Interchangeable endotoxin-binding domains in proteins with opposite lipopolysaccharide-dependent activities

Host defense against microorganisms involves proteins that bind specifically to bacterial endotoxins (LPS), causing different cellular effects. Although LPS-binding protein (LBP) can enhance LPS activities, while bactericidal/permeability-increasing protein (BPI) and Limulus anti-LPS factor (LALF) neutralize LPS, it has been proposed that their LPS-binding domains possess a similar structure. Here, we provide evidence that the LBP/LPS-binding domain is, as in the LALF structure, solvent exposed and therefore available for LPS binding. Our investigations into the activity of LPS-binding domains of different LPS-binding proteins, in the context of LBP, provide the first functional analysis of these domains in a whole protein. We constructed domain exchange hybrid proteins by substituting 12 amino acids of the LBP/LPS-binding domain with those of BPI and LALF and expressed them in Chinese hamster ovary cells. Although discrete point mutations within the LPS-binding domain of LBP disrupted its specific functions, the hybrid proteins were still able to bind LPS and, in addition, retained the wild-type LBP activity of enhancing LPS priming for FMLP-induced oxygen radical production by neutrophils and transferring LPS aggregates to CD14. Although BPI and LALF display opposite activities to LBP, and LALF does not share any sequence homology with LBP, our data provide strong evidence that LBP, BPI, and LALF possess a solvent-exposed, interchangeable LPS binding motif that is functionally independent of LPS transport or neutralization.     

A synthetic lipopolysaccharide-binding peptide based on amino acids 27-39 of serum amyloid P component inhibits lipopolysaccharide-induced responses in human blood

LPS-binding proteins in plasma play an important role in modifying LPS toxicity. Significant properties have already been attributed to the LPS-binding protein (LBP). It accelerates LPS toxicity as well as incorporation into high-density lipoproteins, leading to neutralization of LPS in serum. A search for other LPS-binding components in serum, using LPS-coated magnetic beads, revealed a new LPS-binding protein. N-terminal microsequencing identified this protein as serum amyloid P component (SAP). Purified SAP bound to smooth and rough types of LPS via the lipid A part. SAP inhibited the binding of FITC-labeled ReLPS (LPS from Salmonella minnesota strain R595) to human monocytes and the ReLPS-induced priming of the oxidative burst of human neutrophils only in the presence of low concentrations of LBP. In search for the LPS binding site of SAP, we found that pep27-39, a 13-mer peptide consisting of amino acids 27-39 of SAP, competitively inhibited the binding of LPS to SAP. In addition, pep27-39 significantly inhibited ReLPS-induced responses in phagocytes in the presence of serum, as well as in human whole blood. Carboxamidomethylated pep27-39 showed an even more pronounced reduction of the ReLPS-induced priming of phagocytes in human blood. Performing gel filtration of FITC-labeled ReLPS incubated with soluble CD14, we showed that SAP could not prevent binding of LPS to soluble CD14, in contrast to pep27-39. The ability of pep27-39 to antagonize specifically the effects of LPS in the complex environment of human blood suggests that pep27-39 may be a novel therapeutic agent in the treatment of gram-negative sepsis.     

Serum concentrations of lipopolysaccharide activity-modulating proteins during tuberculosis

Lipopolysaccharide (LPS) is the principal stimulator of host defense against gram-negative bacteria. LPS-binding protein (LBP), bactericidal/permeability-increasing protein (BPI), and soluble CD14 (sCD14) bind LPS and regulate its toxicity. Lipoarabinomannan, a cell wall component of Mycobacterium tuberculosis, resembles LPS with respect to induction of inflammatory responses through recognition by LBP and sCD14. LBP, BPI, and sCD14 were measured in serum of 124 patients with tuberculosis in various stages of disease, in persons who had been in close contact with patients with contagious pulmonary tuberculosis, and in healthy controls. Levels of these LPS toxicity-regulating proteins were elevated in patients with active tuberculosis compared with those in contacts and controls and declined during treatment. The levels of LBP and sCD14 were higher in patients with fever and anorexia. LPS-regulating proteins may play a role in host defense during tuberculosis, presumably through interaction with lipoarabinomannan.     

Relationship between soluble CD14, lipopolysaccharide binding protein, and the alveolar inflammatory response in patients with acute respiratory distress syndrome

The effects of bacterial endotoxin (lipopolysaccharide, LPS) are amplified by lipopolysaccharide binding protein (LBP) and CD14, resulting in cellular activation at very low concentrations of LPS. To investigate the importance of this pathway in acute lung injury, we measured LPS, LBP, and soluble CD14 (sCD14) in the bronchoalveolar lavage fluid (BAL) of 82 patients with acute respiratory distress syndrome (ARDS). LBP and sCD14 increased markedly in BAL of patients with ARDS. sCD14 and LBP each were strongly related to BAL total protein and polymorphonuclear neutrophil (PMN) concentration, whereas LPS concentration was not. Multivariate analyses showed sCD14 to be strongly related to BAL total protein, even after controlling for LPS and LBP concentrations. sCD14 was strongly and independently related to PMN concentration, after controlling for BAL LPS, LBP, and interleukin-8 (IL-8). The BAL LPS concentration was not strongly related to either BAL total protein or BAL PMN. The BAL sCD14 and LBP values were similar in all subgroups of patients with ARDS, and were not related to survival. The serum LBP and sCD14 were elevated in ARDS, but were not related to BAL total protein, LBP, sCD14, PMN, or clinical outcome. Thus, LBP and sCD14 reach high concentrations in the lungs of patients with ARDS, and BAL sCD14 is strongly related to two major indices of lung inflammation: total protein and PMN concentration. CD14-dependent mechanisms may contribute to lung inflammation in ARDS.     

Lipoproteins inhibit macrophage activation by lipoteichoic acid

Regulation of lipid metabolism during infection is thought to be part of host defense, as lipoproteins neutralize endotoxin (LPS) and viruses. Gram-positive infections also induce disturbances in lipid metabolism. Therefore, we investigated whether lipoproteins could inhibit the toxic effects of lipoteichoic acid (LTA), a fragment of gram-positive bacteria. LTA activated RAW264.7 macrophage cells, stimulating production of tumor necrosis factor (TNF) in a dose-dependent matter, but produced less TNF than that seen after LPS activation. High density (HDL) or low density lipoprotein (LDL) alone inhibited the ability of LPS to stimulate TNF production, but had little effect on the activation by LTA. When a maximally effective dose of LTA was mixed with lipoproteins and 10% lipoprotein-depleted plasma (LPDP), the ability of LTA to stimulate macrophage production of TNF was inhibited. HDL, LDL, and the synthetic particle, Soyacal, when mixed with LPDP, were able to inhibit the ability of LTA to activate macrophages. Lipopolysaccharide-binding protein (LBP) substituted for LPDP in catalyzing lipoprotein neutralization of LTA by HDL. Antibody to LBP inhibited the ability of LPDP to induce LTA neutralization by HDL.Thus, lipoproteins can prevent macrophage activation by fragments from both gram-positive and gram-negative microorganisms.-Grunfeld, C., M. Marshall, J. K. Shigenaga, A. H. Moser, P. Tobias, and K. R. Feingold. Lipoproteins inhibit macrophage activation by lipoteichoic acid.     

Lipopolysaccharide stimulates both nuclear localization of the nuclear factor kappa B 50-kDa subunit and loss of the 105-kDa precursor in RAW264 macrophage-like cells

Nuclear factor kappa B (NF-kappa B) is an important regulator of gene expression in cells of the immune system. One such gene, tumor necrosis factor, is induced by bacterial lipopolysaccharide (LPS) in macrophages, and this induction has been shown to be mediated in part by NF-kappa B activation in murine macrophages. In this study, immunochemical analysis was used to follow LPS activation of the NF-kappa B 50-kDa subunit in the RAW264 macrophage-like cell line. The recombinant NF-kappa B 50-kDa subunit was used as an immunogen to produce a rabbit antiserum, which was then affinity-purified using a portion of the NF-kappa B 50-kDa subunit that does not have homology to other members of the c-rel gene family. Untreated macrophages had little NF-kappa B in the nucleus as detected by Western immunoblotting. The protein was predominantly localized in the cytoplasmic fraction. Interestingly, NF-kappa B was found as the 50-kDa mature protein and 105-kDa precursor. After LPS treatment, there was a rapid nuclear translocation of NF-kappa B as detected by immunoblot analysis. There was also a rapid decrease in the amount of the cytoplasmic 105-kDa protein. This may indicate that the 105-kDa protein is a reservoir for the 50-kDa protein and that one of the actions of LPS is to increase the rate of 105-kDa precursor processing.     

Soluble CD14(1-152) confers responsiveness to both lipoarabinomannan and lipopolysaccharide in a novel HL-60 cell bioassay

CD14 is a pattern recognition receptor involved in the interaction with multiple ligands, including LPS from gram-negative bacteria and lipoarabinomannan (LAM) from mycobacteria. While the interactions between LPS and soluble CD14 (sCD14) have been analyzed in detail, LAM/CD14 interactions remain uncharacterized due to the lack of suitable functional assays. We describe herein a novel bioassay for the analysis of CD14/ligand interactions. CD14-negative myeloid HL-60 cells up-regulate endogenous CD14 gene expression when stimulated with LPS in the presence of recombinant soluble CD14(1-348). Using the HL-60 bioassay, we showed that sCD14(1-348) confers responsiveness not only to LPS, but also to LAM. The response to LAM, but not that to LPS, was highly dependent on LPS binding protein (LBP). The N-terminal half of CD14 was sufficient to mediate HL-60 responses to LAM, since HL-60 cells responded with similar efficiency when stimulated with LAM and LBP in the presence of sCD14(1-348) or sCD14(1-152). Thus, the N-terminal 152 amino acids of CD14 contain the site(s) involved in the interaction with LAM and LBP, as well as the residues required for LAM-dependent CD14 signaling.     

Generation by limited proteolysis of a catalytically active 39-kDa protein from the 115-kDa form of phosphatidylinositol-glycan-specific phospholipase D from bovine serum

It has been suggested previously that small amounts of the mature 115-kDa form of phosphatidylinositol (PtdIns)-glycan-specific phospholipase D from bovine serum may exist as a 47-kDa form which can also be generated in vitro by treatment with proteases. In this study, we investigated the possible proteolytic processing by trypsin of partially purified PtdIns-glycan- specific phospholipase D from bovine serum and found that tryptic digestion caused an apparent activation of the enzyme when assayed in the presence of 0.1% (mass/vol.) Triton X-100. Trypsin cleaved the 115-kDa form of PtdIns-glycan-specific phospholipase D into three major polypeptides with molecular masses of 33, 39, and 47 kDa. Under non-denaturing conditions, the polypeptides remained tightly but noncovalently associated with each other. However, in the presence of 6 M urea, the polypeptides could be separated by anion-exchange chromatography. After renaturation, PtdIns-glycan-specific phospholipase D activity was found to be associated with a 39-kDa fragment. Based on its size and its amino acid sequence, the active-site-containing fragment consisted of approximately 275 residues of the N-terminal region of PtdIns-glycan-specific phospholipase D. The active 39-kDa fragment hydrolyzed the PtdIns-glycan-anchors of solubilized acetylcholinesterase from bovine erythrocytes and variant surface glycoprotein from blood stream trypanosomes. However, this fragment was inactive on membrane-associated acetylcholinesterase and PtdIns.     

The induction of acute phase proteins by lipopolysaccharide uses a novel pathway that is CD14-independent

LPS (endotoxin) and proinflammatory cytokines (IL-6, IL-1, and TNF-alpha) are potent inducers of acute phase proteins (APP). Since LPS induces high levels of these cytokines after its interaction with CD14, a protein expressed on the surface of monocytes and neutrophils, it has been assumed that CD14 mediates the LPS induction of APP expression. To test this hypothesis, CD14-deficient and control mice were injected with low doses of LPS, and the expression of several APP that are normally up-regulated by LPS was measured. CD14-deficient mice showed no alteration in the induction of APP, including serum amyloid A, LPS-binding protein, fibrinogen, or ceruloplasmin; in contrast, C3H/HeJ mice, which carry a mutation in the Lps gene, do not up-regulate the expression of these proteins. These studies show that the up-regulation of APP by LPS utilizes a non-CD14 receptor and requires a functional Lps gene.     

Endotoxin and its binding protein in organ failure

Endotoxin (lipopolysaccharide: LPS) infection due to bacterial translocation is intimately involved in the organ failure that occurs after highly invasive interventions such as extensive liver resection, and has attracted a great deal of interest. LPS that has translocated into portal blood binds to LPS-binding protein (LBP) and is transported to the monocytes and Kupffer cells in liver sinusoids where it is activated through the soluble CD14 that is present on their cell membranes or in blood plasma. Inflammatory cytokines such as TNF-alpha and IL-6 are produced and released by monocytes and Kupffer cells that have been activated by LPS-LBP complexes, and the endothelial cell of the sinusoids is damaged by these inflammatory cytokines. The original anticoagulant function of the damaged endothelial cells is reduced, and microcirculatory insufficiency is caused by the formation of microthrombi. It is important to understand this network mechanism and to prevent invasion, and attention has recently been focused on LBP and CD14 because of this point. The increase in LPS as a result of extensive liver resection, which is highly invasive, begins 3 hours after surgery, and peaks at 6 hours. LBP, however, is awaiting the entry of LPS, principally in the periportal hepatocytes. Its production peaks at 12 hours, and this is related to the excessive production of inflammatory cytokines have been produced, expression of LBP must be suppressed within 12 hours to inhibit this excessive reaction, and that will be the task of a future study.     

Lipopolysaccharide-binding protein is present in effluents of patients with Gram-negative and Gram-positive CAPD peritonitis

BACKGROUND: Bacterial peritonitis is a frequent complication during treatment of end-stage renal failure by continuous ambulatory peritoneal dialysis. Local host defence mechanisms including the secretion of proinflammatory cytokines by peritoneal macrophages are of particular importance in the pathogenesis of infectious complications. LPS-binding protein (LBP) and soluble CD14 (sCD14) are serum factors known to regulate the endotoxin-induced cellular immune response. However, it is still unknown whether LBP and sCD14 are also present in the peritoneal effluent of CAPD patients. METHODS: Using specific immunoassays, we examined the concentration of LBP, sCD14 and the proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 in the dialysis effluents of 31 patients with CAPD-associated peritonitis. Twenty patients without peritonitis served as controls. Intraperitoneal LPS concentrations were determined using the limulus amebocyte lysate assay. RESULTS: Bacterial lipopolysaccharide could be detected in 42% of the infected dialysis effluents. In comparison to controls (0.2 +/- 0.05 microg/ml), LBP was significantly elevated in both gram-negative/LPS-positive (1.03 +/- 0.3 microg/ml) and gram-positive infections (0.5 +/- 0.14 microg/ml) (P<0.05). No significant differences were detected concerning the intraperitoneal sCD14 levels in the three patient groups. Levels of TNF-alpha, IL-1beta and IL-6 were significantly increased in the effluents of patients with bacterial peritonitis compared to noninfected controls. Moreover the respective cytokine concentrations were significantly higher in the gram-negative/LPS-positive compared to the gram-positive bacterial infections (P<0.01). CONCLUSION: Our data demonstrate that LBP is significantly elevated in the dialysis effluents of patients with CAPD-associated peritonitis caused by both gram-negative and gram-positive bacteria and might be used as a marker of intraperitoneal infection. Moreover, our findings support the concept that LBP enhances the effects of LPS on cytokine production by peritoneal macrophages. The function of LBP in gram-positive infection remains to be further elucidated.     

Lipopolysaccharide-binding protein is required to combat a murine gram-negative bacterial infection

An invading pathogen must be held in check by the innate immune system until a specific immune response can be mounted. In the case of Gram-negative bacteria, the principal stimulator of the innate immune system is lipopolysaccharide (LPS), a component of the bacterial outer membrane. In vitro, LPS is bound by lipopolysaccharide-binding protein (LBP) and transferred to CD14--the LPS receptor on the macrophage surface--or to high-density lipoprotein (HDL) particles. Transfer to CD14 triggers an inflammatory response which is crucial for keeping an infection under control. Here we investigate how LBP functions in vivo by using LBP-deficient mice. Surprisingly, we find that LBP is not required in vivo for the clearance of LPS from the circulation, but is essential for the rapid induction of an inflammatory response by small amounts of LPS or Gram-negative bacteria and for survival of an intraperitoneal Salmonella infection.     

Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling

Vertebrates and invertebrates initiate a series of defence mechanisms following infection by Gram-negative bacteria by sensing the presence of lipopolysaccharide (LPS), a major component of the cell wall of the invading pathogen. In humans, monocytes and macrophages respond to LPS by inducing the expression of cytokines, cell-adhesion proteins, and enzymes involved in the production of small proinflammatory mediators. Under pathophysiological conditions, LPS exposure can lead to an often fatal syndrome known as septic shock. Sensitive responses of myeloid cells to LPS require a plasma protein called LPS-binding protein and the glycosylphosphatidylinositol-anchored membrane protein CD14. However, the mechanism by which the LPS signal is transduced across the plasma membrane remains unknown. Here we show that Toll-like receptor 2 (TLR2) is a signalling receptor that is activated by LPS in a response that depends on LPS-binding protein and is enhanced by CD14. A region in the intracellular domain of TLR2 with homology to a portion of the interleukin (IL)-1 receptor that is implicated in the activation of the IL-1-receptor-associated kinase is required for this response. Our results indicate that TLR2 is a direct mediator of signalling by LPS.     

CD11/CD18 and CD14 share a common lipid A signaling pathway

The activation of phagocytes by the lipid A moiety of LPS has been implicated in the pathogenesis of Gram-negative sepsis. While two LPS receptors, CD14 and CD11/CD18, have been associated with cell signaling, details of the LPS signal transduction cascade remain obscure. CD14, which exists as a GPI-anchored and a soluble protein, lacks cytoplasmic-signaling domains, suggesting that an ancillary molecule is required to activate cells. The CD11/CD18 integrins are transmembrane proteins. Like CD14, they are capable of mediating LPS-induced cellular activation when expressed on the surface of hamster fibroblasts Chinese hamster ovary (CHO)-K1. The observation that a cytoplasmic deletion mutant is still capable of activating transfected CHO-K1 argues that CD11/CD18 also utilizes an associated signal transducer. We sought to identify further similarities between the signaling systems utilized by CD14 and CD11/CD18. LPS-binding protein, which transfers LPS to CD14, enhanced both LPS-induced cellular activation and binding of Gram-negative bacteria in CD11/CD18-transfected CHO-K1, thus implying that LPS-binding protein can also transfer LPS to CD11/CD18. When synthetic lipid A analogues were analyzed for their ability to function as LPS agonists, or antagonists, in the CHO transfectants, we found the effects were identical regardless of which LPS receptor was expressed. This supports the hypothesis that a receptor distinct from CD14 and CD11/CD18 is responsible for discriminating between the lipid A of LPS and the LPS antagonists. We propose that this receptor, which is the target of the LPS antagonists, functions as the true signal transducer in LPS-induced cellular activation for both CD14 and CD11/CD18.     

Relationship between plasma phospholipid transfer protein activity and HDL subclasses among patients with low HDL and cardiovascular disease

NK lysin is a 9-kDa polypeptide that was originally isolated from porcine intestinal tissue based on its antibacterial activity. It is produced by cytolytic lymphocytes and is cytolytic against a number of different types of tumor cells. Here we report the binding of NK lysin to lipopolysaccharide (LPS) and its anti-LPS activity. NK lysin binds to matrix-coated LPS from Escherichia coli, Pseudomonas aeruginosa, and different strains of Salmonella enterica. Lipid A and polymyxin B inhibited the binding, demonstrating a preferential interaction of NK lysin with the lipid part of LPS. Chromium-labeled lymphoma cells were lysed by NK lysin, and LPS dose-dependently inhibited the cytolysis at equimolar amounts. In the same manner, NK lysin inhibited certain LPS-stimulated effects on mouse bone marrow cells as well as LPS binding to mouse granulocytes. These results suggest that NK lysin may be a another natural LPS-binding protein from lymphocytes that may participate in the endogenous defense response associated with elevated concentrations of LPS.     

Isolation, cloning, and expression of a 70-kilodalton plasminogen binding protein of Borrelia burgdorferi

Surface receptors for plasminogen are expressed by many gram-positive and gram-negative bacteria and may play a role in the dissemination of organisms by binding plasminogen, which upon conversion to plasmin can digest extracellular matrix proteins. Two plasminogen binding proteins have been identified for Borrelia burgdorferi, outer surface protein A and a 70-kDa protein (BPBP). We purified BPBP by plasminogen affinity chromatography and obtained its amino acid sequence by Edman degradation of a tryptic digest. The gene coding for BPBP was isolated from a lambda-ZAP II genomic library with probes developed from sequenced portions of the protein. This gene was expressed in Escherichia coli; the recombinant product was seen by antibody raised against native BPBP and also bound 125I-labeled plasminogen. The experimentally derived amino acid sequences corresponded to the predicted sequence encoded by the BPBP gene. The deduced amino acid sequence for BPBP revealed significant similarity to p30, a 30-kDa protein of B. burgdorferi (54% identity and 65% similarity), to a 60-kDa protein in Borrelia coriaceae (66% identity and 80% similarity), to oligopeptide binding protein A of E. coli (34% identity and 57% similarity), and, more generally, to the periplasmic oligopeptide binding family of proteins.     

Transcriptional regulation of fibroblast growth factor-2 expression in human astrocytes: implications for cell plasticity

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1beta, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter-luciferase constructs identified a unique -555/-513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (-624/-556 bp) essential for PKC and cAMP stimulation. DNA-protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.     

Effects of prior exercise on exercise-induced arterial hypoxemia in young women

Insulin-like growth factor binding proteins (IGFBP) proteases have been proposed to be involved in changes of serum IGFBP pattern during pregnancy. IGFBP-4 and -5 are degraded specifically by proteases in pregnancy serum in vitro, whereas IGFBP-3 proteolytic activity was also detected in nonpregnancy serum. To identify and characterize IGFBP proteases, human pregnancy serum was fractionated by size exclusion chromatography revealing IGFBP-4 protease activities in fractions coeluting with proteins of approximately 600-kDa and 50- to 100-kDa molecular mass. In both fractions, a predominant 50-kDa gelatinase was found, suggesting that parts of the gelatinase activity might aggregate or are complexed with other proteins forming a higher molecular complex. Hydroxyapatite chromatography and chromatofocusing of the 50- to 100-kDa serum fraction showed that the IGFBP-4 protease and the 50-kDa gelatinase activity were copurified. When the 50-kDa gelatinase-containing band was excised from the polyacrylamide gel, it exhibited IGFBP-4 proteolytic activity, resulting in the formation of 17- and 10-kDa fragments. [125I] IGFBP substrate zymography combined with fragment blotting showed that the 1,10-phenanthroline-sensitive 50-kDa protease activity purified by chromatofocusing also cleaved IGFBP-3 and -5. Other proteases detected in pregnancy serum fractions with Mr estimates of 79-, 30-, and 22-kDa degraded IGFBP-3 and -5 but not IGFBP-4. [125I] IGFBP-5 substrate zymography revealed that the 30-kDa IGFBP protease was inhibited by serine protease inhibitors. Whereas 1,10-phenanthroline inhibited the IGFBP proteolytic activity in the solution assay, serine protease inhibitors failed to affect proteolysis, indicating the predominant contribution of the metalloproteinase to IGFBP proteolysis. Tissue inhibitors of matrix metalloproteinases-1 and -2 revealed weak or no inhibition of IGFBP-4 and -5 proteolytic activity, whereas a hydroxamic acid-based inhibitor, potentially inhibiting disintegrin metalloproteases, completely prevented the proteolysis of IGFBPs. Whereas no specific immunoreactivity of the 50-kDa protein with antimatrix metalloproteinase-1, -2, -3, -9, or -13 antibodies was observed, antidisintegrin domain-specific antibodies bound to the 50-kDa gelatinase. These studies provide the first direct biochemical evidence that human pregnancy serum contains a 50-kDa IGFBP protease with properties of a soluble disintegrin metalloproteinase that appears to be potentially involved in regulating IGF bioavailability for placental and fetal growth.     

Membrane components of Treponema denticola trigger proteinase release from human polymorphonuclear leukocytes

Tissue destruction during periodontitis is believed to be primarily brought about by leukocyte proteinases. We postulate that oral spirochetes cause discharge of polymorphonuclear leukocyte (PMN) lysosomal enzymes. Effects of Treponema denticola 53-kDa outer membrane protein, lipopolysaccharide (LPS), and peptidoglycan on degranulation of matrix metalloproteinases (MMP)-8 (collagenase) and -9 (gelatinase), cathepsin G, and elastase by human peripheral blood PMNs were studied by specific enzyme assays and Western blot analysis. T. denticola 53-kDa kDa outer membrane protein was found to be a particularly efficient inducer of MMP-8 release. The induction was comparable with that of phorbol myristate acetate, a known inducer of PMN specific granule discharge. All of the treponemal substances, most notably the 53-kDa protein and LPS, induced release of MMP-9, a component of C-type granules. Both collagenase and gelatinase released from PMNs were mostly in active forms. Release of cathepsin G and elastase was also observed with the 53-kDa protein treatment. The other T. denticola substances did not induce release of these serine proteinases. Lactate dehydrogenase was not released from PMNs by the treatments, indicating that the degranulation was specific and not caused by toxic effects of the substances. This was confirmed by transmission electron microscopy of PMNs treated with the 53-kDa protein that showed rapid vacuole formation and cell shape changes but no disintegration of the cells. Thus, T. denticola may participate in the PMN-dependent extracellular matrix degradation during the course of periodontal inflammation by triggering the secretion and activation of matrix metalloproteinases.