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PubMed2005

Focal adhesion kinase in neutrophil-induced microvascular hyperpermeability.

OBJECTIVE: Recent experimental evidence indicates an essential role of focal adhesion kinase (FAK) in mediating endothelial adhesion, contraction, and migration under physical stress and chemical stimulation. However, the functional impact of FAK on microvascular barrier property during inflammation has not been revealed. The aim of this study was to explore the potential contribution of FAK to neutrophil-dependent microvascular hyperpermeability. METHODS: The apparent permeability coefficient of albumin was measured in intact, isolated porcine coronary venules during stimulation by C5a-activated neutrophils. In parallel, the transendothelial flux of albumin was quantified in cultured venular endothelial cell monolayers exposed to C5a-activated neutrophils. Western blotting and immunocytochemistry were performed to assess FAK tyrosine phosphorylation and distribution in endothelial cells, respectively. To specify the signaling effect of FAK on neutrophil-elicited endothelial hyperpermeability, FAK-related nonkinase (FRNK) was expressed, purified, and directly transfected into the endothelium of venules, and the permeability response to neutrophils was measured during inhibition of FAK. RESULTS: C5a-activated neutrophils induced a time- and concentration-dependent increase in venular permeability. Transfection of venules with FRNK did not alter the basal barrier function but greatly attenuated neutrophil-induced hyperpermeability in a dose-related manner. A similar permeability response to neutrophils was observed in venular endothelial cell monolayers, which was diminished after FRNK transfection. In addition, Western blot analysis showed that activated neutrophils caused a concentration-dependent increase in FAK tyrosine phosphorylation with a time course correlating with that of venular hyperpermeability. Transfection of FRNK blocked neutrophil-evoked FAK tyrosine phosphorylation. Furthermore, immunofluorescence microscopy revealed a significant morphological change of FAK from a punctuated, dot-like pattern under normal conditions to an elongated, dash-like staining that aligned with the longitudinal axis of cells upon neutrophil stimulation. CONCLUSION: The results suggest that focal adhesion kinase significantly contributes to the endothelial hyperpermeability response to neutrophil activation. Phosphorylation of FAK may play an important signaling role in the regulation of microvascular barrier function during inflammation.

Animals↗
PubMed2006

A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis.

Neutrophil recruitment into tissue plays an important role in host defense and disease pathogenesis, including the inflammatory arthritides. A multitude of diverse chemoattractants have been implicated in neutrophil recruitment, suggesting that they have overlapping functions in mediating this critical biological response. However, here we demonstrate a unique, non-redundant role for the leukotriene B4 receptor BLT1 in mediating neutrophil recruitment into the joint in the K/BxN mouse model of inflammatory arthritis. We demonstrate that neutrophil expression of BLT1 was absolutely required for arthritis generation and chemokine production in this model, and that specific BLT1 inhibition reversed established disease. Adoptive transfer of wild-type (WT) neutrophils restored arthritis and chemokine production in BLT1(-/-) mice. Surprisingly, the primary effect of the transferred WT neutrophils into BLT1(-/-) mice was to promote the entry of endogenous BLT1(-/-) neutrophils into the joints of these mice. However, continued joint inflammation was dependent on the presence of WT neutrophils, indicating an ongoing specific requirement for BLT1-activated neutrophils in mediating BLT1(-/-) neutrophil recruitment by other chemoattractants. These experiments demonstrate that neutrophil BLT1 functions in a novel and essential non-cell-autonomous manner to enable the recruitment of additional neutrophils not expressing this receptor, thereby amplifying the inflammatory response in autoantibody-induced arthritis.

Animals↗
PubMed2002

Isoflurane inhibits neutrophil-endothelium interactions in the coronary circulation: lack of a role for adenosine triphosphate-sensitive potassium channels.

UNLABELLED: Isoflurane protects myocardium during ischemia-reperfusion via a mechanism involving the adenosine triphosphate-sensitive potassium channels. We tested the hypothesis that an inhibition of the neutrophil-endothelium interactions by isoflurane contributes to this effect. Polymorphonuclear neutrophils and coronary artery segments were obtained from 35 healthy dogs. Superoxide production by neutrophils, stimulated with platelet-activating factor (PAF; 1.0 microM), was measured spectrophotometrically. Adherence of PAF-activated neutrophils to the endothelium of coronary segments was assessed by direct counting of neutrophils labeled with fluorescent dye. Coronary artery rings were exposed to PAF-activated neutrophils, and, after washing and preconstriction with U46619, they were evaluated for vasorelaxation responses to acetylcholine (endothelium dependent) and sodium nitroprusside (endothelium independent). Measurements were performed in the absence and presence of isoflurane (1 and 2 minimum alveolar anesthetic concentration) both with and without glibenclamide (10 microM). Isoflurane inhibited superoxide production and adherence by neutrophils and abolished neutrophil-induced reductions in coronary vascular relaxation responses to acetylcholine. Glibenclamide did not alter the effects of isoflurane on neutrophils or coronary artery endothelium. In conclusion, isoflurane had an inhibitory action on neutrophil-endothelium interactions and neutrophil-mediated coronary endothelial dysfunction--effects that may be involved in its cardioprotective action in vivo. These inhibitory actions of isoflurane were not mediated by adenosine triphosphate-sensitive potassium channels. IMPLICATIONS: Isoflurane inhibited neutrophil-endothelium interactions and the inflammatory response in vitro via a pathway independent of the adenosine triphosphate-sensitive potassium channels. This action could be involved in the cardioprotection by isoflurane observed in vivo.

Acetylcholine↗
PubMed1998

Hypertonic saline resuscitation diminishes lung injury by suppressing neutrophil activation after hemorrhagic shock.

UNLABELLED: Hypertonic saline (HS) resuscitation after hemorrhage and sepsis has been shown to markedly reduce the development of lung injury in animals, compared with traditional resuscitation with lactated Ringer's (LR). These experiments examined the effect of HS on lung injury after hemorrhage without sepsis. The effects of HS and LR resuscitation on neutrophil trafficking, neutrophil adhesion, and neutrophil oxidative burst were studied. METHODS: BALB/c mice were hemorrhaged to a mean arterial pressure of 40 torr for 1 h. Animals were resuscitated with shed blood and either 4 mL/kg of 7.5% HS or LR in twice the volume of the shed blood. Lung histology was examined 24 h after hemorrhage. Lung myeloperoxidase content and bronchoalveolar lavage fluid neutrophil counts were obtained. Peripheral blood smears were obtained to determine the neutrophil percentage. Peripheral blood neutrophil CD11b expression and neutrophil H2O2 production were assayed by flow cytometry. RESULTS: HS animals had less lung injury than LR animals. The mean myeloperoxidase activity in HS versus LR animals was 1.79+/-1.33 U/100 mg versus 3.0+/-1.33 U/100 mg, respectively. The percentage of neutrophils in the bronchoalveolar lavage fluid of HS animals (3.8%+/-.8) was significantly less than that of LR animals (10.8%+/-2.1). This corresponded to a significantly higher peripheral blood neutrophil count in HS animals compared with LR animals, 41% vs. 20%, respectively. There was no difference in neutrophil expression of the CD11b integrin between the HS and LR groups. The neutrophils of LR animals had basal H2O2 production that was 107% greater than that of controls; HS suppressed this hemorrhage-induced activation by > 60%. HS resuscitation after hemorrhagic shock protects against the development of lung injury. This protection is due, in part, to suppression of the hemorrhage-induced neutrophil oxidative burst. HS resuscitation offers immunomodulatory potential after hemorrhagic shock.

Animals↗
PubMed1994

Neutrophil function in juvenile periodontitis: induction of adherence.

Polymorphonuclear leukocytes (neutrophils) in patients with localized juvenile periodontitis with decreased chemotaxis exhibit increased adherence. The molecular basis of increased adherence of neutrophils in localized juvenile periodontitis is not clear. We show that the neutrophils from localized juvenile periodontitis patients with decreased neutrophil chemotaxis and increased adherence exhibit increased expression of the CD11/CD18 family of adherence molecules, Mac-1, leukocyte function-associated antigen (LFA-1) and p150,95, as compared with neutrophils obtained from systemically and periodontally healthy controls. Treatment of neutrophils obtained from healthy subjects with sera from localized juvenile periodontitis patients resulted in an increased expression of Mac-1, leukocyte function-associated antigen and p150,95 molecules. Sera obtained from health subjects, patients with adult periodontitis or localized juvenile periodontitis patients with normal chemotaxis did not increase the expression of CD11/CD18 molecules on the neutrophils obtained from healthy subjects. The ability of localized juvenile periodontitis sera to induce expression of adherence molecules was at least partially inhibited by pretreatment of localized juvenile periodontitis sera with anti-tumor necrosis factor and anti-interleukin-1 antibodies. Furthermore, increasing concentrations of rh-tumor necrosis factor-alpha and interleukin-1 beta also induced increased expression of the Mac-1 molecule on neutrophils from localized juvenile periodontitis patients and healthy subjects. The increased expression of adherence molecules on neutrophils from localized juvenile periodontitis patients was sustained and could not be reversed by treatment of localized juvenile periodontitis neutrophils with sera obtained from healthy subjects. The upregulation of adherence molecules on neutrophils by localized juvenile periodontitis sera in these experiments emphasizes the physiologic importance of quantitatively small but biologically significant levels of cytokines in the modulation of neutrophil functions in localized juvenile periodontitis.

Adolescent↗
PubMed1997

Electrophysiologic effects of interactions between activated canine neutrophils and cardiac myocytes.

INTRODUCTION: Myocardial ischemia causes neutrophils to bind to activated myocytes and liberate platelet-activating factor (PAF). PAF causes delayed repolarization, early afterdepolarizations (EADs), and arrest of repolarization. We studied the effect of activation of neutrophils bound to canine cardiac myocytes to determine if such activation causes PAF generation and similar changes in transmembrane potentials. METHODS AND RESULTS: Myocytes from canine left ventricle and neutrophils from the same dog were superfused with Tyrode's solution and transmembrane potentials recorded from the former. Neutrophils (100 microL, 10(6)/mL) were added and allowed to bind to the myocytes. Neutrophils were activated with 1% zymosan-activated serum (ZAS). CV-6209 (100 nM) was used to block receptors for PAF. Liberation of PAF by activated neutrophils was quantified with a commercial radioimmunoassay kit. Neutrophils activated with ZAS caused changes in myocyte transmembrane potentials like those induced by PAF: action potential prolongation, runs of EAD, and periods of plateau arrest. PAF receptor blockade prevented neutrophil activation from altering transmembrane potentials. Neutrophils activated with 1% ZAS liberated significant amounts of PAF. CONCLUSIONS: When neutrophils bound to cardiac myocytes are activated by exposure to 1% ZAS, they cause prompt and consistent changes in myocyte electrical activity that could be arrhythmogenic for the in situ heart. These changes are similar to those caused by PAF in pharmacologic studies. Neutrophils activated in this manner generate PAF, and the effects of their activation are prevented by blockade of PAF receptors. We conclude that, during reperfusion of ischemic myocardium, PAF generated by activated neutrophils most likely is a cause of some arrhythmias.

Action Potentials↗
PubMed2002

Granulocyte colony-stimulating factor improves deficient in vitro neutrophil transendothelial migration in patients with advanced liver disease.

Bacterial infections are frequent complications in patients with liver cirrhosis. Cirrhotic patients present abnormalities in both innate and adaptive immune responses, including a deficient neutrophil recruitment to infected sites. The purpose of this study was to assess neutrophil-endothelium interactions in cirrhotic patients and evaluate the effects of G-CSF on this process. We studied neutrophil adhesion and transendothelial migration in 14 cirrhotic patients and 14 healthy controls. We also analyzed neutrophil expression of the adhesion molecules CD62L and CD11b in whole blood by flow cytometry. Cirrhotic patients expressed higher levels of CD11b than healthy controls, whereas CD62L expression was significantly lower, suggesting exposure of neutrophils to activating agents within the bloodstream. Neutrophils from cirrhotic patients showed increased adhesion to both resting and tumor necrosis factor alpha-stimulated microvascular endothelial cells and decreased transendothelial migration. Granulocyte colony-stimulating factor (G-CSF) (100 ng/ml) significantly enhanced neutrophil adhesion to microvascular endothelial cells in healthy controls but not in cirrhotic patients. G-CSF also significantly improved neutrophil transmigration in cirrhotic patients and healthy controls. In conclusion, cirrhotic patients exhibit increased neutrophil adhesion to microvascular endothelium and deficient transendothelial migration. G-CSF enhances neutrophil transendothelial migration in cirrhotic patients despite having no effect on neutrophil adhesion. Therefore, G-CSF may be able to increase neutrophil recruitment into infected sites in these patients.

Adult↗
PubMed2005

Isoflurane prevents platelets from enhancing neutrophil-induced coronary endothelial dysfunction.

We evaluated whether platelets can enhance polymorphonuclear neutrophil-induced coronary endothelial dysfunction, and, after observing this, whether isoflurane can prevent the effect. Neutrophils, coronary artery segments, and platelets were obtained from 25 healthy dogs. Coronary artery rings were exposed to neutrophils activated with platelet-activating factor (1.0 microM), and after washing and preconstriction with U46619, were evaluated for concentration-related responses to acetylcholine, an endothelium-dependent vasorelaxing drug. Superoxide production by activated neutrophils was measured spectrophotometrically. Adherence of the activated neutrophils to the endothelium of coronary segments was assessed by direct counting of neutrophils labeled with fluorescent dye. Measurements were performed in absence and presence of isoflurane (1 minimum alveolar concentration) both with and without platelets. The presence of platelets enhanced the neutrophil-induced rightward shift in the concentration-vasorelaxation response curve to acetylcholine (the concentration of acetylcholine required to elicit 50% of maximal relaxation (-log M) was increased from 6.78 +/- 0.7 to 5.26 +/- 0.6), and it increased superoxide oxide production from 45.0 +/- 4.2 to 54.3 +/- 4.2 nM O2-/5 x 10(6) neutrophils and adherence of activated neutrophils from 204 +/- 10 to 268 +/- 5 neutrophils/mm2. Isoflurane abolished these effects of platelets. In conclusion, platelets enhanced the ability of neutrophils to cause coronary endothelial dysfunction. This effect was prevented by isoflurane. This may be attributable to an inhibitory action on superoxide production by the neutrophils leading to reduced expression of endothelial adhesion molecules and, in turn, reduced neutrophil adherence.

Acetylcholine↗
PubMed2002

Role of galectin-3 as an adhesion molecule for neutrophil extravasation during streptococcal pneumonia.

Recruitment of neutrophils from blood vessels to sites of infection represents one of the most important elements of innate immunity. Movement of neutrophils across blood vessel walls to the site of infection first requires that the migrating cells firmly attach to the endothelial wall. Generally, neutrophil extravasation is mediated at least in part by two classes of adhesion molecules, beta(2) integrins and selectins. However, in the case of streptococcal pneumonia, recent studies have revealed that a significant proportion of neutrophil diapedesis is not mediated by the beta(2) integrin/selectin paradigm. Galectin-3 is a beta-galactoside-binding lectin implicated in inflammatory responses as well as in cell adhesion. Using an in vivo streptococcal pneumonia mouse model, we found that accumulation of galectin-3 in the alveolar space of streptococcus-infected lungs correlates closely with the onset of neutrophil extravasation. Furthermore, immunohistological analysis of infected lung tissue revealed the presence of galectin-3 in the lung tissue areas composed of epithelial and endothelial cell layers as well as of interstitial spaces. In vitro, galectin-3 was able to promote neutrophil adhesion to endothelial cells. Promotion of neutrophil adhesion by galectin-3 appeared to result from direct cross-linking of neutrophils to the endothelium and was dependent on galectin-3 oligomerization. Together, these results suggest that galectin-3 acts as an adhesion molecule that can mediate neutrophil adhesion to endothelial cells. However, accumulation of galectin-3 in lung was not observed during neutrophil emigration into alveoli induced by Escherichia coli infection, where the majority of neutrophil emigration is known to be beta(2) integrin dependent. Thus, based on our results, we propose that galectin-3 plays a role in beta(2) integrin-independent neutrophil extravasation, which occurs during alveolar infection with Streptococcus pneumoniae.

Animals↗
PubMed2003

In vivo impairment of neutrophil recruitment during lentivirus infection.

Evidence indicates that the lentivirus, HIV, infection affects neutrophil response to bacteria and bacterial products in vitro. We used a novel model of rapid onset immunosuppression following infection with a similar lentivirus, feline immunodeficiency virus (FIV), in cats to examine neutrophil function within the microvasculature in vivo and to determine the steps that are impaired in the neutrophil recruitment cascade. In uninfected cats and cats infected neonatally with FIV, the mesentery was exteriorized, but remained autoperfused during intravital microscopy for 4 h. When the tissue was superfused with 10 micro g/ml of LPS for 4 h, intravital microscopy displayed a profound increase in neutrophil rolling at both 8 and 12 wk of age in uninfected cats. At 12 wk of age, FIV-infected animals showed a profound decrease in the number of rolling neutrophils. In vitro studies revealed that neutrophils from infected and uninfected animals rolled equally well on surrogate selectin substrata. In addition, in vivo neutrophil adhesion and emigration out of the vasculature were severely reduced, and in vitro neutrophil chemotaxis from FIV-infected animals was significantly impaired in response to fMLP or IL-8. However, FIV infection of neutrophils could not be detected. In summary, in vivo lentivirus infection with immunosuppression leads to a severe impairment in neutrophil rolling, adhesion, and emigration in response to bacterial stimulants potentially involving both endothelial and neutrophil dysfunction. These in vivo studies also indicate that neutrophil dysfunction should be taken into account when treating infections and tissue injury.

Aging↗
PubMed1995

Rat, mouse and human neutrophils stimulated by a variety of activating agents produce much less nitrite than rodent macrophages.

The role of reactive nitrogen intermediates (RNI) in the antimicrobial activities of neutrophils from various mammalian species is unclear. However, it has been reported that rodent neutrophils possess the inducible form of nitric oxide synthase and that inflammatory neutrophils from rats produce potentially antimicrobial levels of RNI. In the present study, neutrophils from humans, rats and mice were evaluated for production of nitrite, a stable end-product of RNI. Human neutrophil preparations (> 95% neutrophils) isolated from peripheral blood were stimulated for 2-24 hr with agents known to trigger the Ca(2+)-dependent constitutive nitric oxide synthase, or to stimulate synthesis of the inducible nitric oxide synthase. Superoxide dismutase was added to some cultures to decrease the levels of superoxide, a compound reported to react with RNI and yield products other than nitrite. Even though the cells were viable and responsive to stimuli, they did not produce nitrite concentrations indicative of antimicrobial potential. Preparations of inflammatory (casein-elicited) mouse neutrophils also failed to produce high concentrations of nitrite. Inflammatory rat neutrophils (2.5 x 10(6)/ml) produced nitrite concentrations of approximately 40 microM in 24-hr cultures, but plots of nitrite production versus cell number for neutrophil and macrophage preparations indicated that contaminating macrophages could account for all the nitrite production in the neutrophil preparations. Thus, neutrophils from rats, mice and humans seem comparable in their inability to produce high levels of nitrite in response to a variety of stimuli. This suggests that in most circumstances the constitutive nitric oxide synthase known to be present in these cells is limited to the production of low levels of nitric oxide for intercellular signalling. In addition, this raises questions about the presence or functional status of inducible nitric oxide synthase in rodent neutrophils.

Adult↗
PubMed1995

Expression and role of adhesion molecule CD18 on bovine neutrophils.

Expression of CD18 on bovine neutrophils in response to stimulation by zymosan activated serum (ZAS) and phorbol myristate acetate (PMA) and the effects of monoclonal antibodies (MAB) recognizing CD18 or bovine neutrophil surface antigens (S2G8 and S5F8G10) on adherence, chemotactic responses and phagocytosis of bovine neutrophils were evaluated. CD18 expression of neutrophils was increased after ZAS and PMA treatment by 12.2 and 54.2% respectively, and were significantly (p < 0.05, p < 0.01) different from those of untreated neutrophils. CD18 expression by neutrophils from a Holstein-Friesian heifer affected with leukocyte adhesion deficiency was within negative controls when stimulated by ZAS and PMA. Adherence, chemotactic responses, and phagocytosis were significantly decreased (p < 0.01) in neutrophils continuously treated with anti-CD18 MAB (MHM 23). Adherence was also significantly decreased in anti-CD18 pretreated neutrophils. Significant (p < 0.01) differences of chemotactic responses and phagocytosis of neutrophils were found between neutrophils pretreated and continuously treated with anti-CD18 MAB (MHM 23). Monoclonal antibodies to other surface antigens did not significantly alter neutrophil adherence, chemotaxis or phagocytosis. This study demonstrated that CD18 expression on bovine neutrophils is increased significantly by stimulation with ZAS and PMA and that the adhesion molecule CD18 plays an important role in adhesion-related functions.

Animals↗
PubMed1997

Intracellular pool of vascular endothelial growth factor in human neutrophils.

Vascular endothelial growth factor (VEGF ), an endothelial cell mitogen, is a potent angiogenic factor produced by several cell types. Whether human neutrophils are potential producers of VEGF has not yet been described. The present work shows that phorbol-12-myristate 13-acetate (PMA), fMet-Leu-Phe, and tumor necrosis factor-alpha (TNF-alpha) triggered a time-dependent secretion of VEGF by human neutrophils. Cells incubated with 50 ng/mL of PMA released significant amounts of VEGF after 15 minutes. Because the extracellular content of VEGF in human neutrophils supernatants remained constant over a period of 2 to 24 hours and because PMA is a potent inducer of human neutrophil degranulation, the PMA-induced secretion of VEGF may be due to a pre-existing intracellular pool of this molecule. This hypothesis was reinforced by the absence of cycloheximide effect on the PMA-induced secretion of VEGF. The existence of an intracellular pool of VEGF was confirmed by measuring the intracellular content of VEGF in resting neutrophils. A dosedependent inhibition of PMA-induced VEGF secretion was observed when the cells were incubated in the presence of pentoxifylline, a methylxanthine known to inhibit neutrophil degranulation. To confirm the implication of neutrophil degranulation in VEGF release, the effects of two inducers of physiologic degranulation, fMet-Leu-Phe and TNF-alpha, were determined. Both agonists induced a release of VEGF in the absence of cytochalasin B, confirming the involvement of neutrophil degranulation and suggesting the intracellular localization of VEGF in the specific granule fraction. In addition, the kinetics of fMet-Leu-Phe- and TNF-alpha-induced secretion of lactoferrin were similar to those of VEGF release induced by these two both agonists. The subcellular fractionation of human neutrophils showed a granule-specific distribution of the intracellular pool of VEGF in resting neutrophils. The finding that human neutrophils contain an intracellular pool of VEGF, secreted in the extracellular space under PMA-, fMet-Leu-Phe-, and TNF-alpha-induced degranulation, suggests a role for human neutrophils as cellular effectors of physiologic as well as pathologic angiogenesis.

Cells, Cultured↗
PubMed1991

Tumor necrosis factor and endotoxin can cause neutrophil activation through separate pathways.

We investigated the possibility that tumor necrosis factor (TNF) mediates neutrophil activation by endotoxin. The number of C3b receptors on the neutrophil cell-surface was used as the indicator of activation, as assessed by indirect immunofluorescence. Incubation of buffy-coat neutrophils with TNF-alpha for 30 minutes at 37 degrees C caused neutrophil activation, increasing C3b receptor-dependent fluorescence from 340 with buffer alone to 580 with TNF (250 pg/mL). Increasing amounts of anti-TNF IgG progressively inhibited neutrophil activation by TNF (250 pg/mL). Addition of the active dose range of anti-TNF to neutrophils incubating in endotoxin (10 ng/mL) did not affect the degree of endotoxin-mediated neutrophil activation. Mixtures of neutrophils with the 50% suppressive dose of anti-TNF and varying endotoxin concentrations showed the same degree of neutrophil activation as mixtures without the antibody. Thus, an antibody that can inhibit TNF-mediated neutrophil activation does not inhibit endotoxin-mediated neutrophil activation. We conclude that endotoxin and TNF can activate neutrophils through separate pathways.

Antibodies↗
PubMed2003

Synovial fluid neutrophils transcribe and express class II major histocompatibility complex molecules in rheumatoid arthritis.

OBJECTIVE: To investigate a potential interaction between neutrophils and T cells in rheumatoid arthritis (RA), by defining the optimal conditions for induction of class II major histocompatibility complex (MHC) expression on peripheral blood neutrophils in vitro and investigating the capacity for neutrophils to express class II MHC molecules in RA. METHODS: Surface expression of class II MHC and costimulatory molecules by peripheral blood and synovial fluid (SF) neutrophils obtained from healthy controls and patients with RA was measured by flow cytometry and fluorescence microscopy. Intracellular class II MHC protein and messenger RNA (mRNA) were detected by Western blotting and Northern blotting, respectively. RESULTS: Freshly isolated peripheral blood neutrophils from controls did not express surface class II MHC; expression was induced by culture with appropriate cytokines. Freshly isolated peripheral blood neutrophils from patients with RA expressed mRNA, but there was no surface expression of class II MHC. Freshly isolated SF neutrophils from patients with RA contained high levels of class II MHC mRNA, did not express surface class II MHC, but did have large intracellular amounts of this protein as detected by Western blotting. After culture for 20 hours in vitro, SF neutrophils from RA patients expressed large amounts of surface class II MHC but very low levels of costimulatory molecules CD80 and CD86. Fluorescence microscopy localized surface class II MHC to discrete areas on the neutrophil. Class II MHC-expressing neutrophils stimulated T cell proliferation. CONCLUSION: Peripheral blood neutrophils from patients with RA but not healthy controls express class II MHC mRNA. SF neutrophils in RA synthesize and express large amounts of class II MHC but not costimulatory molecules. This might underlie a novel interaction with T cells that is important in terms of disease pathology.

Arthritis, Rheumatoid↗
PubMed1992

Platelet-activating factor inhibits proteoglycan synthesis and enhances neutrophil-mediated proteoglycan degradation in cartilage explants.

OBJECTIVE: Platelet-activating factor (PAF), which stimulates the release of tissue-destructive enzymes and reactive oxygen metabolites from neutrophils, was investigated for its role in neutrophil-mediated cartilage breakdown. METHODS: Bovine cartilage explants were incubated with or without human neutrophils, PAF, and other reagents. Cartilage damage was measured as either proteoglycan degradation (percent release of 35S-labeled proteoglycan from 35S-labeled cartilage) or inhibition of proteoglycan synthesis (rate of incorporation of 35S into proteoglycan). RESULTS: PAF increased neutrophil-mediated proteoglycan degradation in the 2-20 microM range. Three specific PAF-receptor antagonists, WEB2086, CV3988, and CV6209, reversed this effect of PAF. These antagonists also reduced the enhancement of neutrophil-mediated cartilage damage caused by granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF alpha). The results suggest that there may be a positive feedback mechanism whereby cytokine-primed neutrophils produce PAF, which amplifies the release of other tissue-damaging substances from neutrophils. In the absence of neutrophils, PAF (2-20 microM) inhibited the synthesis of proteoglycan by bovine cartilage. Neutrophils also inhibit proteoglycan synthesis, but PAF probably is not involved in this effect of neutrophils because the PAF receptor antagonists had no consistent effect. CONCLUSION: PAF increases neutrophil-mediated cartilage proteoglycan degradation in vitro. GM-CSF and TNF alpha enhancement of neutrophil damage to cartilage is partly due to PAF. PAF alone inhibits cartilage proteoglycan synthesis.

Animals↗
PubMed1988

Thrombin-induced adherence of neutrophils to cultured endothelial monolayers: increased endothelial adhesiveness.

We examined the effects of alpha-thrombin on the adherence of neutrophils to endothelial cell monolayers. Endothelial cells derived from the ovine pulmonary artery and ovine neutrophils were used. Thrombin (10(-8) M) resulted in a time-dependent increase in neutrophil adherence to the endothelium. The response was concentration-dependent with a maximal response at 10(-8) M. Thrombin did not induce neutrophil adherence either to plastic or to endothelial cell-derived matrix. The adherence response was inhibited in the presence of alpha-thrombin that had been inactivated with anti-thrombin III (1U:1U) or with hirudin (1 U/ml). However, the addition of either anti-thrombin III or hirudin simultaneously with alpha-thrombin to the cultured endothelial monolayers did not prevent neutrophil adherence. The monoclonal antibody MoAb 60.3, which precipitates a complex of four neutrophil surface glycoproteins (CDw18) was used to further characterize the reaction. MoAb 60.3 decreased the thrombin-induced adherence of neutrophils to the endothelial monolayer. Addition of 10(-8) M thrombin to the endothelial monolayer for 60 min, followed by washing the endothelium with fresh medium, caused resting neutrophils to adhere to the endothelial monolayers. MoAb 60.3 decreased neutrophil adherence to the washed endothelium. The factor(s) responsible for adherence was partially transferable. Medium obtained from incubating endothelial monolayers with thrombin (10(-8) M) for 60 min, adding hirudin to the medium to inactivate thrombin, and transferring it to untreated endothelial monolayers, elicited neutrophil adherence. The response was less than that obtained with thrombin alone (22.9 +/- 2.3% vs. 12.9 +/- 3.3%). The results indicate that the catalytic site of the thrombin molecule is responsible for the adherent activity. Thrombin elicits a rapid activation of endothelial cells with a response that involves the expression of endothelial adhesion sites and sites that interact with the neutrophil CDw18 adhesive glycoprotein complex. In addition, soluble transferable factor(s) which are generated by the endothelium also contribute to thrombin-induced neutrophil adherence.

Animals↗
PubMed1993

Flow cytometry reveals different lag times in rapid cytoplasmic calcium elevations in human neutrophils in response to N-formyl peptide.

Flow cytometric analyses were performed to study intracellular single-cell calcium transients ([Ca2+]i) in suspended human neutrophils during the initial phase of N-formyl peptide stimulation. Thereby, two neutrophil populations became apparent. Early maximally Ca(2+)-responding (high fluorescence) neutrophils and not-yet Ca(2+)-responding (low fluorescence) neutrophils, but no neutrophils with intermediate levels of [Ca2+]i were detected. Within 7 s the number of low fluorescence neutrophils decreased and the number of high fluorescence neutrophils increased maximally. This suggests that [Ca2+]i transients occurred abruptly in individual neutrophils within a time interval below 1 s. At lower N-formyl peptide concentrations the lag times of individual neutrophils and the interval time of maximal activation of the [Ca2+]i-responding neutrophil population increased, however the percentage of [Ca2+]i-responding cells decreased. Surprisingly, no influence of the N-formyl peptide concentration on the [Ca2+]i-induced fluorescence signal of the individual cell was observed: it was always in an almost maximal range or not responding. In parallel, binding studies performed with fluorescein-labeled N-formyl peptide revealed that the heterogeneity of [Ca2+]i-responding cells cannot be explained by different receptor occupancy. In summary, this study demonstrates that [Ca2+]i transients induced by N-formyl peptides in suspended individual human neutrophils occur very rapidly in an almost "all-or-none manner" and that the mean increasing fluorescence signal of a calcium indicator within a whole neutrophil population results from varying lag times of the individual cells, rather than from the mean simultaneous progress of many cells.

Calcium↗