Lipoteichoic acid and M protein: dual adhesins of group A streptococci.

The roles of lipoteichoic acid (LTA) and M protein in the adherence of group A streptococci to human cells were investigated. Both M+ and M- streptococci bound to pharyngeal and buccal epithelial cells in similar numbers. Streptococcal attachment was inhibited by LTA, but not by the pepsin-extracted, amino-terminal half of M protein (pep M), suggesting that M protein does not mediate attachment to these cells. However, a purified, recombinant, intact M protein did block attachment of streptococci to buccal cells. Using synthetic peptides, the inhibitory domain was localized to a region of intact M protein that is within or near the bacterial cell wall. Evidence is presented to suggest that on the surface of streptococci this region of the M protein is probably not accessible for interactions with host cell receptors and that M protein does not mediate attachment to buccal or pharyngeal cells. In contrast, approximately 10-times more M+ streptococci bound to Hep-2 cells than did M- streptococci and pep M protein blocked binding of streptococci to Hep-2 cells. The data suggest that at least two streptococcal adhesins, LTA and M protein, are involved in the adherence of streptococci to certain cells and that the relative contributions of these adhesins to the attachment process depends on the type of host cells used to study adherence.     

Role of cellular lipoteichoic acids in mediating adherence of serotype III strains of group B streptococci to human embryonic, fetal, and adult epithelial cells.

Lipoteichoic acids (LTA) of serotype III strains of group B streptococci (GBS) were shown to mediate adherence of these organisms to human embryonic (HEC), fetal (HFC), and adult buccal (HBEC) epithelial cells. The binding of GBS was temperature dependent, and maximum attachment occurred at 37 degrees C. HEC, HFC, and HBEC preincubated with purified LTA significantly inhibited attachment of GBS, whereas the group B and type III antigens had no effect. Under phosphate-limiting conditions in which cell-associated LTA could not be detected in these organisms, bacterial adherence did not take place. GBS (virulent) that were isolated from infected infants and previously shown to have significantly higher quantities of cell-associated LTA in comparison to GBS strains from asymptomatically colonized infants adhered with greater binding avidity to HEC and HFC and in greater numbers than to HBEC. It was determined that the mechanism of LTA-mediated adherence of GBS to HBEC differed from adherence to embryonic and fetal cells for both virulent and asymptomatic GBS strains bound to HBEC in a similar manner, enhanced by the lipid portion of the LTA. In contrast, the binding of GBS to HEC and HFC was mediated by hydrophobic as well as specific interactions due to the glycerolphosphate polymer of LTA. These results indicate that possible receptor sites for LTA present on cells in prenatal stages of development may differ from those of adult cells, which may result in increased susceptibility of newborn infants to group B streptococcal disease. The implications of LTA-mediated adherence of GBS and their possible role as virulence factors are discussed.     

Specific binding of the human S protein (vitronectin) to streptococci, Staphylococcus aureus, and Escherichia coli.

Specific binding of the 125I-labeled human S protein (vitronectin) which has been shown to be identical with serum-spreading factor, was observed with group A, C, and G streptococci as well as with Staphylococcus aureus and Escherichia coli. The specific binding of S protein to group A, C, and G streptococci was high, whereas the binding to S. aureus and E. coli cultures was moderate. In contrast, group B streptococci and a number of other bacterial species tested did not interact with S protein. The binding of S protein to bacteria was saturable and could be inhibited only by unlabeled S protein but not by albumin. Trypsinization and heat treatment of bacteria destroyed the S-protein binding capacity for group G streptococci, S. aureus, and E. coli but not for group A and C streptococci. Likewise, unlabeled human fibronectin and heparin inhibited the binding of labeled S protein to group G streptococci, S. aureus, and E. coli, but did not influence the binding to group A and C streptococci. Double-reciprocal plots of S-protein binding to group G streptococci indicated that fibronectin inhibited the binding in a competitive manner, while heparin acts in a noncompetitive manner. Moreover, the binding of S protein to G streptococci could be partially by the synthetic peptide Gly-Arg-Gly-Asp-Ser, which contains the cell attachment site of S protein. Trypsin-treated S protein had similar binding activity as untreated S protein for group G streptococci, S. aureus, and E. coli, but showed reduced binding to group A and C streptococci. The present data are indicative of two different types of bacterial binding sites in S protein. The binding to group G streptococci, S. aureus, and E. coli is mediated in part through a domain in the S protein containing the sequence Arg-Gly-Asp, whereas a different site is responsible for the binding to group A and C streptococci.     

Comparative analysis of the localization of lipoteichoic acid in Streptococcus agalactiae and Streptococcus pyogenes.

The cellular locations of deacylated lipoteichoic acid (dLTA) and lipoteichoic acid (LTA) were examined in late-exponential-phase cells of a serotype III strain of Streptococcus agalactiae (group B streptococci [GBS]) isolated from an infant with late-onset meningitis and compared with a fresh clinical isolate of Streptococcus pyogenes (group A streptococci [GAS]). LTA and dLTA were found to be associated with the protoplast membranes of both organisms, with only dLTA found in mutanolysin cell wall digests. Both organisms released dLTA during growth, but only the GAS released substantial levels of LTA into the culture medium. However, penicillin treatment (5 micrograms/ml for 60 min) of GBS resulted in the recovery of LTA in cell wall digests as well as in the culture medium. These results suggest that under normal growth conditions, the hydrophobic region (glycolipid) of LTA remains associated with the cytoplasmic membrane of GBS and unavailable for hydrophobic interactions at the cell surface with epithelial cells. In contrast, release of LTA into the environment by the GAS allows the fatty acid moieties to interact with hydrophobic domains on the surface of epithelial cells. These results may help explain the marked differences in the specificity of binding between these two major streptococcal pathogens for human fetal and adult epithelial cells.     

Highly purified lipoteichoic acid activates neutrophil granulocytes and delays their spontaneous apoptosis via CD14 and TLR2

Lipoteichoic acid (LTA) is a major component of the cell membrane of gram-positive bacteria. Although LTA has become increasingly recognized as an immunomodulator, its effect on polymorphonuclear neutrophil granulocytes (PMN) is still not clear. The interaction between LTA and PMN, however, is of particular importance, as PMN are the first leukocytes that migrate to the site of infection and encounter bacterial pathogens. In the present study, the interaction of highly purified human PMN with endotoxin-free LTA from Staphylococcus aureus was investigated. After exposure to LTA, neutrophil granulocytes acquired typical activated cell morphology. LTA had a marked activating effect on the functions of PMN as well. Shedding of CD62L, degranulation, and priming for formyl-Met-Leu-Phe-mediated oxidative burst were induced in PMN upon exposure to LTA. Moreover, LTA treatment induced the release of proinflammatory cytokines such as interleukin-8, tumor necrosis factor alpha, and granulocyte-colony stimulating factor by PMN. The effects of LTA on PMN were found to be associated with nuclear factor-kappaB activation. Of particular interest was that LTA inhibited the spontaneous apoptosis and therefore, increased the lifespan of PMN. Experiments using blocking antibodies revealed that CD14 and Toll-like receptor 2 (TLR2) but not TLR4 play a major role in LTA-mediated effects on PMN. These data clearly show that LTA, a component of gram-positive bacteria, directly activates neutrophil granulocytes, the primary effector cells in the first line of defense against infectious challenge.     

Kinetic and chemical analyses of the biologic significance of lipoteichoic acids in mediating adherence of serotype III group B streptococci.

The mechanism(s) involved in the binding of lipoteichoic acid (LTA), isolated from virulent, asymptomatic, or avirulent serotype III strains of group B streptococci, to human embryonic epithelial cells (HEC), human fetal epithelial cells (HFC), and human adult buccal epithelial cells was investigated. It was determined that the binding of purified [3H]LTA to human adult buccal epithelial cells differed from the binding to HEC and HFC. LTA from all group B streptococcus strains bound to human adult buccal epithelial cells in a similar manner and was enhanced by the lipid portion of the polymer; in contrast, [3H]LTA binding to HEC and HFC was mediated by hydrophobic as well as specific interactions due to the glycerolphosphate backbone of LTA. Binding avidity of the LTAs to HEC and HFC varied depending on the bacterial strain. Polymers from asymptomatic and avirulent strains were easily dissociated from cell surfaces with unlabeled virulent LTA through competitive interactions; however, 10-fold greater levels of the same material were required to displace virulent [3H]LTA from HEC and HFC surfaces. These observed differences in binding avidity were shown to be due to longer LTA chains (30 to 35 glycerolphosphate units) in virulent strains when compared with LTA chains (10 to 12 glycerolphosphate units) of asymptomatic and avirulent strains. Thus, LTA appears to enhance the ability of virulent group B streptococci to bind to HEC and HFC with stronger avidity by virtue of the increased length of the cell-associated polymers synthesized by these strains. Mild enzymatic treatment of HEC and HFC with trypsin or periodate abolished LTA binding, which suggests the presence of a certain glycoprotein receptor(s) for LTA which does not appear to be present on human adult buccal epithelial cells. These data may therefore partially explain the increased susceptibility of newborn infants to group B streptococcal infections.     

Phagocytosis of Escherichia coli mediated by mannose resistant non-fimbrial haemagglutinin (NFA-1).

We examined the interaction between a urinary isolate of E. coli carrying non-fimbrial MR adhesin (NFA-1) and human polymorphonuclear leukocytes (PMN). We found that the organisms attach to PMN and the attachment was dose dependent and saturable. It was inhibited by isolated NFA-1, but not by mannoside. Recombinant plasmid encoding NFA-1 adhesin conferred binding capacity on a non-adhesive strain. The attachment of the bacteria carrying NFA-1 to PMN was associated with oxidative burst and inhibited by isolated NFA-1. The data taken together suggest that NFA-1 mediates the attachment of the bacteria to PMN. The attachment is followed by ingestion and killing of the organisms, but at a slower rate than with bacteria bound via type 1 fimbriae.     

Insertional inactivation of genes responsible for the D-alanylation of lipoteichoic acid in Streptococcus gordonii DL1 (Challis) affects intrageneric coaggregations

Most human oral viridans streptococci participate in intrageneric coaggregations, the cell-to-cell adherence among genetically distinct streptococci. Two genes relevant to these intrageneric coaggregations were identified by transposon Tn916 mutagenesis of Streptococcus gordonii DL1 (Challis). A 626-bp sequence flanking the left end of the transposon was homologous to dltA and dltB of Lactobacillus rhamnosus ATCC 7469 (formerly called Lactobacillus casei). A 60-kb probe based on this flanking sequence was used to identify the homologous DNA in a fosmid library of S. gordonii DL1. This DNA encoded D-alanine-D-alanyl carrier protein ligase that was expressed in Escherichia coli from the fosmid clone. The cloned streptococcal dltA was disrupted by inserting an ermAM cassette, and then it was linearized and transformed into S. gordonii DL1 for allelic replacement. Erythromycin-resistant transformants containing a single insertion in dltA exhibited a loss of D-alanyl esters in lipoteichoic acid (LTA) and a loss of intrageneric coaggregation. This phenotype was correlated with the loss of a 100-kDa surface protein reported previously to be involved in mediating intrageneric coaggregation (C. J. Whittaker, D. L. Clemans, and P. E. Kolenbrander, Infect. Immun. 64:4137-4142, 1996). The mutants retained the parental ability to participate in intergeneric coaggregation with human oral actinomyces, indicating the specificity of the mutation in altering intrageneric coaggregations. The mutants were altered morphologically and exhibited aberrant cell septa in a variety of pleomorphs. The natural DNA transformation frequency was reduced 10-fold in these mutants. Southern analysis of chromosomal DNAs from various streptococcal species with the dltA probe revealed the presence of this gene in most viridans streptococci. Thus, it is hypothesized that D-alanyl LTA may provide binding sites for the putative 100-kDa adhesin and scaffolding for the proper presentation of this adhesin to mediate intrageneric coaggregation.     

On the Interaction between β2-Microglobulin and Group A Streptococci

beta 2-microglobulin (beta 2m) was found to interact with many group A streptococcal strains. The interaction appeared to require multipoint attachment, since monomeric beta 2m in solution showed no binding, whereas both beta 2m monomers bound to liposomes, and beta 2m in aggregates showed affinity for the bacteria. Aggregated HLA antigens (-A, -B and -C) and aggregated beta 2m exhibited the same binding patterns when tested in binding experiments with various group A streptococcal strains. Furthermore, beta 2m aggregates in excess completely blocked the binding of aggregated HLA antigens, thereby demonstrating that beta 2m is able to interact with streptococcal surface structures also when it is part of the HLA antigen complex. M protein-positive group A streptococcal strains bound significantly more beta 2m than M protein-negative variants of these strains. Purified M 12 protein partly inhibited the binding of radiolabelled beta 2m aggregates to whole streptococci, and in gel filtration and affinity chromatography experiments, the M 12 protein interacted with beta 2m. These various data suggest that the interaction between beta 2m and group A streptococci could be mediated by M protein. Lipoteichoic acid (LTA) is a constituent of the streptococcal cell wall that has been reported to form complexes with M protein at the bacterial cell surface. However, LTA did not influence the interaction between beta 2m and streptococci, suggesting that the binding of beta 2m to streptococcal M protein represents a pure protein-protein interaction. In vivo such an interaction could be established between infecting streptococci and host cells. Among 45 strains of different M types large differences in beta 2m binding were recorded, whereas among 60 strains of the classical nephritogenic M types 12 and 49, all were highly beta 2m-reactive, which points towards a role for beta 2m in streptococcal pathogenicity.     

Binding of lipoteichoic acid of group A streptococci to isolated human erythrocyte membranes.

The spontaneous binding of group A streptococcal lipoteichoic acid (LTA) to mammalian cell membranes was studied in isolated membranes of human erythrocytes. The binding of radiolabeled LTA to erythrocyte membranes was dependent on membrane concentration and time. Binding approached a maximum within 30 min of incubation. The bound LTA could be displaced by adding a 50-fold excess of unlabeled LTA. The displaced LTA was eluted from a column of Sepharose 6B in a position identical to that of authentic LTA, suggesting that binding did not alter the size of the molecule. A dissociation constant of 42 micrometers was calculated, and only one population of approximately 5.5 X 10(6) binding sites per erhtyrocyte membrane was detected. Since these results suggested that erythrocyte membranes possess specific binding sites for LTA, an attempt was made to localize the putative receptors to the outside or the inside surface of the erhtyrocyte membrane. Assays of the binding of LTA to resealed right-side-out and inside-out membrane ghosts demonstrated that the outside surface was able to bind over 10 times more LTA than the inside surface. These results support the concept that the membranes possess specific binding sites for LTA and inciate that these binding sites are located almost entirely on the outside surface of erythrocyte membranes.     

Inhibition of pulmonary neutrophil trafficking during endotoxemia is dependent on the stimulus for migration

In rat models of Gram-negative pneumonia, pulmonary emigration of neutrophils (polymorphonuclear leukocytes [PMNs]) is blocked when rats are made endotoxemic by an intravenous administration of endotoxin (lipopolysaccharide [LPS]). To test whether dysfunctional PMN migratory responses in the endotoxemic rat are specific for airway endotoxin, we gave rats intrapulmonary stimuli known to elicit different adhesion pathways for pulmonary PMN migration. Sprague-Dawley rats were treated intravenously with either saline or LPS and then instilled intratracheally with either sterile saline, LPS from Escherichia coli, interleukin (IL)-1, hydrochloric acid (HCl), zymosan-activated serum (ZAS), or lipoteichoic acid (LTA). Three hours later, accumulation of PMNs and protein in bronchoalveolar lavage fluid (BALF) were assessed. BALF PMN accumulation in response to intratracheal treatment with LPS (100%), IL-1 (100%), ZAS (40%), and LTA (58%) was inhibited by endotoxemia. In rats given intratracheal HCl, BALF PMN numbers were unaffected by intravenous LPS. The pattern of inhibition of migration suggests that intravenous LPS only inhibits migration in response to stimuli for which migration is CD18-dependent. In contrast to PMN migration, BALF protein accumulation was inhibited by intravenous LPS only when IL-1 or LPS was used as the intratracheal stimulus. To characterize further the differential responses to the various airway stimuli, the appearance in BALF of tumor necrosis factor-alpha (TNF-alpha) and the PMN chemokine macrophage inflammatory protein (MIP)-2 was measured. Accumulation of PMNs in BALF correlated with the BALF concentrations of MIP-2 (r = 0.846, P < 0.05) and TNF (r = 0.911; P < 0.05). The ability of intravenous LPS to inhibit pulmonary PMN migration correlated weakly with MIP-2 (r = 0.659; P < 0.05) and with TNF (r = 0.413; P > 0.05) concentrations in BALF. However, this correlation was strengthened for TNF (r = 0.752; P < 0.05) when data from IL-1-treated animals were excluded. Thus, the presence in BALF of inflammatory mediators that are known to promote CD18-mediated migration correlates with endotoxemia-related inhibition of PMN migration. Furthermore, the pattern of inhibition of pulmonary PMN migration during endotoxemia is consistent with the CD18 requirement of each migratory stimulus.     

Deposition of circulating streptococcal lipoteichoic acid in mouse tissues.

The tissue binding properties of streptococcal lipoteichoic acid (LTA) were studied using normal and passively immunized BALB/c mice. After intraperitoneal injection in non-immunized mice, 3H-LTA concentrations in blood, heart, kidney and liver were highest between 24 and 30 h post-injection. LTA deposits in heart remained high for the next 24 h, whereas other tissue levels decreased. Constant amounts of 3H-LTA were detected in urine throughout the 48 h period. In passively immunized mice, the amount of tissue deposition of 3H-LTA was inversely proportional to the ratio of antibodies to LTA. Autoradiography revealed focal deposits of 3H-LTA in heart, kidney and liver. These observations indicate that LTA, released by streptococci growing at remote body sites, can be carried by the blood to internal organs where it can accumulate and participate in pathogenesis.     

Leukotriene A4 modulates generation of leukotriene B4 and sulphidopeptide leukotrienes by human neutrophils.

We investigated the influence of exogenous leukotriene A4 (LTA4) on the reactivity of polymorphonuclear leucocytes (PMN). PMN were either prestimulated with LTA4 or incubated simultaneously with LTA4 and the Ca ionophore A23187 or sodium fluoride (NaF). The Ca ionophore A23187 and NaF induced generation of LTB4 from PMN was significantly diminished in the presence of LTA4 while the formation of LTC4 was enhanced. In contrast, preincubation of cells with LTA4 followed by subsequent stimulation with NaF synergistically increased the LTB4 generation from PMN. LTA4, either alone or in combination with the calcium ionophore A23187 or NaF, decreases GTPase activity in human PMN. This decrease was abolished when LTA4 pretreated cells were subsequently stimulated with NaF, but not with calcium ionophore A23187, suggesting a regulatory role of LTA4 on G-proteins. The results demonstrate dual functions of LTA4: it serves as a substrate for the generation of leukotrienes and also regulates the susceptibility of human PMN for subsequent response.     

Conversion of leukotriene A4 by neutrophils and platelets from patients with atopic dermatitis.

The generation of arachidonic acid-derived inflammatory mediators from unstimulated and stimulated neutrophils (PMN) and platelets in the presence of exogenous LTA4 has been studied in patients with atopic dermatitis (AD) as well as in healthy volunteers. PMN were stimulated with the interleukins IL-3, IL-8, C5a, and the Ca-ionophore A23187. In addition, NaF and thrombin were used to stimulate platelets. The release of leukotriene (LT)B4, 20-COOH- and 20-OH-LTB4, cysteinyl-leukotrienes and 12-HETE was measured. The proinflammatory mediator release from PMN and platelets of patients with AD was significantly higher as compared to the control group. The spontaneous conversion of LTA4 by PMN and platelets was markedly enhanced in patients with AD. Different results with receptor-specific and non-specific stimuli (Ca-ionophore A23187) in the presence of exogenous LTA4 were obtained. The results indicate a higher state of activation for enzymes involved in leukotriene formation. Furthermore, the production of 12-HETE by platelets from patients with AD was enhanced in unstimulated and stimulated cells. Our data emphasize that neutrophils and platelets may play an important role in the pathogenesis of AD by an increased responsiveness to receptor-specific stimuli and cell-cell interaction via LTA4.     

Lipopolysaccharide binding proteins on polymorphonuclear leukocytes: comparison of adult and neonatal cells.

We have previously shown that polymorphonuclear leukocytes (PMN) from cord blood of normal full-term infants have a decreased priming response to lipopolysaccharide (LPS) compared with PMN of adults. Because the reason for this difference is poorly understood, we compared LPS binding on PMN from adults and newborns by using a photoactivatable iodinated LPS (from Escherichia coli O111:B4), coupled to 2-(p-azidosalicylamido)-1,3'-dithopropionate (LPS-ASD) to covalently link LPS to the PMN membrane. We incubated 2 x 10(4) adult or neonatal PMN with 125I-ASD-LPS (100 ng/ml) together with unlabelled LPS (0 to 100,000 ng/ml) for 20 min at 4 degrees C. The maximum total 125I-ASD-LPS binding to newborn PMN (1,004 +/- 103 cpm) was lower than that binding to adult PMN (3,583 +/- 444 cpm; P < 0.01 with respect to newborn PMN). However, the concentration of unlabelled LPS that displaced 50% of the maximum specifically bound 125I-ASD-LPS was similar for PMN from adult and newborn infants (-4.85 +/- 0.04 and -5.13 +/- 0.14 log g of LPS per ml, respectively; P > 0.05). We further assessed the membrane binding of 125I-ASD-LPS to PMN by using membrane extracts analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. LPS binding proteins were found at approximately 73, 55 to 57, and 25 kDa in both adult and neonatal PMN. However, PMN from newborn infants had markedly lower membrane-associated 125I-ASD-LPS at the 55- to 57- and 25-kDa protein bands as indicated by the intensity of the autoradiograph. Binding of LPS at these bands was specific for the lipid A portion of LPS, since purified unlabelled lipid A displaced 125I-ASD-LPS in a dose-dependent manner. Thus, PMN from newborn infants bind less LPS than do PMN from adults, even though the sites for LPS membrane binding appear to be the same.     

Interaction of lipoteichoic acid of group A streptococci with human platelets.

The interaction of group A streptococcal lipoteichoic acid (LTA) with mammalian cell membranes was studied in human platelets. The binding of LTA to platelets was platelet concentration and time dependent. Binding approached a maximum within 10 min of incubation. The bound LTA could be displaced by adding a 50-fold excess of unlabeled LTA. An association constant of 1.9 X 10(-7) M was calculated, and only one population of binding sites was detected. Immuno-ferritin labeling of LTA-treated platelets demonstrated a patchy distribution of LTA binding sites on the platelet surface. LTA inhibited collagen- and alpha1 chain-induced platelet aggregation, but not the platelet release reaction, suggesting that the LTA and collagen binding sites on human platelets are distinct. Apparently, LTA binds to platelets and interferes with collagen-induced aggregation although collagen is still able to attach to binding sites to trigger the release reaction.     

Binding of monoclonal IgM rheumatoid factor to streptococci via the antibody combining site

Radiolabelled monoclonal IgM rheumatoid factors, four from patients with type II essential cryoglobulinaemia and one originating from a patient with rheumatoid arthritis, were tested for binding to group A, B, C and G streptococci and Escherichia coli. Two of the preparations exhibited different binding patterns for the streptococci, whereas the remaining three were not reactive. The binding of one of the factors to group A streptococci type 15 was inhibited by F(ab')2-fragments of anti-idiotypic antibodies but not anti-IgM Fc or F(ab')2 of normal rabbit IgG, indicating that the reaction was mediated via the antibody combining sites.     

Amplification of extracellular nucleotide-induced leukocyte(s) degranulation by contingent autocrine and paracrine mode of leukotriene-mediated chemokine receptor activation

Extracellular nucleotide-induced stimulation and activation of peripheral blood leukocytes and subsequent degranulation play a critical role in immediate-type hypersensitivity reaction and other inflammatory diseases. The extracellular nucleotides stimulate a P2Y receptor(s) on human PMN with the pharmacological profile similar to that of the P2Y2 receptor. Upon activation of P2Y2, arachidonic acid, formed from the membrane bound lipids by phospholipase A2, which subsequently metabolized by 5-lipoxygenase to form the leukotrienes. Of the several leukotrienes generated, LTB(4) is a potent pro-inflammatory chemokine. Upon its release LTB(4) binds to the PMN in a paracrine manner and also other leukocytes such as monocytes at the site of vascular injury, leading to an accelerated rate of degranulation. It is known that LTA(4) formed in the 5-lipoxygenase pathway in PMN could be released from PMN by receptor-mediated transport. Upon its release, the monocytes, erythrocytes, platelet, endothelial or smooth muscle cells can take up LTA(4). The endogenous LTA(4) hydrolase form the LTB(4) from LTA(4) in erythrocytes, platelet, endothelial or smooth muscle cells. As in PMN, LTB(4) is released from these cells via receptor-mediated transport to the extracellular milieu. Thus, released LTB(4) most likely acts as potentially accelerating factor in PMN and MN degranulation through its receptor-specific binding. It is not known whether any LTB(4) receptor exists in cytoplasm in any given cell type and also, the existence of any other signaling cascade for the extracellular nucleotide-induced leukocyte degranulation. Thus, it is convincing that the extracellular nucleotides released from the activated platelets and other damaged cell types exacerbate the inflammatory response by leukotriene generation. In turn the leukotriene will act in both autocrine and paracrine manner to amplify the degranulation processes in leukocytes invoked by extracellular nucleotides.     

Lipoteichoic acid is the major cell wall component responsible for surface hydrophobicity of group A streptococci.

The contribution of lipoteichoic acid (LTA) to the hydrophobic surface properties of group A streptococci was investigated in aqueous dextran-polyethylene glycol two-phase systems. Enzymatic digestions were performed to characterize the hydrophobic surface structure. The results obtained indicated that LTA is a major factor responsible for the hydrophobic character of the cell surface of group A streptococci. This was further supported by the similarity of partition in polymer two-phase systems between whole group A streptococci and tritiated LTA extracted from a group A streptococcal strain. Surface LTA was also determined on intact organisms by a new method measuring the adsorption of antibodies to LTA to the bacterial surface. A correlation was found between the content of surface LTA and the hydrophobicity of the group A streptococci. We conclude that surface-associated LTA is the major factor determining surface hydrophobicity of group A streptococci.     

A role for the laminin receptor in leukocyte chemotaxis

Previous studies on the mechanism of leukocyte traversal of basement membranes showed that rabbit peritoneal exudate polymorphonuclear cells (PMN) preferentially used laminin, a major constituent of basement membrane, to attach to another component, type IV collagen. PMN also responded chemotactically to nanomolar levels of laminin. We have now determined that PMN possess a receptor for laminin. Scatchard analysis using 125I laminin indicates a single class of saturable high affinity binding sites (kd = 6.15 nM/L) on PMN and 3.6 X 10(4) sites per cell. A chymotryptically derived fragment of laminin, C1, which lacks both the long arm and the globular end regions of the short arm (i.e., matrix binding sites) but retains the laminin receptor binding region, gave similar results. Immunoperoxidase studies using monoclonal antibodies to the laminin receptor (mAbLR) indicated the presence of the receptor on the surface of PMN. These cells responded chemotactically to nanomolar levels of C1 and laminin, a result consonant with binding data. PMN chemotaxis to a formyl peptide was markedly inhibited by mAbLR, suggesting that the laminin receptor may be required for PMN chemotaxis in general. Our results suggest that PMN extravasation across basement membranes is aided both by reversible attachment of the cells to laminin in the matrix and by chemotaxis to a gradient of soluble intact and possibly degraded laminin. These characteristics have much in common with those of highly metastatic tumor cells.