Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.
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To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased. (+info)
Lipoxygenase products increase monocyte adhesion to human aortic endothelial cells.
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The development of atherosclerosis is accelerated in individuals with type 2 diabetes. Adhesion of monocytes to the vascular endothelium is a key initial step in atherogenesis. We have previously shown that monocyte adhesion to human aortic endothelial cells (HAECs) cultured long-term in high-glucose medium (25 mmol/L, 2 passages) is increased compared with cells grown in normal glucose (5 mmol/L). One potential mechanism for increased monocyte adhesion to HAECs under hyperglycemic conditions is via the 12-lipoxygenase (12-LO) pathway. In this study, we demonstrated in HAECs that the major LO metabolite of arachidonic acid was the 12-LO product, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], which was increased severalfold in HAECs cultured under high-glucose conditions. Furthermore, treatment of HAECs with 12(S)-HETE induced monocyte, but not neutrophil, adhesion an average of 3-fold (range of 1.5- to 5-fold) compared with untreated cells (75+/-5 versus 26+/-1 monocytes per field, respectively, P<0.001). Expression of the adhesion molecules vascular cell adhesion molecule-1, E-selectin, and intercellular adhesion molecule-1 was not significantly increased. However, both glucose and 12(S)-HETE induced a 60% increase in HAEC surface expression of connecting segment-1 (ie, CS-1) fibronectin, a ligand for very late-acting antigen-4 (VLA-4). The antibodies used to block monocyte integrin VLA-4 and leukocyte function-related antigen-1, a monocytic counterreceptor for intercellular adhesion molecule-1, inhibited the ability of both 12-LO products and high glucose to induce monocyte adhesion. These results definitively demonstrate for the first time in HAECs that the 12-LO pathway can induce monocyte-endothelial cell interaction and that the effects of glucose may be mediated, at least in part, through this pathway. Thus, these results suggest that the 12-LO pathway may play a role in the increased susceptibility of diabetics to atherosclerosis. (+info)
Particles binding beta(2)-integrins mediate intracellular production of oxidative metabolites in human neutrophils independently of phagocytosis.
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Complement-opsonised particles are readily ingested by human neutrophils through a complement receptor-mediated process leading to phagolysosome fusion and production of oxidative metabolites. To investigate the complement receptor 3 (CR3)-associated signal system involved, cells were challenged with protein A-positive, heat-killed Staphylococcus aureus to which antibodies with specificity for the subunits of the beta(2)-integrins, i.e. anti-CD11b (the alpha subunit of CR3) and anti-CD18 (the beta subunit of CR3), were bound through their Fc moiety. Despite not being ingested by the neutrophils, the surface associated anti-CD18- and anti-CD11b-coated particles were able to activate the neutrophil NADPH-oxidase. Also anti-CD11a- (the alpha subunit of LFA-1) and to a lesser extent anti-CD11c- (the alpha subunit of CR4) coated particles were able to trigger the NADPH-oxidase. The NADPH-oxidase was activated without extracellular release of reactive oxygen species. The activity was inhibited by cytochalasin B, suggesting a necessary role for the cytoskeleton in the signalling pathway that activates the oxidase. We show that particle-mediated cross-linking of beta(2)-integrins on the neutrophil surface initiates a signalling cascade, involving cytoskeletal rearrangements, leading to an activation of the NADPH-oxidase without phagosome formation or extracellular release of reactive oxygen species. (+info)
Expression of beta-integrin adhesion molecules in non-Hodgkin's lymphoma: correlation with clinical and evolutive features.
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PURPOSE: To analyze beta-integrin expression in non-Hodgkin's lymphomas (NHLs) in order to assess its distribution among histologic subtypes and correlate with clinical features and outcome. PATIENTS AND METHODS: The expression of alpha2 through alpha6 and beta1 common chains of very late activation antigen (VLA ) molecules and alphaL (CD11a) and beta2 common (CD18) chains of leukocyte function-associated antigen 1 molecule were studied in 137 patients with NHL. Immunostaining was performed by a streptavidin-biotin alkaline phosphatase method, and integrin expression was semiquantitatively assessed. Correlation with clinical features was analyzed in 80 patients consecutively diagnosed as having immunocytoma (five cases), follicular lymphoma (19 cases), mantle-cell lymphoma (MCL; four cases), diffuse large-cell lymphoma (DLCL; 40 cases), lymphoblastic lymphoma (LL; six cases), anaplastic Ki-1-positive lymphoma (one case), and other peripheral T-cell lymphoma (five cases). RESULTS: MCL cells did not show alpha2 and alpha6 expression, whereas most expressed weak to moderate levels of alpha3, alpha4, and alpha5. LL mostly showed alpha2 to alpha5 expression, whereas alpha6 was observed in seven of 11 cases (higher proportion than that shown in other subgroups). Alpha chains of VLA molecules were present more frequently in T-cell than in B-cell lymphomas. Patients with moderate/strong alpha4, CD11a, and beta2 common chain expression presented more frequently with advanced stage and bone marrow infiltration. Moderate/strong alpha4, alpha5, and beta1 common chain expression correlated with extranodal involvement. In the subset of B-cell DLCL patients, negative/weak expression of alpha3 and alpha4 chains was related to a higher complete response rate. Moreover, negative or weak expression of alpha2, alpha3, alpha4, and beta1( )common chain had favorable significance for overall and failure-free survivals. CONCLUSION: In NHL, beta-integrin expression is related to histologic subtype. The expression pattern of these molecules probably influences disease dissemination and patients' prognoses. (+info)
Migration of CD18-deficient neutrophils in vitro: evidence for a CD18-independent pathway induced by IL-8.
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Neutrophils isolated from a child with severe leukocyte adhesion deficiency 1 (LAD1) had a complete absence of expression of the CD11/CD18 beta2 integrin family of adhesion molecules, and were shown to be deficient in the in vitro adhesion and migration properties. However, we found that interleukin-8 (IL8), a potent chemoattractant for neutrophils, and sputum sol phase induced these LAD1 neutrophils to migrate through an endothelial cell layer in vitro, and confirmed that this migration was CD18-independent. These findings add to evidence of CD18-independent mechanisms of neutrophil recruitment, in particular neutrophil infiltration into the lungs, where IL8 may be an important recruitment factor. (+info)
Phenotypic changes in neutrophils related to anti-inflammatory therapy.
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Previous work from the group has shown that non-steroidal anti-inflammatory agents given to volunteers and patients inhibit PMN function possibly by affecting the developing neutrophil during the differentiation process. In this study indomethacin treatment in vivo reduced neutrophil chemotaxis and proteolytic degradation of fibronectin, with a maximal effect after 14 days. Stimulated neutrophil adherence to fibronectin was also reduced but this was not due to quantitative changes in beta(2) integrin expression or function. L-Selectin expression on resting and stimulated neutrophils was increased after 14 days and there was a small decrease in plasma levels of soluble L-selectin. These effects, however, could not be reproduced by treatment of neutrophils with indomethacin in vitro, suggesting they are due to effects on differentiating/maturing PMNs. In an attempt to interpret these changes, studies were performed with dexamethasone, which is known to alter neutrophil function and kinetics. Dexamethasone treatment reduced chemotaxis and increased superoxide generation after 1 day and was associated with increased expression of activated beta(2) integrins and reduced L-selectin expression on resting neutrophils. This suggests the appearance of mainly 'activated' cells as a result of demargination and indicates that the effects of indomethacin are distinctive and not related to changes in compartmentalisation. (+info)
Selective recruitment of T-cell subsets to the udder during staphylococcal and streptococcal mastitis: analysis of lymphocyte subsets and adhesion molecule expression.
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During bacterial infection of the bovine mammary gland, large numbers of leukocytes migrate into the udder, resulting in the establishment of a host response against the pathogen. Currently, the specific leukocyte populations mediating this immune response are not well defined. In the studies described here, we analyzed blood and milk from healthy cows and cows with naturally occurring mastitis to determine if distinct alphabeta and gammadelta T-lymphocyte subsets were involved in the response of the udder to a mastitis pathogen and if the type of mastitis pathogen influenced the subset composition of these responding leukocytes. Although blood samples from cows with confirmed staphylococcal and streptococcal mastitis were characterized by increased numbers of gammadelta T cells, the most dramatic changes in leukocyte distributions occurred in milk samples from these cows, with a 75% increase in alphabeta T-cell levels and a 100% increase in gammadelta T-cell levels relative to the levels in milk samples from healthy animals. Interestingly, the increase in alphabeta T-cell numbers observed in milk from cows with staphylococcal mastitis was primarily due to increased numbers of CD4(+) T cells, while the increase in alphabeta T-cell numbers observed in cows with streptococcal mastitis was due to a parallel increase in both CD4(+) and CD8(+) T-cell numbers. The increased numbers of gammadelta T cells in milk from cows with staphylococcal and streptococcal mastitis were due to a selective recruitment of a distinct gammadelta T-cell subset (GD3.1(+)), while no change in the numbers of GD197(+) gammadelta T cells was observed. We also analyzed adhesion protein expression on blood and milk leukocytes and found that, in comparison to the situation for healthy cows, L-selectin was down-regulated and CD18 was up-regulated on leukocytes from cows with mastitis. Thus, shedding of L-selectin and up-regulation of CD18 by neutrophils may provide a sensitive indicator of early inflammatory responses during bovine mastitis. Overall, these studies suggest that distinct alphabeta and gammadelta T-cell subsets are involved in the host defense of the udder against mastitis infection and that selective recruitment of these T-cell subsets depends on the infectious agent involved. (+info)
15-Deoxy-Delta12,1412,14-prostaglandin J2 inhibits the beta2 integrin-dependent oxidative burst: involvement of a mechanism distinct from peroxisome proliferator-activated receptor gamma ligation.
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15-Deoxy-Delta12,14-PGJ2 (dPGJ2) is a bioactive metabolite of the J2 series that has been identified as a ligand for peroxisome proliferator-activated receptor gamma (PPARgamma) and has received attention for its potential antiinflammatory effects. Because neutrophils express cell-surface receptors for PGs, the effect of dPGJ2 was tested on an inflammatory response that should not require PPARgamma, the oxidative burst made by adherent human neutrophils. dPGJ2 inhibited adhesion-dependent H2O2 production with an IC50 of 1. 5 microM when neutrophils were stimulated with TNF, N-formylnorleucylleucylphenylalanine, or LPS. Inhibition by dPGJ2 occurred during the lag phase, before generation of peroxide, suggesting blockade of an early signaling step. Indeed, dPGJ2 blocked adhesion of neutrophils to fibrinogen in response to TNF or LPS with an IC50 of 3-5 micro+dPGJ2 was more potent at inhibiting the adhesion-dependent oxidative burst than several other PGs tested. Further, dPGJ2 did not appear to act through either the DP receptor or receptors for PGE2. PG receptors modulate cAMP levels, and the inhibition of adhesion and oxidative burst by dPGJ2 was enhanced in the presence of 3-isobutyl-1-methylxanthine, a cAMP phosphodiesterase inhibitor. A potent PPARgamma agonist (AD-5075) did not inhibit peroxide production or adhesion, nor did it change the IC50 for dPGJ2 inhibition. These studies suggest that dPGJ2 may interact with an unknown receptor on neutrophils, distinct from PPARgamma, to modulate the production of reactive oxygen intermediates. (+info)