Relative contribution of endothelial cell and polymorphonuclear neutrophil activation in their interactions in systemic inflammatory response syndrome

OBJECTIVE: To examine the relative contribution of polymorphonuclear neutrophil (PMN) vs endothelial cell (EC) activation on the adherence and subsequent killing of ECs by PMNs. DESIGN: In vitro comparative studies of PMN-EC adherence and cytotoxicity. SETTING: Research laboratory and the surgical intensive care unit of a tertiary-level university hospital. PATIENTS: Patients with systemic inflammatory response syndrome admitted to the surgical intensive care unit and hospitalized preoperative noninfected surgical patients. INTERVENTION: None. METHODS: Polymorphonuclear neutrophils were isolated from 21 healthy volunteers, 22 preoperative patients, and 30 patients from the surgical intensive care unit with systemic inflammatory response syndrome. The PMNs were activated with lipopolysaccharide, 100 ng/mL (Escherichia coli 0111:b4), for 40 minutes at 37 degrees C before the adherence and cytotoxicity assays. Human umbilical vein endothelial monolayers were stimulated with tumor necrosis factor alpha, 25 ng/mL, and interleukin 1 beta, 15 U/mL, for 3 hours. The PMNs or EC cells were labeled with sodium chromate Cr 51 and used in a standard adherence or killing assay as required. RESULTS: Control and preoperative patient PMN treatment with lipopolysaccharide produced a modest increase in adherence. The PMNs from patients with systemic inflammatory response syndrome showed moderately increased human umbilical vein endothelial cell adherence, and this could not be augmented further with lipopolysaccharide stimulation. There was a marked increase in PMN adherence to EC after EC activation in all study groups (P < .001). Similar to the adherence data, human umbilical vein endothelial cell cytotoxicity was significantly increased in all groups after human umbilical vein endothelial cell activation (P < .01) but not after PMN stimulation with lipopolysaccharide. CONCLUSION: These data suggest that stimulation of ECs is far more important in producing increased adherence and cytotoxicity of EC than PMN stimulation with lipopolysaccharide in all study groups. Therapeutic efforts in patients with systemic inflammatory response syndrome should be focused on the EC.     

Polymorphonuclear leukocytes induce PDGF release from IL-1beta-treated endothelial cells: role of adhesion molecules and serine proteases

Polymorphonuclear leukocytes (PMNs) and endothelial cells interact at sites of vascular injury during inflammatory response and during the development of atherosclerotic lesions. Such close proximity leads to the modulation of several of the biological functions of the 2 cell types. Because we have shown previously that PMNs enhance release of growth factors from resting endothelial cells, we decided to evaluate whether coincubation of PMNs with interleukin-1beta (IL-1beta)-stimulated human umbilical vein endothelial cells (HUVEC) could further modulate mitogen release from HUVEC. We found that PMN-HUVEC coincubation resulted in a 10-fold increase in mitogen release, compared with HUVEC alone (14+/-6 versus 1.3+/-0.1). When PMNs were incubated with IL-1beta-treated HUVEC, a further increase in mitogen release (up to 35-fold) was observed. The mitogenic activity was immunologically related to platelet-derived growth factor (PDGF) because the activity was abolished by an anti-PDGF antibody. PDGF-AB antigen, detected in low concentrations in conditioned medium from HUVEC alone, was increased 4-fold when IL-1beta or PMNs were incubated with HUVEC and dramatically upregulated (up to 40-fold) when PMNs were cocultured with IL-1beta-treated HUVEC. The presence of the protease inhibitor eglin C abolished mitogenic activity generation, suggesting a role for PMN-derived elastase and cathepsin G. Indeed, purified elastase and cathepsin G mimicked PMN-induced mitogen release from HUVEC. Because PMNs firmly adhered to IL-1beta-treated HUVEC, we investigated the role of cell-cell adhesion in mitogen release. Adhesion and PDGF release were inhibited by approximately 60% in the presence of anti-CD11a/CD18 and anti-intercellular adhesion molecule-1 monoclonal antibodies. This study suggests a new role for PMNs and their interaction with endothelium in pathological conditions in which intimal hyperplasia is a common feature.     

Thyroid function in a population with an extra iodine intake]

Polymorphonuclear leukocyte (PMN) superoxide (.O2-) production has been implicated in the pathogenesis of cardiopulmonary bypass (CPB)-related end organ injury. PMN "priming" has been described as an event which enhances the release of .O2- following a second, activating insult. We hypothesized that PMN priming occurs during CBP and is temporally related to the plasma level of complement (C3a), interleukin (IL)-6, and IL-8. PMNs were isolated from 10 CPB patients pre-bypass (preCPB), 5 min after protamine administration (PROT), and at 6 and 24 h post-CPB. PMN .O2- production was measured by a cytochrome c reduction assay in the presence or absence of either phorbol 12-myristate-13-acetate (PMA, 0.4 microgram/ml) or N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 microM) and also after priming with 2000 nM platelet-activating factor (PAF) followed by activation with either PMA or FMLP. Plasma levels of C3a, IL-6, and IL-8 were determined by enzyme-linked immunosorbent assay. PMA-activated PMN .O2- production was significantly elevated at 6 h post-CPB compared to pre-CPB levels (11.04 +/- 0.9 vs 7.62 +/- 0.57, P = 0.009), indicating that CPB is associated with in vivo PMN priming. When PMNs were primed in vitro with PAF and then activated with PMA or FMLP, .O2- release at 6 h post-CPB was also significantly greater than pre-CPB levels (16.04 +/- 0.74 vs 12.2 +/- 0.92, P = 0.038; and 17.33 +/- 1.38 vs 13.33 +/- 1.35, P < 0.05), indicating that CPB acts synergistically with PAF to prime PMNs. Levels of C3a rose significantly over pre-CPB levels at PROT (P = 0.001), and IL-6 and IL-8 rose over pre-CPB levels at 6 h post-CPB (P = 0.01 and P = 0.006, respectively). These findings demonstrate that CPB not only directly primes PMNs, but also potentiates priming of PMNs by PAF. This "primed" PMN state, which coincided with the increased plasma levels of inflammatory mediators, may suggest a mechanism of predisposition to organ dysfunction following CPB.     

Postinjury neutrophil priming and activation: an early vulnerable window

BACKGROUND: Generation of extracellular, cytotoxic superoxide anion (O2-) by polymorphonuclear neutrophils (PMNs) contributes to an unbridled inflammatory response that can precipitate multiple organ failure (MOF). Release of O2- is markedly enhanced when activated PMNs have been previously "primed" by inflammatory mediators, such as those expressed after trauma. We therefore hypothesized that PMN priming occurs as an integral part of the early inflammatory response to trauma. METHODS: PMNs were obtained from 17 high-risk patients with torso trauma at 3, 6, 12, 24, 48, and 72 hours after injury, as well as from 10 healthy donors, and the in vitro release of O2- was quantitated with a kinetic, superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay. PMN O2- release was measured in the presence and absence of 1 mumol/L N-formyl-methionyl-leucyl-phenylalanine (fMLP) and after priming and activation with 20 nmol/L platelet-activating factor (PAF) and 1 mumol/L fMLP, respectively. RESULTS: In vitro PMN O2- release was used to determine whether postinjury PMNs were (1) activated in vivo, (2) primed in vivo, or (3) primable in vitro. Unstimulated PMNs from trauma patients spontaneously expressed modest amounts of O2- in vitro from 6 to 48 hours after injury, suggesting endogenous activation. Also, fMLP-activated PMNs collected between 3 and 24 hours after injury expressed more O2- than controls (p < or = 0.02), indicating in vivo, trauma-related priming. Furthermore, postinjury PMNs were maximally primed in vivo (i.e., in vitro exposure to PAF before fMLP activation failed to significantly enhance O2- release) as compared to PMNs treated with fMLP. CONCLUSIONS: These data indicate that major torso trauma (first hit) primes and activates PMNs within 3 to 6 hours after injury. Consequently, we postulate that postinjury priming of PMNs may create an early vulnerable window during which a second hit (e.g., a secondary operation or delayed hemorrhage) activates exuberant PMN O2- release, rendering the injured patient at high risk for MOF.     

Accelerated healing of gingival incisions by the helium-neon diode laser: a preliminary study

Platelet-activating factor (PAF) concordantly primes neutrophils (PMNs) for superoxide generation and elastase release. beta-Adrenergic stimulation of PMNs enhances cAMP-dependent protein kinase A (PKA) activity and has been shown to inhibit PAF-mediated NADPH-oxidase activity. PMN superoxide generation is thought to play a predominate microbicidal role, whereas elastase is known to mediate untoward PMN-endothelial interactions. We hypothesized that beta-adrenergic neutrophil stimulation has disparate effects on PAF-mediated PMN superoxide generation versus elastase release. Human PMNs were isolated using a standard Ficoll/Hypaque gradient. PMNs were then primed with PAF (200 nM) and activated with fMLP (1 microM). Subsets of PMNs were pretreated for 5 min with a beta agonist (10(-4) M isoprotereno) or an adenylate cyclase agonist (10(-5) M forskolin). Superoxide generation was determined by superoxide dismutase inhibitive cytochrome c reduction. Elastase activity was measured by the cleavage of n-methoxylsuccinyl-A-A-P-V-p-nitroanilide. Pretreatment with isoproterenol and forskolin yielded superoxide generation of 3.2 +/- 0.6 and 3.1 +/- 1.2 nmole/2.5 x 10(5) PMN/min compared to 9.0 +/- 0.6 nmole/2.5 x 10(5) PMN/min for PAF/fMLP alone, whereas isoproterenol and forskolin did not significantly affect PAF-mediated neutrophil elastase release, 22.4 +/- 5.3 and 24.0 +/- 3.6%, respectively, compared to 39.4 +/- 9.1% for PAF/fMLP alone. Disparate PMN signal transduction for superoxide generation versus elastase release may explain the SICU clinical paradox, in which patients are both susceptible to infection and vulnerable to PMN-mediated multiple organ failure.     

Herbal medicines, phytoestrogens and toxicity: risk:benefit considerations

Expression of DAF (CD55) is enhanced on colonic epithelial cells of patients with ulcerative colitis (UC), and stool DAF concentrations are increased in patients with active disease. Cytokines are known to modulate DAF expression in various human cells, and lesions of UC reveal altered profiles of cytokine production. In this study, we evaluate the effects of various cytokines, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, and interferon-gamma (IFN-gamma), on the synthesis and kinetics of DAF protein in HT-29 human intestinal epithelial cells. Using flow cytometry and an ELISA, we found that HT-29 cells constitutively express DAF on the cell surface and spontaneously release DAF into the culture supernatant under standard culture conditions. When the culture supernatant was centrifuged at 100000g, nearly a half of DAF was precipitated, indicating that one half of the released DAF was present as a membrane-bound form and the other half as a soluble form. Analysis of the culture supernatant of biotin surface-labelled HT-29 cells suggested that the soluble form DAF was derived by secretion from within the cell or by cleavage from the cell surface. Among the cytokines, IL-4 markedly, and IL-1beta moderately, enhanced the expression and the release of DAF. Actinomycin D, cycloheximide, and brefeldin A inhibited the increase in DAF release induced by IL-4 and IL-1beta stimulation. These results suggest that DAF is released from intestinal epithelial cells in response to cytokine stimulation and that IL-4 and IL-1beta are possible cytokines involved in DAF release into the colonic lumen of patients with UC.     

Neutrophil chemotaxis in response to TGF-beta isoforms (TGF-beta 1, TGF-beta 2, TGF-beta 3) is mediated by fibronectin

TGF-beta isoforms regulate numerous cellular functions including cell growth and differentiation, the cellular synthesis and secretion of extracellular matrix proteins, such as fibronectin (Fn), and the immune response. We have previously shown that TGF-beta 1 is the most potent chemoattractant described for human peripheral blood neutrophils (PMNs), suggesting that TGF-beta s may play a role in the recruitment of PMNs during the initial phase of the inflammatory response. In our current studies, we demonstrate that the maximal chemotactic response was attained near 40 fM for all mammalian TGF-beta isoforms. However, there was a statistically significant difference in migratory distance of the PMNs: TGF-beta 2 (556 microM) > TGF-beta 3 (463 microM) > TGF-beta 1 (380 microM) (beta 2: beta 3, p < or = 0.010; beta 3: beta 1, p < or = 0.04; beta 2: beta 1, p < or = 0.0012). A mAb to the cell binding domain (CBD) of Fn inhibited the chemotactic response to TGF-beta 1 and TGF-beta 3 by 63% and to TGF-beta 2 by 70%, whereas the response to FMLP, a classic chemoattractant, was only inhibited by 18%. In contrast, a mAb to a C-terminal epitope of Fn did not retard migration (< 1.5%). The Arg-gly-Asp-ser tetrapeptide inhibited chemotaxis by approximately the same extent as the anti-CBD (52 to 83%). Furthermore, a mAb against the VLA-5 integrin (VLA-5; Fn receptor) also inhibited TGF-beta-induced chemotaxis. These results indicate that chemotaxis of PMNs in response to TGF-beta isoforms is mediated by the interaction of the Arg-gly-Asp-ser sequence in the CBD of Fn with an integrin on the PMN cell surface, primarily the VLA-5 integrin. TGF-beta isoforms also elicited the release of cellular Fn from PMNs; we observed a 2.3-fold increase in Fn (389 to 401 ng/ml) in the supernatants of TGF-beta-stimulated PMNs compared with unstimulated cells (173.6 ng/ml). The concentration of TGF-beta required to cause maximal release of Fn from PMNs (4000 fM) is a concentration at which TGF-beta is no longer chemotactic, suggesting that PMNs only use Fn that is constitutively expressed for migration. At higher concentrations of TGF-beta, the Fn released may accumulate basal to the cell, ultimately retarding cellular migration and modulating the chemotactic response.     

Adherent neutrophils activate endothelial myosin light chain kinase: role in transendothelial migration

Increased vascular endothelial cell (EC) permeability and neutrophilic leukocyte (PMN) diapedesis through paracellular gaps are cardinal features of acute inflammation. Activation of the EC contractile apparatus is necessary and sufficient to increase vascular permeability in specific models of EC barrier dysfunction. However, it is unknown whether EC contraction with subsequent paracellular gap formation is required for PMN transendothelial migration in response to chemotactic factors. To test this possibility, we assessed migration of human PMNs across confluent bovine pulmonary arterial EC monolayers. Transendothelial PMN migration in the absence of a chemotactic gradient was minimal, whereas abluminal addition of leukotriene B4 (LTB4; 5 microM) resulted in significantly increased PMN migration. Reductions in EC myosin light chain kinase (MLCK) activity by EC monolayer pretreatment with specific MLCK inhibitors (KT-5926 or ML-7) or by increases in cAMP-protein kinase A activity (cholera toxin) significantly reduced PMN transmigration (30-70% inhibition). In contrast, pretreatment with the myosin-associated phosphatase inhibitor calyculin resulted in the accumulation of phosphorylated myosin light chains, EC contraction, and significantly enhanced PMN migration. Finally, the interaction of PMNs with 32P-labeled EC monolayers was shown to directly increase EC myosin phosphorylation in a time-dependent fashion. Taken together, these results are consistent with the hypothesis that the phosphorylation status of EC myosin regulates PMN migration and further indicate that EC MLCK is activated by chemoattractant-stimulated PMNs. Neutrophil-dependent activation of the EC contractile apparatus with subsequent paracellular gap formation may be a key determinant of transendothelial PMN migration responses to chemotactic agents.     

Release of monocyte chemoattractants by polymorphonuclear leukocytes stimulated by their adhesion to stratum corneum opsonized via complement activation, measured with a human acute monocytic leukemic cell line, THP-1

Stratum corneum (SC) exposed to living tissues, induces inflammation characterized by the formation of mixed cell granulomas consisting of infiltrative polymorphonuclear leukocytes (PMNs) and monocytes/macrophages. In this study, to clarify the mechanism for the later monocyte accumulation in SC-induced granulomas, we evaluated monocyte chemotactic activity induced by PMNs treated with serum-opsonized SC by using a human acute monocytic leukemic cell line, THP-1. When the supernatant was obtained from a PMN suspension cultured with opsonized plantar SC, higher THP-1 chemotactic activity was detected as compared with that cultured with non-opsonized SC. Although some concentrations of the chemokines, MIP-1alpha and MIP-1beta, were detected in supernatants obtained from the PMN suspensions cultured with plantar SC than in the control suspensions of PMN alone, their production by PMN was not influenced by the opsonization procedure. In contrast, MCP-1 was found to be secreted from PMN suspensions constitutively, showing no correlation to this THP-1 chemotactic activity. Moreover, HPLC analysis of PMN suspensions indicated that factors with far higher molecular weight values than these chemokines are involved in the chemotaxis of THP-1 cells.     

Kinetics of leukocyte-induced changes in endothelial barrier function

1. Extravasation of polymorphonuclear leukocytes (PMN) and associated plasma leakage are key events in the inflammatory process. The kinetics of PMN-induced changes in endothelial barrier function were studied by means of confluent monolayers of bovine aorta or human umbilical vein endothelial cells (EC), cultured on permeable membranes and mounted in a two-compartment diffusion chamber. The model permitted continuous measurement of transendothelial electrical resistance (TEER), and analysis of protein efflux and PMN migration across the EC monolayer. 2. Transendothelial chemotactic stimulation (fMLP or LTB4) of PMN resting on EC in the upper compartment induced a prompt decline in TEER, followed by an increase in protein flux and transmigration of PMN. Adding the chemoattractant together with PMN in the upper compartment provoked adhesion of PMN, fall in TEER and increase in protein permeability, but no transmigration of PMN, whereas inhibition of PMN adhesion to EC by pretreatment with anti-CD18 mAb prevented all responses to chemotactic stimulation. 3. Chemoattractant-induced adhesion of PMN to the EC monolayer induced a rapid rise in EC cytosolic free Ca2+, similar to that obtained by direct stimulation of EC with histamine, indicating an active response of EC to PMN activation and adhesion. 4. In summary, continuous recording of transendothelial electrical resistance in the in vitro model described permits rapid and sensitive analysis of leukocyte activation-induced effects on EC barrier function. The kinetics and specificity of the EC and PMN responses to chemoattractant stimulation suggest that activated PMN, via adhesion-dependent events, have a direct effect on EC junctional integrity independent of whether transmigration occurs or not.     

Apical secretion of a pathogen-elicited epithelial chemoattractant activity in response to surface colonization of intestinal epithelia by Salmonella typhimurium

Modeling Salmonella-epithelial cell interaction in vitro has led to the realization that epithelial cells are crucial in orchestrating neutrophil (PMN) responses, in part by stimulating basolateral release of epithelial chemokines, including IL-8. However, such basolaterally released chemokines, while likely important in orchestration of PMN movement across the subepithelial matrix, are unlikely to be responsible for the final step of transepithelial migration of PMN and entry into the apical compartment. We now show that S. typhimurium attachment to T84 cell apical epithelial membranes induces polarized apical secretion of a pathogen-elicited epithelial chemoattractant (PEEC) bioactivity. Experiments employing semipurified PEEC indicate that it is released in a polarized apical fashion and is sufficient to explain the observed final step of transepithelial migration of PMN induced by Salmonella-apical membrane interaction. By preliminary physical characterization and profiles of PMN activation, PEEC appears to be a novel PMN chemotactic bioactivity. This 1- to 3-kDa nominal molecular mass chemokine-like bioactivity directly stimulates PMN via a pertussis toxin-sensitive receptor and elicits a Ca2+ signal. While these latter features are shared by most other chemokines, analysis of PEEC-elicited PMN activation reveals that, unlike these other agonists, PEEC, even at saturating concentrations, elicits chemotactic activity in the absence of stimulation of superoxide production and/or release of primary and/or secondary granules. These data suggest that the apically released PEEC activity appears to represent a novel epithelial-derived chemoattractant that directs PMN movement across epithelial monolayers.     

Down-regulation of CXCR2 expression on human polymorphonuclear leukocytes by TNF-alpha

TNF-alpha is implicated in the initiation of cytokine cascades in various inflammatory settings. To assess the interactions of multiple cytokines at the level of inflammatory effector cells, we examined the effects of TNF-alpha on the expression of two IL-8Rs (CXCR1 and CXCR2) on polymorphonuclear leukocytes (PMNs). TNF-alpha decreased the surface expression of CXCR2 in a dose- and time-dependent manner. In contrast, CXCR1 expression was not affected by TNF-alpha. The release of CXCR2 into the supernatant of TNF-alpha-treated PMNs was detected by immunoblotting and immuno-slot-blot analyses, suggesting that the down-regulation of CXCR2 was caused mainly by shedding from the cell surface. The CXCR2 down-regulation was inhibited by PMSF and aprotinin, supporting the hypothesis that the shedding was mediated by serine protease(s). The intracellular Ca2+ mobilization and chemotaxis in response to IL-8 were suppressed by the pretreatment of PMNs with TNF-alpha, indicating that the decrease in CXCR2 was reflected in the decreased functional responses to IL-8. In contrast, the O2- release, which is mediated by CXCR1, was not suppressed by TNF-alpha. The treatment of whole blood with TNF-alpha also caused a significant reduction in CXCR2 and markedly suppressed intracellular Ca2+ mobilization and chemotaxis in response to IL-8, while enhancing the O2- release. These findings suggest that TNF-alpha down-regulates CXCR2 expression on PMNs and modulates IL-8-induced biologic responses, leading to the intravascular retention of PMNs with an enhanced production of reactive oxygen metabolites.     

In vitro correlates of the L. casei animal model of Kawasaki disease

The induction of coronary arteritis in mice by Lactobacillus casei cell wall (CW) is thought to represent an animal model of Kawasaki disease. Treatment of vascular endothelial cells (EC) in vitro with supernatants from CW stimulated human mononuclear cells (MNC) enhanced adherence of human polymorphonuclear leukocyte (PMN) to human EC, and EC expression of intercellular adhesion molecule-1 (ICAM-1) but not HLA-DR. Supernatants contained high concentrations of tumor necrosis factor-alpha (TNF-alpha) and PMN adherence correlated directly with the concentration of TNF-alpha. Intravenous human gamma globulin (IVGG) preparations did not block the effect of cytokine containing MNC supernates upon EC, ICAM-1 expression by EC, or PMN adherence to prestimulated EC. However, both EC ICAM-1 expression and enhanced PMN adherence to EC by CW induced MNC supernatants were blocked by anti-TNF-alpha treatment. The initial coronary inflammatory reaction in the mouse model appears to involve PMN adherence to vascular EC that have been activated by TNF-alpha released by MNC after stimulation with CW.     

Reduction by cefodizime of the pulmonary inflammatory response induced by heat-killed Streptococcus pneumoniae in mice

It has recently become apparent that overwhelming inflammatory reactions contribute to the high mortality rate associated with pneumococcal infection in immunocompetent hosts. Cefodizime (CEF) is an antibiotic that seems to be endowed with immunomodulating properties. To investigate the influence of CEF on the pulmonary inflammatory response induced by Streptococcus pneumoniae, we infected mice with repeated intranasal inoculations of 10(7) CFU of heat-killed fluorescein isothiocyanate-labeled bacteria, which are insensitive to the killing properties of the drug. CEF downregulated but did not abolish the strong polymorphonuclear leukocyte (PMN) recruitment induced by S. pneumoniae. PMN recruitment was not primarily mediated by leukotriene B4 in this model. The drug did not interfere with intrinsic mechanisms of phagocytosis by PMNs and alveolar macrophages. CEF totally abrogated the pneumococcus-induced tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) secretion in bronchoalveolar lavage fluid. The drug also prevented IL-6 release in lung homogenates and partly inhibited TNF-alpha, but it did not interfere with IL-1alpha secretion in the lungs of infected mice. The fractional and selective downregulation of inflammatory cells and cytokines by CEF suggests cell-specific and intracellular specific mechanisms of interaction of the drug. The immunomodulatory properties of CEF may help restrain excessive inflammatory reactions, thus contributing to the reported good clinical efficacy of the drug against lower respiratory tract infections.     

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.     

Substantiation of MPEL of acoustic exposure in accordance with its informational value]

Our previous studies have proved that a great number of polymorphonuclear neutrophils (PMN) adhered to endothelial lining and induced endothelial cell (EC) injury. This study was attempted to investigate the change in adhesive force between PMN and EC in response to burn patients' serum within 24 hours and effects of antibody against CD11/CD18 on PMN-EC adhesive force. Burn serum was isolated from 4 burn patients (III degrees 20%-50% TBSA) within 48 hours after burn injury. PMNs were isolated from 8 volunteers. Adhesive force between a PMN and a HUVEC was calculated by means of micropipette technique at 1 h, 3 h, 6 h, 12 h, 24 h after PMN or HUVEC incubated with burn serum or normal serum. Five individual PMN-HUVEC pairs were measured for each sample. All data were analyzed statistically using the t test. RESULTS: 1. The adhesive force between a single PMN-HUVEC pair increased sharply after PMN incubated with burn serum, which reached the maximun level at 1 h after incubation and remained at this level in the following 23 hours. However, the adhesive force was reduced by 78.5%, 72.5%, 75.2% at 1 h, 6 h, 24 h, respectively after the PMNs were pretreated with CD11/CD18 mAb; 2. The adhesive force between a single burn serum stimulated-HUVEC and PMN pair increased gradually, reached peak at 12 h and remained at this level in the following 12 hours. CONCLUSIONS: Burn serum could induce PMN-EC adhesion, with related to PMN aggregation in internal organs and EC damage after burn injury. Antibodies against CD11/CD18 could partially block PMN-EC adhesion and might reduce EC injury induced by activated PMNs.     

Mg2+ coordination in catalytic sites of F1-ATPase

Cytokines are hormone-like proteins which mediate and regulate inflammatory and immune responses. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) and inflammatory cytokines on regulation of interleukin-6 (IL-6) production by human gingival fibroblasts (HGF). The HGF cell lines used in this study, H-CL and F-CL, were established by the explant technique from healthy gingival tissue. Cultured cells were grown to confluency and incubated with various concentrations of LPS from Escherichia coli or Porphyromonas gingivalis or with the recombinant human cytokine tumor necrosis factor alpha (TNF-alpha), IL-1alpha, or IL-1beta. Culture supernatants were collected at various times and assessed for IL-6 production by enzyme-linked immunosorbent assay. Total RNA was isolated from the harvested cells and used to assess levels of IL-6 mRNA by the RNase protection assay. Both LPS preparations induced IL-6 production (1 to 4 ng of IL-6 per ml) by both HGF cell lines. Although TNF-alpha stimulated IL-6 production by HGF, > 10-fold-larger amounts were induced with IL-1alpha and IL-1beta. Furthermore, the addition of both IL-1alpha and TNF-alpha to cultured cells resulted in approximately 600- to 800-fold-higher levels of IL-6 than seen in control cultures, suggesting that these cytokines synergistically induced IL-6 production by HGF. IL-6 message in cultured cells was upregulated 20-fold by TNF-alpha, 1,000-fold by IL-1alpha and IL-1beta, and 1,400-fold by IL-1alpha plus TNF-alpha. IL-1alpha and TNF-alpha alone upregulate IL-6 production in a dose- and time-dependent fashion. The addition of IL-1alpha and TNF-alpha to cultured HGF cells resulted in a synergistic induction of IL-6 after 8 h of incubation and when greater than 10 pg of this combination per ml was used. Our studies show that inflammatory cytokines are hundreds of times more potent than LPS in stimulating IL-6 production by HGF.     

Ciliary neurotrophic factor potentiates the beta-cell inhibitory effect of IL-1beta in rat pancreatic islets associated with increased nitric oxide synthesis and increased expression of inducible nitric oxide synthase

Proinflammatory cytokines are implicated as effector molecules in the pathogenesis of IDDM. Interleukin-6 (IL-6) alone or in combination with IL-1beta inhibits glucose-stimulated insulin release from isolated rat pancreatic islets by unknown mechanisms. Here we investigated 1) if the effects of IL-6 are mimicked by ciliary neurotrophic factor (CNTF), another member of the IL-6 family of cytokines signaling via gp130, 2) the possible cellular mechanisms for these effects, and 3) if islet endocrine cells are a source of CNTF. CNTF (20 ng/ml) potentiated IL-1beta-mediated (5-150 pg/ml) nitric oxide (NO) synthesis from neonatal Wistar rat islets by 31-116%, inhibition of accumulated insulin release by 34-49%, and inhibition insulin response to a 2-h glucose challenge by 31-36%. CNTF potentiated IL-1beta-mediated NO synthesis from RIN-5AH cells by 83%, and IL-1beta induced islet inducible NO-synthase (iNOS) mRNA expression fourfold. IL-6 (10 ng/ml) also potentiated IL-1beta-mediated NO synthesis and inhibition of insulin release, whereas beta-nerve growth factor (NGF) (5 or 50 ng/ml) had no effect. mRNA for CNTF was expressed in rat islets and in islet cell lines. In conclusion, CNTF is constitutively expressed in pancreatic beta-cells and potentiates the beta-cell inhibitory effect of IL-1beta in association with increased iNOS expression and NO synthesis, an effect shared by IL-6 but not by beta-NGF. These findings indicate that signaling via gp130 influences islet NO synthesis associated with iNOS expression. We hypothesize that CNTF released from destroyed beta-cells during the inflammatory islet lesion leading to IDDM may potentiate IL-1beta action on the beta-cells.