Profound differences in leukocyte-endothelial cell responses to lipopolysaccharide versus lipoteichoic acid

We have investigated the effects of LPS from Escherichia coli, lipoteichoic acid (LTA), and peptidoglycan (PepG) from Staphylococcus aureus, and live S. aureus on leukocyte-endothelial interactions in vivo using intravital microscopy to visualize muscle microvasculature. Systemic vs local administration of LPS induced very different responses. Local administration of LPS into muscle induced significant leukocyte rolling, adhesion, and emigration in postcapillary venules at the site of injection. LPS given systemically dramatically dropped circulating leukocyte counts and increased neutrophils in the lung. However, the drop in circulating leukocytes was not associated with leukocyte sequestration to the site of injection (peritoneum) nor to peripheral microvessels in muscles. Unlike LPS, various preparations of LTA had no systemic and very minor local effect on leukocyte-endothelial interactions, even at high doses and for prolonged duration. LPS, but not LTA, potently activated human endothelium to recruit leukocytes under flow conditions in vitro. Endothelial adhesion molecule expression was also increased extensively with LPS, but not LTA. Interestingly, systemic administration of live S. aureus induced leukocyte-endothelial cell responses similar to LPS. PepG was able to induce leukocyte-endothelial interactions in muscle and peritoneum, but had no effect systemically (no increase in neutrophils in lungs and no decrease in circulating neutrophil counts). These results demonstrate that: 1) LPS has potent, but divergent local and systemic effects on leukocyte-endothelial interactions; 2) S. aureus can induce a systemic response similar to LPS, but this response is unlikely to be due to LTA, but more likely to be mediated in part by PepG.     

Atrial natriuretic peptide protects against Staphylococcus aureus-induced lung injury and endothelial barrier dysfunction

Lung inflammation and alterations in endothelial cell (EC) permeability are key events to development of acute lung injury (ALI). Protective effects of atrial natriuretic peptide (ANP) have been shown against inflammatory signaling and endothelial barrier dysfunction induced by gram-negative bacterial wall liposaccharide. We hypothesized that ANP may possess more general protective effects and attenuate lung inflammation and EC barrier dysfunction by suppressing inflammatory cascades and barrier-disruptive mechanisms shared by gram-negative and gram-positive pathogens. C57BL/6J wild-type or ANP knockout mice (Nppa-/-) were treated with gram-positive bacterial cell wall compounds, Staphylococcus aureus-derived peptidoglycan (PepG) and/or lipoteichoic acid (LTA) (intratracheal, 2.5 mg/kg each), with or without ANP (intravenous, 2 μg/kg). In vitro, human pulmonary EC barrier properties were assessed by morphological analysis of gap formation and measurements of transendothelial electrical resistance. LTA and PepG markedly increased pulmonary EC permeability and activated p38 and ERK1/2 MAP kinases, NF-κB, and Rho/Rho kinase signaling. EC barrier dysfunction was further elevated upon combined LTA and PepG treatment, but abolished by ANP pretreatment. In vivo, LTA and PepG-induced accumulation of protein and cells in the bronchoalveolar lavage fluid, tissue neutrophil infiltration, and increased Evans blue extravasation in the lungs was significantly attenuated by intravenous injection of ANP. Accumulation of bronchoalveolar lavage markers of LTA/PepG-induced lung inflammation and barrier dysfunction was further augmented in ANP-/- mice and attenuated by exogenous ANP injection. These results strongly suggest a protective role of ANP in the in vitro and in vivo models of ALI associated with gram-positive infection. Thus ANP may have important implications in therapeutic strategies aimed at the treatment of sepsis and ALI-induced gram-positive bacterial pathogens.     

The Escherichia coli heat labile toxin binds to Golgi membranes and alters Golgi and cell morphologies using ADP-ribosylation factor-dependent processes

The fate of the catalytic subunit of the Escherichia coli heat labile toxin (LTA(1)) was studied after expression in mammalian cells to assess the requirement for ADP-ribosylation factor (ARF) binding to localization and toxicity and ability to compete with endogenous ARF effectors. A progression in LTA(1) localization from cytosol to binding Golgi stacks to condensation of Golgi membranes was found to correlate with the time and level of LTA(1) expression. At the highest levels of LTA(1) expression the staining of LTA and both extrinsic and lumenal Golgi markers all became diffuse, in a fashion reminiscent of the actions of brefeldin A. Thus, LTA(1) binds to the Golgi and can alter its morphology in two distinct ways. However, point mutants of LTA(1) that are defective in the ability to bind activated ARF were also unable to bind Golgi membranes or modify Golgi morphology. Co-expression of mutants of ARF3 that regained binding to these same mutant LTA(1) proteins restored the localization and activities of the toxin. Thus, binding to ARF is required both for the localization of the toxin to the Golgi and for effects on Golgi membranes. A correlation was also seen between the ability of LTA mutants to bind ARF and the increase in cellular cAMP levels. These results demonstrate the importance of ARF binding to the toxicity and cellular effects of the ADP-ribosylating bacterial toxin and reveal that mutants defective in binding ARF retain basal ADP-ribosylation activity but are the least toxic LTA(1) mutants yet described, making them the best candidates for development as mucosal adjuvants.     

Distinct and essential morphogenic functions for wall‐ and lipo‐teichoic acids in Bacillus subtilis

Teichoic acids (TAs) are anionic polymers that constitute a major component of the cell wall in most Gram‐positive bacteria. Despite decades of study, their function has remained unclear. TAs are covalently linked either to the cell wall peptidoglycan (wall TA (WTA)) or to the membrane (lipo‐TA (LTA)). We have characterized the key enzyme of LTA synthesis in Bacillus subtilis, LTA synthase (LtaS). We show that LTA is needed for divalent cation homoeostasis and that its absence has severe effects on cell morphogenesis and cell division. Inactivation of both LTA and WTA is lethal and comparison of the individual mutants suggests that they have differentiated roles in elongation (WTA) and division (LTA). B. subtilis has four ltaS paralogues and we show how their roles are partially differentiated. Two paralogues have a redundant role in LTA synthesis during sporulation and their absence gives a novel absolute block in sporulation. The crystal structure of the extracytoplasmic part of LtaS, solved at 2.4‐Å resolution, reveals a phosphorylated threonine residue, which provides clues about the catalytic mechanism and identifies the active site of the enzyme.     

Formation of Molecular Complexes Between a Structurally Defined M Protein and Acylated or Deacylated Lipoteichoic Acid of Streptococcus pyogenes

The orientation of lipoteichoic acid (LTA) molecules on the surface of bacterial cells undoubtedly is determined by the ability of the LTA, during its transit through the cell wall, to bind via its polyglycerophosphate backbone or its glycolipid moieties to other constituents of the cytoplasmic membrane and the cell wall. We have investigated the possibility that LTA may become anchored to the cell surface by binding through its polyanionic backbone to positively charged regions of cell wall proteins. LTA was found to prevent the precipitation of partially purified HCl extracts of several strains of streptococci as well as a structurally defined streptococcal M protein molecule (pep M24) in 83% solutions of ethanol. The formation of complexes between LTA and M protein was demonstrated further by immunoelectrophoresis of pep M24 protein with increasing concentrations of radiolabeled LTA and by using antiserum against pep M24 to develop precipitin arcs. Pep M24 electrophoresed alone produced a single precipitin arc close to the origin. In contrast, when electrophoresed as a mixture with LTA or deacylated LTA, the M protein produced a second precipitin arc toward the anode coinciding with the area of migration of the radioactive LTA. Increasing concentrations of LTA or deacylated LTA shifted increasing amounts of the pep M24 antigen to the region of the second arc. Maleylation of M protein to block the positively charged free amino groups before mixing it with LTA prevented the formation of complexes. The complexes formed by the M protein with LTA, but not with deacylated LTA, showed the capacity to bind bovine serum albumin; LTA had been shown previously to bind to the fatty acid binding sites on bovine serum albumin. These results indicate that the LTA molecule is able to bind via its polyanionic backbone to positively charged residues of surface proteins of cells of S. pyogenes. The implications of such interaction as to the orientation of LTA molecules on the surface of cells of S. pyogenes are discussed.     

Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes

Lipoteichoic acid (LTA) is an important cell wall polymer in Gram-positive bacteria. The enzyme responsible for polyglycerolphosphate LTA synthesis is LtaS, first described in Staphylococcus aureus. Four LtaS orthologues, LtaS(BS) , YfnI, YqgS and YvgJ, are present in Bacillus subtilis. Using an in vitro enzyme assay, we determined that all four proteins are Mn(2+) -dependent metal enzymes that use phosphatidylglycerol as a substrate. We show that LtaS(BS) , YfnI and YqgS can produce polymers, suggesting that these three proteins are bona-fide LTA synthases while YvgJ functions as an LTA primase, as indicated by the accumulation of a GroP-Glc(2) -DAG glycolipid. Western blot analysis of LTA produced by ltaS(BS) , yfnI, yqgS and yvgJ single, triple and the quadruple mutant, showed that LTA production was only abolished in the quadruple and the YvgJ-only expressing mutant. B. subtilis strains expressing YfnI in the absence of LtaS(BS) produced LTA of retarded mobility, presumably caused by an increase in chain length as suggested by a structural analysis of purified LTA. Taken together, the presented results indicate that the mere presence or absence of LTA cannot account for cell division and sporulation defects observed in the absence of individual enzymes and revealed an unexpected enzymatic interdependency of LtaS-type proteins in B. subtilis.     

Lipopolysaccharide- and lipoteichoic acid-induced tolerance and cross-tolerance: distinct alterations in IL-1 receptor-associated kinase

Human Toll-like receptor (TLR) 4 and TLR2 receptors recognize LPS or lipoteichoic acid (LTA), respectively. Prolonged exposure of human macrophages/monocytes to bacterial LPS induces a state of adaptation/tolerance to subsequent LPS challenge. Inflammatory gene expressions such as IL-1beta and TNF-alpha are selectively repressed, while certain anti-inflammatory genes such as secretory IL-1R antagonist are still induced in LPS-adapted/tolerant cells. In this report, we demonstrate that LPS-tolerized human promonocytic THP-1 cells develop cross-tolerance and no longer respond to LTA-induced IL-1beta/TNF-alpha production, indicating that disruption of common intracellular signaling is responsible for the decreased IL-1beta/TNF-alpha production. We observe that down-regulation of IL-1R-associated kinase (IRAK) protein level and kinase activity closely correlates with the development of cross-tolerance. IRAK protein levels and kinase activities in LPS-tolerized cells remain low and hyporesponsive to subsequent LPS or LTA challenges. We also demonstrate that THP-1 cells with prolonged LTA treatment develop LTA tolerance and do not express IL-1beta/TNF-alpha upon further LTA challenge. Strikingly, cells tolerized with LTA are only refractory to subsequent LTA challenge and can still respond to LPS stimulation. Correspondingly, stimulation of TLR2 by LTA, although activating IRAK, does not cause IRAK degradation. IRAK from LTA-tolerized cells can be subsequently activated and degraded by further LPS challenge, but not LTA treatment. Our studies reveal that LTA-induced tolerance is distinct compared with that of LPS tolerance, and is likely due to disruption of unique TLR2 signaling components upstream of MyD88/IRAK.     

Lipoteichoic acid and toll-like receptor 2 internalization and targeting to the Golgi are lipid raft-dependent

Lipoteichoic acid (LTA), a key cell wall component of Gram-positive bacteria, seems to function as an immune activator with characteristics very similar to lipopolysaccharide from Gram-negative bacteria. It has been shown that LTA binds CD14 and triggers activation via Toll-like receptor 2, but whether the activation occurs at the cell surface or internalization is required to trigger signaling has yet to be demonstrated. In this work we have investigated LTA binding and internalization and found that LTA and its receptor molecules accumulate in lipid rafts and are subsequently targeted rapidly to the Golgi apparatus. This internalization seems to be lipid raft-dependent because raft-disrupting drugs inhibited LTA/Toll-like receptor 2 colocalization in the Golgi. Similarly to lipopolysaccharide, LTA activation occurs at the cell surface, and the observed trafficking is independent of signaling.     

Pathways Involved in the Synergistic Activation of Macrophages by Lipoteichoic Acid and Hemoglobin

Lipoteichoic acid (LTA) is a Gram-positive cell surface molecule that is found in both a cell-bound form and cell-free form in the host during an infection. Hemoglobin (Hb) can synergize with LTA, a TLR2 ligand, to potently activate macrophage innate immune responses in a TLR2- and TLR4-dependent way. At low levels of LTA, the presence of Hb can result in a 200-fold increase in the secretion of IL-6 following macrophage activation. Six hours after activation, the macrophage genes that are most highly up-regulated by LTA plus Hb activation compared to LTA alone are cytokines, chemokines, receptors and interferon-regulated genes. Several of these genes exhibit a unique TLR4-dependent increase in mRNA levels that continued to rise more than eight hours after stimulation. This prolonged increase in mRNA levels could be the result of an extended period of NF-κB nuclear localization and the concurrent absence of the NF-κB inhibitor, IκBα, after stimulation with LTA plus Hb. Dynasore inhibition experiments indicate that an endocytosis-dependent pathway is required for the TLR4-dependent up-regulation of IL-6 secretion following activation with LTA plus Hb. In addition, interferon-β mRNA is present after activation with LTA plus Hb, suggesting that the TRIF/TRAM-dependent pathway may be involved. Hb alone can elicit the TLR4-dependent secretion of TNF-α from macrophages, so it may be the TLR4 ligand. Hb also led to secretion of high mobility group box 1 protein (HMGB1), which synergized with LTA to increase secretion of IL-6. The activation of both the TLR2 and TLR4 pathways by LTA plus Hb leads to an enhanced innate immune response.     

Dissociation between cholesterol secretion and plasma lipid transfer activity in rabbits

Human and rabbit plasma contains a lipid transfer protein that transfers cholesteryl esters and triglycerides among the plasma lipoproteins and may also have a role in the movement of lipids into and out of cells. Little is known about the regulation of the activity of the lipid transfer protein, but in the rabbit, hypercholesterolemia is associated with increased plasma lipid transfer activity (LTA). Perfused rabbit livers secrete LTA, and hepatic cholesterol secretion is increased in rabbits with diet-induced hypercholesterolemia. Thus, experiments were performed with rabbits to determine if LTA is regulated by a concerted hepatic secretion of lipoprotein protein cholesterol and LTA. Rabbits were fed chow or chow plus coconut oil (14% wt/wt), and plasma lipids, LTA, and the rate of secretion of cholesterol into plasma were determined. Coconut oil feeding increased plasma cholesterol by 68%, LTA by 42%, and hepatic cholesterol secretion by 69%. Mevinolin (75 mg/day), an inhibitor of cholesterol biosynthesis, lowered LTA and plasma cholesterol without affecting the rate of secretion of cholesterol into plasma. These studies provide further evidence that, in the rabbit, plasma cholesterol and LTA are closely related, and the association is not likely to be caused by a concerted hepatic secretion of cholesterol and LTA.     

Identification of a new quaternary association for legume lectins

Lotus tetragonolobus lectin (LTA) is a fucose-specific legume lectin. Although several studies report a diverse combination of biological activities for LTA, little is known about the mechanisms involved in l-fucosyl oligosaccharide recognition. The crystal structure of LTA at 2.0A resolution reveals a different legume lectin tetramer. Its structure consists of a homotetramer composed of two back-to-back GS4-like dimers arranged in a new mode, resulting in a novel tetramer. The LTA N-linked carbohydrate at Asn4 and the unusual LTA dimer-dimer interaction are related to its particular mode of tetramerization. In addition, we used small angle X-ray scattering to investigate the quaternary structure of LTA in solution and to compare it to the crystalline structure. Although the crystal structure of LTA has revealed a conserved metal-binding site, its l-fucose-binding site presents some punctual differences. Our investigation of the new tetramer of LTA and its fucose-binding site is essential for further studies related to cross-linking between LTA and complex divalent l-fucosyl carbohydrates.     

Two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in Listeria monocytogenes

Lipoteichoic acid (LTA) is an important cell wall polymer in Gram-positive bacteria and often consists a polyglycerolphosphate backbone chain that is linked to the membrane by a glycolipid. In Listeria monocytogenes this glycolipid is Gal-Glc-DAG or Gal-Ptd-6Glc-DAG. Using a bioinformatics approach, we have identified L. monocytogenes genes predicted to be involved in glycolipid ( lmo2555 and lmo2554 ) and LTA backbone ( lmo0644 and lmo0927 ) synthesis. LTA and glycolipid analysis of wild-type and mutant strains confirmed the function of Lmo2555 and Lmo2554 as glycosyltransferases required for the formation of Glc-DAG and Gal-Glc-DAG. Deletion of a third gene, lmo2553 , located in the same operon resulted in the production of LTA with an altered structure. lmo0927 and lmo0644 encode proteins with high similarity to the staphylococcal LTA synthase LtaS, which is responsible for polyglycerolphosphate backbone synthesis. We show that both proteins are involved in LTA synthesis. Our data support a model whereby Lmo0644 acts as an LTA primase LtaP and transfers the initial glycerolphosphate onto the glycolipid anchor, and Lmo0927 functions as LTA synthase LtaS, which extends the glycerolphosphate backbone chain. Inactivation of LtaS leads to severe growth and cell division defects, underscoring the pivotal role of LTA in this Gram-positive pathogen.     

Parallel changes in plasma cholesterol and lipid transfer activity in pregnant rabbits

The relationship between the concentration of plasma cholesterol and the lipid transfer activity (LTA) of lipoprotein-deficient plasma (d greater than 1.21) was studied in two models of pregnancy in the rabbit. Plasma cholesterol and the protein-mediated transfer of cholesteryl ester and triglyceride were monitored throughout gestation, 48 hr after parturition, and during lactation in New Zealand white (NZW) and heterozygous WHHL rabbits. Lipoprotein cholesterol was determined prior to and 48 hr after parturition. For both NZW and heterozygous WHHL rabbits, the progressive hypocholesterolemia of gestation was associated with parallel changes in LTA. Similarly, the rapid postpartum increase in plasma cholesterol was paralleled by increased LTA for both strains. In relation to basal values, the relative changes in plasma cholesterol and LTA were virtually identical. These data provide further evidence that in the rabbit plasma cholesterol and LTA are closely related.     

Lymphocytes binding and T cell mitogenic properties of group A streptococcal lipoteichoic acid.

We previously reported that lipoteichoic acid (LTA) of group A streptococci binds spontaneously to mammaliam cell membranes via lipid moieties ester-linked to the LTA molecule. We now describe biochemical and immunologic evidence that LTA binds to human and murine lymphocytes as an early event in the induction of mitogenesis in T lymphocytes. The biochemical studies showed that binding of radiolabeled LTA to lymphocytes was lymphocyte-concentration, and temperature dependent, and it reached a maximum in 15 min. Binding was reversible and specific with a dissociation constant of 89 micrometer for adult lymphocytes and 57 micrometer for cord blood lymphocytes. Immunologic studies showed that the LTA was mitogenic only for T lymphocytes. Dose response curves of lymphocyte mitogenesis induced by LTA and the binding of LTA to intact lymphocytes were shown to be related. The results suggest that LTA binds to specific receptor sites on T lymphocytes to trigger the mitogenic response.     

Differential metabolism of exogenous and endogenous arachidonic acid in human neutrophils.

Leukotrienes can be produced by cooperative interactions between cells in which, for example, arachidonate derived from one cell is oxidized to leukotriene A(4) (LTA(4)) by another and this can then be exported for conversion to LTB(4) or cysteinyl leukotrienes (cys-LTs) by yet another. Neutrophils do not contain LTC(4) synthase but are known to cooperate with endothelial cells or platelets (which do have this enzyme) to generate cys-LTs. Stimulation of human neutrophils perfusing isolated rabbit hearts resulted in production of cys-LTs, whereas these were not seen with perfused hearts alone or isolated neutrophils. In addition, the stimulated, neutrophil-perfused hearts generated much greater amounts of total LTA(4) products, suggesting that the hearts were supplying arachidonate to the neutrophils and, in addition, that this externally derived arachidonate was preferentially used for exported LTA(4) that could be metabolized to cys-LTs by the coronary endothelium. Stable isotope-labeled arachidonate and electrospray tandem mass spectrometry were used to differentially follow metabolism of exogenous and endogenous arachidonate. Isolated, adherent neutrophils at low concentrations (to minimize transcellular metabolism between them) were shown to generate higher proportions of nonenzymatic LTA(4) products from exogenous arachidonate (deuterium-labeled) than from endogenous (unlabeled) sources. The endogenous arachidonate, on the other hand, was preferentially used for conversion to LTB(4) by the LTA(4) hydrolase. This result was not because of saturation of the LTA(4) hydrolase, because it occurred at widely differing concentrations of exogenous arachidonate. Finally, in the presence of platelets (which contain LTC(4) synthase), the LTA(4) synthesized from exogenous deuterium-labeled arachidonate was converted to cys-LTs to a greater degree than that from endogenous sources. These experiments suggest that exogenous arachidonate is preferentially converted to LTA(4) for export (not intracellular conversion) and raises the likelihood that there are different intracellular pathways for arachidonate metabolism.     

Interaction of the pneumococcal amidase with lipoteichoic acid and choline

The choline-containing lipoteichoic acid (LTA, Forssman Antigen) of Streptococcus pneumoniae suppresses the activity of the pneumococcal autolysin, an N-acetyl-muramoyl-L-alanine-amidase (amidase) in aqueous solution [H?ltje and Tomasz (1975) Proc. Natl Acad. Sci. USA 72, 1690-1694]. The interaction between LTA and enzyme was used to establish a purification by affinity chromatography on LTA-Sepharose. The amidase could be eluted from the column with choline only. This implies that binding of the enzyme to LTA is mediated via the choline residues of the LTA. Upon binding to the LTA-Sepharose, the amidase converted from the applied E-form (an inactive form of the amidase) to the active C-form, a process which up to now was known to be mediated only by the pneumococcal choline-containing wall teichoic acid. Similar interactions between LTA and amidase seemed to occur in membrane fractions derived from choline-grown cells: the membrane-associated enzyme was present in the C-form and could be detached completely with choline, suggesting that the amidase is bound to the membrane attached LTA rather than being a membrane protein itself. This was supported by the absence of amidase activity in membrane fractions derived from ethanolamine-grown pneumococci, in which choline containing LTA is absent. The LTA-Sepharose-associated amidase was not inhibited, but retained its activity. The enzyme was also not inhibited by lipase-digested LTA. Both are conditions where the LTA is not present in micelles, unlike in aqueous solution. Therefore, mere binding to the LTA is probably not responsible for the inhibitory effect, but inhibition is a manifestation of an inaccessibility of the substrate for the amidase when bound to micellar LTA. When the interactions between choline and amidase were investigated, it was found that high choline concentrations (2%) inhibited the enzyme completely. Even in vivo, 2% choline in the culture medium led to phenotypically amidase-deficient pneumococci. Furthermore, in vitro, low choline concentrations (0.1%) suppressed the wall-mediated conversion. On the other hand, with high choline concentrations (2%) conversion took place in the absence of cell walls. Depending on how the amidase has been converted, the apparent Mr of the resulting C-amidase was different: the cell-wall-converted enzyme was of high Mr, whereas the choline-converted and the LTA-Sepharose-eluted enzyme showed an apparent low molecular mass known for the E-form, when analyzed on sucrose gradients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Not lipoteichoic acid but lipoproteins appear to be the dominant immunobiologically active compounds in Staphylococcus aureus

Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of TLR2. However, we previously demonstrated that LTA fraction prepared from bacterial cells contains lipoproteins, which activate cells via TLR2. In this study, we investigated the immunobiological activity of LTA fraction obtained from S. aureus wild-type strain, lipoprotein diacylglycerol transferase deletion (delta lgt) mutant, which lacks palmitate-labeled lipoproteins, and its complemented strain and evaluated the activity of LTA molecule. LTA fraction was prepared by butanol extraction of the bacteria followed by hydrophobic interaction chromatography. Although all LTA fractions activated cells through TLR2, the LTA from delta lgt mutant was 100-fold less potent than those of wild-type and complemented strains. However, no significant structural difference in LTA was observed in NMR spectra. Further, alanylation of LTA molecule showed no effect in immunobiological activity. These results showed that not LTA molecule but lipoproteins are dominant immunobiologically active TLR2 ligand in S. aureus.     

Monocyte and macrophage activation by lipoteichoic Acid is independent of alanine and is potentiated by hemoglobin

Lipoteichoic acids (LTAs) are Gram-positive bacterial cell wall components that elicit mononuclear cell cytokine secretion. Cytokine-stimulating activity is thought to be dependent on retaining a high level of ester-linked D-alanine residues along the polyglycerol phosphate backbone. However, Streptococcus pyogenes LTA essentially devoid of D-alanine caused human and mouse cells to secrete as much IL-6 as LTA with a much higher D-alanine content. Furthermore, hemoglobin (Hb) markedly potentiates the stimulatory effect of various LTAs on mouse macrophages or human blood cells, regardless of their d-alanine content. LTA and Hb appear to form a molecular complex, based on the ability of each to affect the other's migration on native acrylamide gels, their comigration on these gels, and the ability of LTA to alter the absorption spectra of Hb. Because S. pyogenes is known to release LTA and secrete at least two potent hemolytic toxins, LTA-Hb interactions could occur during streptococcal infections and might result in a profound alteration of the local inflammatory response.