown that Gram-positive bacteria can release large amounts of LTA when grown in subinhibitory concentrations of penicillin [43 ]. We have found that S. pyogenes may release up to 150 µg/ml LTA during growth in the presence of subinhibitory concentrations of penicillin (unpublished results). These observations may account for increased levels of anti-LTA in patient serum or at local infection sites during the natural course of antibiotic treatment. Thus, it is not unreasonable to envisage a situation whereby macrophages become treated with LTA in a patient with elevated anti-LTA antibodies. The degree to which such a situation might contribute to shock remains to be seen. In preliminary studies, we found that the LTA-anti-LTA system caused the release of cytokines from a mouse macrophage RAW cell line (unpublished results), suggesting that the in vivo relevance of our findings can now be tested in a murine model.

Our findings suggest that if LTA does sensitize macrophages in vivo, and anti-LTA leads to stimulation, it may be possible to abrogate the development of shock with short, PGP-reactive peptides.


   ACKNOWLEDGEMENTS
 
This work was partially supported by a grant from the Wasserman Foundation and The Kurt Leon Foundation (I. O.) and by Merit Review funds from the U.S. Department of Veterans Affairs (D. H.). The work performed by A. G. is in partial fulfillment of the requirements for a Ph.D. degree from the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

Received October 17, 2000; revised June 6, 2001; accepted June 6, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wicken, A. J., Knox, K. W. (1975) Lipoteichoic acids: a new class of bacterial antigen Science 187,1161-1167[Free Full Text]
  2. Fischer, W. (1990) Bacterial phospholipids and lipoteichoic acids Kates, M. eds. Glycolipids, Phosphoglycolipids and Sulfoglycolipids ,123-234 Plenum New York.
  3. Hasty, D. L., Ofek, I., Courtney, H. S., Doyle, R. J. (1992) Multiple adhesins of Streptococci Infect. Immun. 60,2147-2152[Free Full Text]
  4. Ginsburg, I., Rozengarten, O., Dishon, T., Szabo, M., Bergner-Rabinowitz, S., Ofek, I. (1972) Antibodies to the cell-sensitizing antigen of Group A streptococci in the sera of patients. International congress series no. 317, streptococcal disease and the community Haverkorn, M. J. eds. Proc. 5th Int. Symp. Streptococcus Pyogenes ,97-100 Amsterdam Excerpta Medica.
  5. Markham, J. L., Knox, K. W., Schamschula, R. G. (1973) Antibodies to lipoteichoic acids in man Arch. Oral Biol. 18,313-319[Medline]
  6. Neeman, N., Ginsburg, I. (1972) Cell sensitizing antigen of group A streptococci II. Immunological and biological properties Isr. J. Med. Sci. 8,1807-1816[Medline]
  7. Wicken, A. J., Knox, K. W. (1980) Bacterial cell surface amphiphiles Biochim. Biophys. Acta 604,1-26[Medline]
  8. Hamada, S., Yamamoto, T., Koga, T., McGhee, J. R., Michalek, S. M., Yamamoto, S. (1985) Chemical properties and immunobiological activities of Streptococcal lipoteichoic acids Zentralbl. Bakteriol. Mikrobiol. Hyg. 259,228-243
  9. Yamamoto, A., Usami, H., Nagamuta, M., Sugawara, Y., Hamada, S., Yamamoto, T., Kato, K., Kokeguchi, S., Kotani, S. (1985) The use of lipoteichoic acid (LTA) from Streptococcus pyogenes to induce a serum factor causing tumor necrosis Br. J. Cancer 51,739-742[Medline]
  10. Himanen, J. P., Pyhala, L., Olander, R. M., Merimskaya, O., Kuzina, T., Lysyuk, O., Pronin, A., Sanin, A., Sarvas, M. (1993) Biological activities of lipoteichoic acid and peptidoglycan-teichoic acid of Bacillus subtilis 168 (Marburg) J. Gen. Microbiol. 139,2659-2665[Medline]
  11. Usami, H., Yamamoto, A., Sugawara, Y., Hamada, S., Yamamoto, T., Kato, K., Kokeguchi, S., Takada, H., Kotani, S. (1987) A nontoxic tumor necrosis factor induced by streptococcal lipoteichoic acids Br. J. Cancer 56,797-799[Medline]
  12. De Kimpe, S. J., Kengatharan, M., Thiemermann, C., Vane, J. R. (1995) The cell wall components peptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to cause shock and multiple organ failure Proc. Natl. Acad. Sci. USA 92,10359-10363[Abstract/Free Full Text]
  13. Kengatharan, K. M., De Kimpe, S. J., Thiermermann, C. (1996) Role of nitric oxide in the circulatory failure and organ injury in a rodent model of gram-positive shock Br. J. Pharmacol. 119,1411-1421[Medline]
  14. Kengatharan, K. M., De Kimpe, S. J., Robson, C., Foster, S. J., Thiermermann, C. (1998) Mechanism of Gram-positive shock: identification of peptidoglycan and lipoteichoic acid moieties essential in the induction of nitric oxide synthase, shock, and multiple organ failure J. Exp. Med. 188,305-315[Abstract/Free Full Text]
  15. Bhakdi, S., Klonisch, T., Nuber, P., Fischer, W. (1991) Stimulation of monokine production by lipoteichoic acids Infect. Immun. 59,4614-4620[Abstract/Free Full Text]
  16. Keller, R., Fischer, W., Keist, R., Basseti, S. (1992) Macrophage response to bacteria: induction of marked secretory and cellular activities by lipoteichoic acids Infect. Immun. 60,3664-3672[Abstract/Free Full Text]
  17. Morath, S., Geyer, A., Hartung, T. (2001) Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus J. Exp. Med. 193,393-397[Abstract/Free Full Text]
  18. Hashimoto, M., Imamura, Y., Yasuoka, J., Kotani, S., Kusumoto, S., Suda, Y. (1999) A novel cytokine-inducing glycolipid isolated from the lipoteichoic acid fraction of Enterococcus hirae ATCC 9790: a fundamental structure of the hydrophilic part Glycoconj. J. 16,213-221[Medline]
  19. Ginsburg, I., Fligiel, S. E. G., Ward, P. A., Varani, J. (1988) Lipoteichoic acid-antilipoteichoic acid complexes induce superoxide generation by human neutrophils Inflammation 12,525-548[Medline]
  20. Mancuso, G., Tomasello, F., Ofek, I., Teti, G. (1994) Anti-lipoteichoic acid antibodies enhance release of cytokines by monocytes treated with lipoteichoic acid Infect. Immun. 62,1470-1473[Abstract/Free Full Text]
  21. Smith, G. P. (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface Science 228,1315-1317[Abstract/Free Full Text]
  22. Rodi, D. J., Markowski, L. (1999) Phage-display technology—finding a needle in a vast molecular haystack Curr. Opin. Biotech. 10,87-93[Medline]
  23. Nissim, A., Hoogenboom, H. R., Tomlinson, I. M., Flynn, G., Midgley, C., Lane, D., Winter, G. (1994) Antibody fragments from a ‘single pot’ phage display library as immunochemical reagents EMBO J 13,692-698[Medline]
  24. Ofek, I., Beachey, E. H., Jefferson, W., Campbell, G. L. (1975) Cell membrane-binding properties of group A streptococcal lipoteichoic acid J. Exp. Med. 141,990-1003[Abstract/Free Full Text]
  25. Simpson, W. A., Ofek, I., Beachey, E. H. (1980) Fatty acid binding sites of serum albumin as membrane receptor analogs for streptococcal lipoteichoic acid Infect. Immun. 29,119-122[Abstract/Free Full Text]
  26. Fischer, W., Koch, H. U., Haas, R. (1983) Improved preparation of lipoteichoic acids Eur. J. Biochem. 133,523-530[Medline]
  27. Simpson, W. A., Ofek, I., Beachey, E. H. (1980) Binding of streptococcal lipoteichoic acid to the fatty acid binding sites on serum albumin J. Biol. Chem. 255,6092-6097[Abstract/Free Full Text]
  28. Suda, Y., Tochio, H., Kawano, K., Takada, H., Yoshida, T., Kotani, S., Kusumoto, S. (1995) Cytokine inducing glycolipids in the lipoteichoic acid fraction from Enterococcus hirae ATCC 9790 FEMS Immunol. Med. Microbiol. 12,97-112[Medline]
  29. Courtney, H., Ofek, I., Simpson, W. A., Beachey, E. H. (1981) Characterization of lipoteichoic acid binding to polymorphonuclear leukocytes of human blood Infect. Immun. 32,625-631[Abstract/Free Full Text]
  30. Tomlinson, I. M., Walter, G., Marks, J. D., Llewelyn, M. B., Winter, G. (1992) The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops J. Mol. Biol. 227,776-798[Medline]
  31. Williams, S. C., Winter, G. (1993) Cloning and sequencing of human immunoglobulin V gene segment Eur. J. Immunol. 23,1456-1461[Medline]
  32. Harrison, J. L., Williams, S. C., Winter, G., Nissim, A. (1996) Screening of phage antibody libraries Methods Enzymol 267,83-109[Medline]
  33. Sanger, F., Nicklen, S., Coulson, A. R. (1977) DNA sequencing with chain terminating inhibitors Proc. Natl. Acad. Sci. USA. 74,5463-5467[Abstract/Free Full Text]
  34. Keisari, Y. (1996) Human mononuclear phagocytes in tissue culture Jones, G. E. eds. Methods in Molecular Medicine: Human Cell Culture Protocols Totowa New Jersey.
  35. Ofek, I., Cohen, S., Rahmani, R., Kabha, K., Herzig, Y., Rubinstein, E. (1994) Antibacterial synergism of polymyxin B nonapeptide and hydrophobic antibiotics in experimental gram-negative infections in mice Antimicrob. Agents Chemther. 38,374-377[Abstract/Free Full Text]
  36. Atherton, E., Sheppard, R. C. (1989) Solid Phase Peptide Synthesis: A Practical Approach IRL Oxford.
  37. Chothia, C., Lesk, A. M. (1987) Canonical structures for the hypervariable regions of immunoglobulins J. Mol. Biol. 196,901-917[Medline]
  38. Freudenberg, M. A., Galanos, C. (1991) Tumor necrosis factor alpha mediates lethal activity of killed gram-negative and gram-positive bacteria in D-galactosamine-treated mice Infect. Immun. 59,2110-2115[Abstract/Free Full Text]
  39. Ulevitch, R. J., Tobias, P. S. (1995) Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin Annu. Rev. Immunol. 13,437-457[Medline]
  40. Hashimoto, M., Yasukoa, J., Suda, Y., Takada, H., Yoshida, T., Kotani, S., Kusumoto, S. (1997) Structural feature of the major but not cytokine-inducing molecular species of lipoteichoic acid J. Biochem. 121,779-786[Abstract/Free Full Text]
  41. Ullrich, A., Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity Cell 61,203-212[Medline]
  42. Levinson, A. I., Dziarski, A., Zweiman, B., Dziarski, R. (1983) Staphylococcal peptidoglycan: T-cell-dependent mitogen and relatively T-cell-independent polyclonal B-cell activator of human lymphocytes Infect. Immun. 39,290-296[Abstract/Free Full Text]
  43. Nealon, T. J., Beachey, E. H., Courtney, H. S., Simpson, W. A. (1986) Release of fibronectin-lipoteichoic acid complexes from group A streptococci with penicillin Infect. Immun. 51,529-535[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
D. L. Hasty, S. Meron-Sudai, K. H. Cox, T. Nagorna, E. Ruiz-Bustos, E. Losi, H. S. Courtney, E. A. Mahrous, R. Lee, and I. Ofek
Monocyte and Macrophage Activation by Lipoteichoic Acid Is Independent of Alanine and Is Potentiated by Hemoglobin
J. Immunol., May 1, 2006; 176(9): 5567 - 5576.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
F. C. Neuhaus and J. Baddiley
A Continuum of Anionic Charge: Structures and Functions of D-Alanyl-Teichoic Acids in Gram-Positive Bacteria
Microbiol. Mol. Biol. Rev., December 1, 2003; 67(4): 686 - 723.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gargir, A.
Right arrow Articles by Nissim, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gargir, A.
Right arrow Articles by Nissim, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS