Effects of an aqueous extract from leaves of Ligustrum vulgare on mediators of inflammation in a human neutrophils model.

Leaves of Ligustrum vulgare (common privet) have been used for treatment of oropharyngeal inflammations or as antirheumatic, diuretic, and hypotensive agents in folk medicine in southern Europe. Taking into account that neutrophils are involved in the inflammation, the aim of the study was to determine the effect of an aqueous extract prepared from leaves of Ligustrum vulgare on neutrophil functions. The extract was characterized by the HPLC-DAD-MSn method. The inhibition of reactive oxygen species production by formyl-met-leu-phenylalanine- or phorbol 12-myristate 13-acetate-stimulated neutrophils was determined using luminol- or lucigenin-dependent chemiluminescence. The effect on myeloperoxidase, metalloproteinase 9, and interleukin 8 production by neutrophils was measured by an enzyme-linked immunosorbent assay. Neutrophil elastase release was established spectrophotometrically. The expression of adhesion molecules on neutrophils was analyzed with flow cytometry. The main compounds detected were flavonoids, phenylpropanoids, hydroxycinnamates, and secoiridoids. The inhibition of oxidative burst by the extract was comparable in both stimuli models (formyl-met-leu-phenylalanine: IC50 = 18.2 ± 4.0 µg/mL; phorbol 12-myristate 13-acetate: IC50 = 19.8 ± 3.0 µg/mL). The extract in the concentration range of 5-50 µg/mL inhibited neutrophil elastase release by 23.9-34.1 % and myeloperoxidase release by 24.2-37.4 %. The inhibitory effect on metalloproteinase 9 and interleukin 8 production was around 20 %. The extract in the highest concentration modulated the expression of L-selectin and ß2 integrin. Our results partly support the traditional use of common privet leaves as an anti-inflammatory agent.

The effect of midazolam on neutrophil mitogen-activated protein kinase.

BACKGROUND: Neutrophil p38 mitogen-activated protein kinase (MAPK) is a key enzyme in the intracellular signalling pathway that is responsible for many neutrophil functions, which are important in neutrophil-endothelial interaction. The imidazole compounds are inhibitors of this enzyme system. The objectives of this in-vitro investigation were to examine the effect of midazolam on neutrophil p38 MAPK activation (phosphorylation) following in-vitro ischaemia-reperfusion injury, and the expression of adhesion molecule CD11b/CD18. METHODS: In-vitro injury was produced by incubating the neutrophils with N-formyl-methionyl-leucyl-phenylalanine. Neutrophils were treated with either 10 or 50 times the therapeutic plasma concentrations of midazolam and SB203580 (known inhibitor of p38 MAPK). The concentrations of phosphorylated p38 MAPK and expression of neutrophil adhesion molecules CD11b/CD18 were measured. Flow cytometry was used to estimate adhesion molecule expression. RESULTS: The concentration of phosphorylated p38 MAPK was less in neutrophils subjected to ischaemia-reperfusion and treated with midazolam either 10 microg ml [13.6 (3.2) ng ml] or 50 microg ml [12.4 (3.6) ng ml], or SB203580 [13 (2.6) ng ml] than those subjected to ischaemia-reperfusion alone [18 (3.18) ng ml] at a P value of less than 0.05.Following ischaemia-reperfusion injury, CD11b/CD18 expression (expression mean channel fluorescence) on neutrophils was greater when compared with controls. The magnitudes of CD11b and CD18 expression on ischaemia-reperfusion-injured neutrophils were decreased by midazolam (10 microg ml) as compared with control of 10.3 (2.6) vs. 14 (3.1) microg ml and 28.3 (12.9) vs. 44 (12.1) microg ml, respectively, at a P value of less than 0.05. Similarly, the expression of CD11b and CD18 was less in ischaemia-reperfusion-injured neutrophils treated with inhibitor of 10.3 (2.8) vs. 14 (3.18) microg ml and 29.5 (12.5) vs. 44.3 (12.3) microg ml when compared with controls at a P value of less than 0.05. CONCLUSION: Midazolam diminishes in-vitro ischaemia-reperfusion-induced phosphorylation of p38 MAPK in neutrophils. This decrease in p38 MAPK activation results in decreased neutrophil CD11b/CD18 molecule expression.

Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by beta3 integrin.

The beta3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various beta3 integrins with cytoplasmic tail mutations in beta3-deficient OC precursors. We find that S752 in the beta3 cytoplasmic tail is required for growth factor-induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional beta3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on beta3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional beta3. Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin. Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.

GA-binding protein and p300 are essential components of a retinoic acid-induced enhanceosome in myeloid cells.

Expression of CD18, the beta chain of the leukocyte integrins, is transcriptionally regulated by retinoic acid (RA) in myeloid cells. Full RA responsiveness of the CD18 gene requires its proximal promoter, which lacks a retinoic acid response element (RARE). Rather, RA responsiveness of the CD18 proximal promoter requires ets sites that are bound by GA-binding protein (GABP). The transcriptional coactivator, p300, further increases CD18 RA responsiveness. We demonstrate that GABPalpha, the ets DNA-binding subunit of GABP, physically interacts with p300 in myeloid cells. This interaction involves the GABPalpha pointed domain (PNT) and identifies p300 as the first known interaction partner of GABPalpha PNT. Expression of the PNT domain, alone, disrupts the GABPalpha-p300 interaction and decreases the RA responsiveness of the CD18 proximal promoter. Chromatin immunoprecipitation and chromosome conformation capture demonstrate that, in the presence of RA, GABPalpha and p300 at the proximal promoter recruit retinoic acid receptor/retinoid X receptor from a distal RARE to form an enhanceosome. A dominant negative p300 construct disrupts enhanceosome formation and reduces the RA responsiveness of CD18. Thus, proteins on the CD18 proximal promoter recruit the distal RARE in the presence of RA. This is the first description of an RA-induced enhanceosome and demonstrates that GABP and p300 are essential components of CD18 RA responsiveness in myeloid cells.

Identification of novel agonists of the integrin CD11b/CD18.

We report the identification of novel small molecule agonists of integrin CD11b/CD18, which increased, in a dose-dependent manner, the adhesion of the integrin CD11b/CD18 expressing cells to two physiologically relevant ligands: Fibrinogen and iC3b. Compound 6 showed an ex vivo EC(50) of 10.5 microM and in vitro selectivity for binding to the recombinant alphaA-domain of CD11b/CD18. In silico docking experiments suggest that the compounds recognized a hydrophobic cleft in the ligand-binding alphaA-domain, implying an allosteric mechanism of modulation of integrin affinity by this novel compound.

Tonic protein kinase A activity maintains inactive beta2 integrins in unstimulated neutrophils by reducing myosin light-chain phosphorylation: role of myosin light-chain kinase and Rho kinase.

Activation of beta2 integrins is necessary for neutrophil adhesion and full activation of neutrophil effector functions. We demonstrated previously that inhibition of protein kinase A (PKA) activity in quiescent neutrophils is sufficient to increase beta2-integrin cell surface expression, affinity, and adhesion. Thus, a tonic level of PKA activity prevents inappropriate activation of beta2 integrins in unstimulated neutrophils. Myosin light-chain (MLC) phosphorylation is an important regulator of leukocyte integrin function and adhesion. Moreover, PKA regulates MLC phosphorylation via inhibiting MLC kinase (MLCK) and MLC dephosphorylation via effects on the Rho kinase (ROCK)/MLC phosphatase pathway. We hypothesize that the tonic inhibitory effect of PKA on beta2-integrin activation neutrophils operates via its inhibition of MLC phosphorylation. We demonstrate here that inhibition of PKA activity with KT5720 activated beta2 integrins and adhesion coincident with an increase in MLC serine 19 (Ser 19) phosphorylation. KT5720-induced activation of beta2 integrins, adhesion, and MLC Ser 19 phosphorylation was abolished by pretreatment with the MLCK inhibitor ML-7 and specific MLCK inhibitory peptides but not the ROCK inhibitor Y-27632. These findings demonstrate that tonic PKA activity prevents activation of beta2 integrins and adhesion by inhibiting MLC phosphorylation via a MLCK-dependent but ROCK-independent pathway.

Extracellular survivin up-regulates adhesion molecules on the surface of leukocytes changing their reactivity pattern.

Rheumatoid arthritis (RA) is an autoimmune disease with joints as a principal target of inflammation. We have shown recently that the extracellular expression of the antiapoptotic protein survivin is associated with a destructive course of RA. Here, we address the potential impact of extracellular survivin on peripheral blood leukocytes (PBL). The binding of survivin to the surface of human PBL as well as the expression of adhesion molecules were assessed by FACS. The expression of adhesion molecules on leukocytes as a function of circulating survivin was analyzed in blood of 24 patients with RA and compared with eight healthy individuals. We show that extracellular survivin expresses immunomodulatory properties. It binds to the surface of the majority of granulocytes and a significant part of lymphocytes and monocytes inducing the activation of alpha-chains of beta-integrins and their ligand ICAM-1. Survivin-induced expression of alpha-chains of beta 2-integrins is regulated by p38 MAPK and PI-3K but not by the NF-kappaB signaling pathway. Clinical relevance of our findings is supported by the in vivo association of high circulating survivin levels with an increased expression of CD11c on monocytes and granulocytes in RA patients. The results of our study demonstrate that extracellular survivin affects the phenotype of leukocytes having a possible impact on homing of inflammatory cells during arthritis.

VIP differentially activates beta2 integrins, CR1, and matrix metalloproteinase-9 in human monocytes through cAMP/PKA, EPAC, and PI-3K signaling pathways via VIP receptor type 1 and FPRL1.

The neuropeptide vasoactive intestinal peptide (VIP) regulates the exocytosis of secretory granules in a wide variety of cells of neuronal and non-neuronal origin. In human monocytes, we show that the proinflammatory effects of VIP are associated with stimulation of exocytosis of secretory vesicles as well as tertiary (gelatinase) granules with, respectively, up-regulation of the membrane expression of the beta2 integrin CD11b, the complement receptor 1 (CD35), and the matrix metalloproteinase-9 (MMP-9). Using the low-affinity formyl peptide receptor-like 1 (FPRL1) antagonist Trp-Arg-Trp-Trp-Trp-Trp (WRW4) and the exchange protein directly activated by cAMP (EPAC)-specific compound 8CPT-2Me-cAMP and measuring the expression of Rap1 GTPase-activating protein as an indicator of EPAC activation, we found that the proinflammatory effect of VIP is mediated via the specific G protein-coupled receptor VIP/pituitary adenylate cyclase-activating protein (VPAC1) receptor as well as via FPRL1: VIP/VPAC1 interaction is associated with a cAMP increase and activation of a cAMP/p38 MAPK pathway, which regulates MMP-9, CD35, and CD11b exocytosis, and a cAMP/EPAC/PI-3K/ERK pathway, which regulates CD11b expression; VIP/FPRL1 interaction results in cAMP-independent PI-3K/ERK activation with downstream integrin up-regulation. In FPRL1-transfected Chinese hamster ovary-K1 cells lacking VPAC1, VIP exposure also resulted in PI-3K/ERK activation. Thus, the proinflammatory effects of VIP lie behind different receptor interactions and multiple signaling pathways, including cAMP/protein kinase A, cAMP/EPAC-dependent pathways, as well as a cAMP-independent pathway, which differentially regulates p38 and ERK MAPK and exocytosis of secretory vesicles and granules.

Resistance of melanized yeast cells of Paracoccidioides brasiliensis to antimicrobial oxidants and inhibition of phagocytosis using carbohydrates and monoclonal antibody to CD18.

Paracoccidioides brasiliensis, a thermal dimorphic fungal pathogen, produces a melanin-like pigment in vitro and in vivo. We investigated the involvement of carbohydrates and monoclonal antibody to CD18, on phagocytosis inhibition, involving macrophage receptors and the resistance of melanized fungal cells to chemically generated nitric oxide (NO), reactive oxygen species (ROS), hypochlorite and H2O2. Our results demonstrate that melanized yeast cells were more resistant than nonmelanized yeast cells to chemically generated NO, ROS, hypochlorite and H2O2, in vitro. Phagocytosis of melanized yeast cells was virtually abolished when mannan, N-acetyl glucosamine and anti-CD18 antibody were added together in this system. Intratracheal infection of BALB/c mice, with melanized yeast cells, resulted in higher lung colony forming units, when compared to nonmelanized yeast cells. Therefore, melanin is a virulence factor of P. brasiliensis.

The attenuation effect of 3,4-dihydroxy-phenyl lactic acid and salvianolic acid B on venular thrombosis induced in rat mesentery by photochemical reaction.

3,4-dihydroxy-phenyl lactic acid (DLA) and salvianolic acid B (SAB) are two major water-soluble components of Salvia miltiorrhiza (SM). Previous works have revealed the ability of DLA and SAB to scavenge oxygen free radicals, inhibiting the expression of adhesion molecules CD11b/CD18 in neutrophil. Cardiotonic pills (CP), which is a traditional Chinese medicine compound preparation containing DLA and SAB, was found to inhibit venular thrombosis induced by photochemical reaction (PR) in rat mesentery. The present study addressed the effect of DLA and SAB on PR-induced thrombosis in rat mesentery by utilizing a microcirculation dynamic viewing system. The result demonstrated that both DLA and SAB delayed thrombus-initiation time, while DLA also prolonged thrombus half-size time. The experiments explored the mechanism underlying that the dihydrorhodamine 123 (DHR) fluorescence in the mesenteric venular walls after PR challenge was diminished by pretreatment with either DLA or SAB, the expression of CD18 in neutrophils elicited by PR was depressed by administration of DLA, while mast cell degranulation in rat mesentery induced by PR was damped by SAB. The antioxidant potential of the two substances is likely to be responsible for their most beneficial effects on thrombosis, through either directly scavenging the peroxides produced and/or indirectly depressing the expression of adhesion molecules in neutrophil.

Decreased monocyte shedding of the migration inhibitor soluble CD18 in alcoholic hepatitis.

OBJECTIVES: During alcoholic hepatitis (AH) monocytes traverse the vascular boundaries and massively invade the liver. In principle, tissue extravasation can be limited through shedding of CD18 integrins from leukocytes, including monocytes. The soluble (s) product sCD18 conceals adhesion receptors on the endothelium, which reduces monocyte extravasation. In AH, monocytes are dysfunctional, but whether this involves their self-generated anti-migration is unknown. Our aim was, therefore, to investigate monocyte CD18 dynamics in AH. METHODS: We studied 50 AH patients and 20 healthy controls. We measured monocyte expression and conformational activation of CD18, plasma (P)-sCD18, stimulated in vitro CD18 shedding and P-sCD18 in a short-term chronic-binge mouse model. RESULTS: AH-derived monocytes had a 30-60% higher expression of active CD18 receptors (p < 0.01), but the sCD18 concentration per monocyte was reduced in vivo by 30% and in vitro by 120% (p < 0.01). Ethanol reduced the in vitro shedding of CD18 in the patients only. TNFα increased sCD18 concentration per monocyte, but less so in the patients (p < 0.04). P-sCD18 per monocyte was inversely related to disease severity. In early alcoholic liver disease, P-sCD18 was decreased in the mouse model. CONCLUSIONS: The monocyte CD18 integrins are highly activated in AH and the single monocyte shedding of CD18 was decreased favoring tissue extravasation. Alcohol in itself and altered monocyte responsiveness to TNFα may explain this lowered shedding. TRANSLATIONAL IMPACT: The contribution of this mechanism to the excessive monocyte liver infiltration in AH should be further explored as it may serve as a potential therapeutic target to limit liver inflammation.

The effects of ethanol on beta2-integrin and l-selectin on the surface of leukocytes in human whole blood.

BACKGROUND: Acute alcohol intoxication is associated with increased susceptibility to infection. In host defense, the expression of adhesion molecules such as beta2-integrin and l-selectin on leukocytes is involved in leukocyte migration to inflamed organ tissue. To elucidate the mechanisms underlying the immunosuppressive effects of ethanol, we investigated whether ethanol pretreatment may influence the changes in adhesion molecule expression induced by lipopolysaccharide (LPS) or interleukin (IL)-8 in human whole blood. METHODS: Ethanol was added to samples of human whole blood (final concentration: 0%, 0.2%, 0.4%, and 0.8%). Samples were assigned to an unstimulated group and an LPS-stimulated group. In another set of experiments, stimulation was induced by IL-8. After fluorescence labeling of alphaM-subunit of beta2-integrin (CD11b) and l-selectin (CD62L), the expression of CD11b and CD62L were measured using flow cytometry. RESULTS: Stimulation with LPS significantly upregulated CD11b expression (5.9 +/- 0.9 to 16.3 +/- 1.8, p < 0.05). Ethanol inhibited this LPS-induced upregulation of CD11b (p < 0.001). Stimulation with IL-8 significantly upregulated CD11b expression (5.3 +/- 1.7 to 7.5 +/- 2.7, p < 0.01) and this IL-8-induced upregulation of CD11b was also inhibited by ethanol pretreatment (p < 0.001). In contrast, ethanol did not modify CD62L expression in either unstimulated or stimulated groups. CONCLUSION: The impairment of CD11b expression on leukocytes suggests that alcohol intake interferes with the migration of leukocytes to sites of inflammation, which may explain, in part, why alcohol intoxication increases susceptibility to infection.

Cytoplasmic tails of beta 1, beta 2, and beta 7 integrins differentially regulate LFA-1 function in K562 cells.

The beta 2 integrin lymphocyte function-associated antigen 1 (LFA-1) mediates activation-dependent adhesion of lymphocytes. To investigate whether lymphocyte-specific elements are essential for LFA-1 function, we expressed LFA-1 in the erythroleukemic cell line K562, which expresses only the integrin very late antigen 5. We observed that LFA-1-expressing K562 cannot bind to intercellular adhesion molecule 1-coated surfaces when stimulated by phorbol 12-myristate 13-acetate (PMA), whereas the LFA-1-activating antibody KIM185 markedly enhanced adhesion. Because the endogenously expressed beta 1 integrin very late antigen 5 is readily activated by PMA, we investigated the role of the cytoplasmic domain of distinct beta subunits in regulating LFA-1 function. Transfection of chimeric LFA-1 receptors in K562 cells reveals that replacement of the beta 2 cytoplasmic tail with the beta 1 but not the beta 7 cytoplasmic tail completely restores PMA responsiveness of LFA-1, whereas a beta 2 cytoplasmic deletion mutant of LFA-1 is constitutively active. Both deletion of the beta 2 cytoplasmic tail or replacement by the beta 1 cytoplasmic tail alters the localization of LFA-1 into clusters, thereby regulating LFA-1 activation and LFA-1-mediated adhesion to intercellular adhesion molecule 1. These data demonstrate that distinct signaling routes activate beta 1 and beta 2 integrins through the beta-chain and hint at the involvement of lymphocyte-specific signal transduction elements in beta 2 and beta 7 integrin activation that are absent in the nonlymphocytic cell line K562.

Role of alpha4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium.

Myocardial damage due to reperfusion of ischemic tissue is caused primarily by infiltrating neutrophils. Although leukocyte beta2 integrins (CD18) play a critical role, significant neutrophil emigration persists when CD18 is neutralized or absent. This study examined the role of leukocyte beta1 integrin (alpha4) and its endothelial ligand VCAM-1 in CD18-independent neutrophil migration across cardiac endothelium. In a mouse model of myocardial ischemia and reperfusion, we show that compared with wild-type mice, neutrophil infiltration efficiency was reduced by 50% in CD18-null mice; in both types of mice, myocardial VCAM-1 staining increased after reperfusion. In wild-type mice, antibodies against CD18, ICAM-1 (an endothelial ligand for CD18), or VCAM-1 given 30 minutes before ischemia did not block neutrophil emigration at 3 hours reperfusion. Although anti-VCAM-1 attenuated neutrophil emigration by 90% in CD18-null mice, it did not diminish myocardial injury. To determine if CD18-independent neutrophil emigration was a tissue-specific response, we used isolated peripheral blood neutrophils from wild-type or CD18-null mice and showed neutrophil migration across lipopolysaccharide-activated cultured cardiac endothelium is CD18-independent, whereas migration across endothelium obtained from inferior vena cava is CD18-dependent. Consistent with our in vivo findings, migration of CD18-deficient neutrophils on cardiac endothelial monolayers is blocked by antibodies against alpha4 integrin or VCAM-1. We conclude tissue-specific differences in endothelial cells account, at least partially, for CD18-independent neutrophil infiltration in the heart.

Targeting CCR2 or CD18 inhibits experimental in-stent restenosis in primates: inhibitory potential depends on type of injury and leukocytes targeted.

A central role for leukocytes in neointimal hyperplasia after arterial injury is suspected. However, the relative importance of neutrophils and monocytes in balloon or stent-induced injury are not well understood, and mechanistic targeting of leukocyte recruitment or function is crude. We determined the temporal and spatial distribution of different leukocytes after balloon and stent-induced injury in primate iliac arteries. Based on these data, we targeted neutrophil and monocyte recruitment selectively after angioplasty or stent implantation and demonstrated that monocyte-specific blockade achieved via blockade of the MCP-1 receptor CCR2, was effective at reducing neointimal hyperplasia after stenting. In contrast, combined neutrophil and monocyte blockade achieved by targeting the leukocyte beta(2)-integrin beta-subunit CD18 was required to reduce neointimal hyperplasia after balloon injury. Distinct patterns of leukocyte infiltration in balloon versus stent-injured arteries predict distinct mechanisms for antiinflammatory strategies targeting neutrophils or monocytes in primates and may assist design of effective clinical strategies for optimizing vascular interventions.

Dietary glutamine supplementation reduces cellular adhesion molecule expression and tissue myeloperoxidase activity in mice with gut-derived sepsis.

OBJECTIVES: This study investigated the effects of glutamine (Gln) on plasma intracellular adhesion molecule-1 levels and leukocyte integrin (CD11a/CD18 and CD11b/CD18) expressions in gut-derived sepsis. Myeloperoxidase (MPO) activities in organs were also analyzed to identify the extent of tissue injury resulting from neutrophil infiltration. METHODS: Mice were randomly assigned to a normal group (NC), a control group, or a Gln group. The NC group was fed standard chow diet; the control group was fed a common semipurified diet; and the Gln group received a diet in which part of the casein was replaced by Gln, which provided 25% of total amino acid nitrogen. After 3 wk, sepsis was induced by cecal ligation and puncture (CLP) in the control and Gln groups. Mice in the experimental groups were killed at 0, 6, 12, and 24 h after CLP. Mice in the NC group were killed when CLP was performed. Blood and organ samples were collected for further analysis. RESULTS: Plasma intracellular adhesion molecule-1 levels were significantly lower in the Gln group than in the control group at 6, 12, and 24 h after CLP. Expressions of lymphocyte CD11a/CD18 were significantly higher, whereas polymorphonuclear lymphocyte expressions of CD11b/CD18 were lower in the Gln group than in the corresponding control group at 6 and 12 h after CLP. In comparisons of MPO activities in various organs, the Gln group had lower MPO activities at 6 and 12 h in the lung, at 6, 12, and 24 h in the liver, at 12 and 24 h in the kidneys, and at 12 h in the intestine than those in the control group. CONCLUSIONS: Results of this study demonstrate that a Gln-supplemented enteral diet increased lymphocyte CD11a/CD18 expressions, whereas neutrophil CD11b/CD18 expressions, circulating intracellular adhesion molecule-1 levels, and MPO activities in various organs decreased with gut-derived sepsis. These findings suggest that, under septic conditions, Gln administration may enhance lymphocyte function, attenuate interactions between polymorphonuclear lymphocytes and endothelium, and thus may decrease neutrophil infiltration into tissues.

Inhibition of proinflammatory activities of major periodontal pathogens by aqueous extracts from elder flower (Sambucus nigra).

BACKGROUND: Prolonged induction of excessive levels of inflammatory mediators contributes to the pathogenesis of chronic disease states, such as periodontitis. It is thus important to develop safe and effective anti-inflammatory strategies for therapeutic reasons. In this study, we determined the ability of aqueous extracts from elder flower (Sambucus nigra) to inhibit the proinflammatory activity of major virulence factors from the periodontal pathogens Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans. METHODS: Monocytes/macrophages or neutrophils were incubated with whole cells of P. gingivalis, A. actinomycetemcomitans, or purified components thereof (lipopolysaccharide and fimbriae) in the absence or presence of elder flower extract and were assayed for cytokine production, integrin activation, or induction of the oxidative burst. RESULTS: The elder flower extract was found to potently inhibit all proinflammatory activities tested. Investigation of the underlying mechanisms revealed that the anti-inflammatory extract inhibited activation of the nuclear transcription factor kappaB and of phosphatidylinositol 3-kinase. CONCLUSION: The elder flower extract displays useful anti-inflammatory properties that could be exploited therapeutically for the control of inflammation in human periodontitis.

Transient blockade of the CD11d/CD18 integrin reduces secondary damage after spinal cord injury, improving sensory, autonomic, and motor function.

The early inflammatory response to spinal cord injury (SCI) causes significant secondary damage. Strategies that nonselectively suppress inflammation have not improved outcomes after SCI, perhaps because inflammation has both adverse and beneficial effects after SCI. We have shown that the selective, time-limited action of a monoclonal antibody (mAb) to the CD11d subunit of the CD11d/CD18 integrin, delivered intravenously during the first 48 hr after SCI in rats, markedly decreases the infiltration of neutrophils and delays the entry of hematogenous monocyte-macrophages into the injured cord. We hypothesized that this targeted strategy would lead to neuroprotection and improved neurological outcomes. In this study the development of chronic pain was detected in rats by assessing mechanical allodynia on the trunk and hindpaws 2 weeks to 3 months after a clinically relevant clip-compression SCI at the twelfth thoracic segment. The anti-CD11d mAb treatment reduced this pain by half. Motor performance also improved as rats were able to plantar-place their hindpaws and use them for weight support instead of sweeping movements only. Improved cardiovascular outcome was shown after SCI at the fourth thoracic segment by significant decreases in autonomic dysreflexia. Locomotor performance was also improved. These functional changes correlated with significantly greater amounts and increased organization of myelin and neurofilament near the lesion. The improved neurological recovery after the specific reduction of early inflammation after SCI demonstrates that this selective strategy increases tissue at the injury site and improves its functional capacity. This early neuroprotective treatment would be an ideal foundation for building later cell-based therapies.

Differentiation of circulating endothelial progenitor cells to a cardiomyogenic phenotype depends on E-cadherin.

Progenitor cells may contribute to cardiac regeneration. Here, we investigated the role of cadherins and integrins for differentiation of human adult circulating endothelial progenitor cells (EPCs) into cardiomyocytes (CM) in a co-culture system. N- and E-cadherin were expressed in EPCs and were localized at the interface between EPCs and CM. Incubation of a blocking antibody against E-cadherin reduced the expression of CM marker protein in EPCs. Blocking antibodies against N- or P-cadherin or the beta1- and beta2-integrins were not effective. These data suggested that cell-to-cell communication mediated by E-cadherin contributes to the acquirement of a cardiomyogenic phenotype of human endothelial progenitor cells.

The role of CD11/CD18 integrins in the reverse passive Arthus reaction in rat dermal tissue.

The CD11/CD18 leukocyte integrins are necessary for tissue localization of neutrophils, an early requisite event in inflammation. We have analyzed the contribution of CD11a/CD18 and CD11b/CD18 to local neutrophil accumulation and tissue injury in the reverse passive Arthus reaction in the rat dermis. Experimental groups comprised animals that received an intravenous infusion of (1) recombinant neutrophil inhibitory factor (NIF), a hookworm-derived antagonist of CD11b/CD18; (2) monoclonal antibody to CD11a/CD18 (TA-3); (3) a combination of these agents; (4) a monoclonal antibody to CD18 (WT.3); or (5) saline. Administration of recombinant NIF or anti-CD11a/CD18 monoclonal antibody alone produced a slight reduction in neutrophil accumulation but did not affect edema formation. In contrast, a combination of these antagonists yielded a significant reduction in neutrophil accumulation and a modest reduction in edema, equivalent to levels observed with either anti-CD18 antibodies or animals that were rendered neutropenic. These results indicate that neutrophil infiltration in rat dermal tissue in the reverse passive Arthus reaction is dependent predominantly on the leukocyte integrins CD11a/CD18 and CD11b/CD18 and that either of these integrins is sufficient for neutrophil trafficking in this inflammatory setting.