Cannabinoid receptor 2 deficiency exacerbates inflammation and neutrophil recruitment.

Cannabinoid receptor (CB)2 is an immune cell-localized GPCR that has been hypothesized to regulate the magnitude of inflammatory responses. However, there is currently no consensus as to the mechanism by which CB2 mediates its anti-inflammatory effects in vivo. To address this question, we employed a murine dorsal air pouch model with wild-type and CB2-/- 8-12-wk-old female and male C57BL/6 mice and found that acute neutrophil and lymphocyte antigen 6 complex, locus Chi monocyte recruitment in response to Zymosan was significantly enhanced in CB2-/- mice. Additionally, levels of matrix metalloproteinase 9 and the chemokines C-C motif chemokine ligand (CCL)2, CCL4, and C-X-C motif chemokine ligand 10 in CB2-/- pouch exudates were elevated at earlier time points. Importantly, using mixed bone marrow chimeras, we revealed that the proinflammatory phenotype in CB2-/- mice is neutrophil-intrinsic rather than stromal cell-dependent. Indeed, neutrophils isolated from CB2-/- mice exhibited an enhanced migration-related transcriptional profile and increased adhesive phenotype, and treatment of human neutrophils with a CB2 agonist blocked their endothelial transmigration. Overall, we have demonstrated that CB2 plays a nonredundant role during acute neutrophil mobilization to sites of inflammation and, as such, it could represent a therapeutic target for the development of novel anti-inflammatory compounds to treat inflammatory human diseases.-Kapellos, T. S., Taylor, L., Feuerborn, A., Valaris, S., Hussain, M. T., Rainger, G. E., Greaves, D. R., Iqbal, A. J. Cannabinoid receptor 2 deficiency exacerbates inflammation and neutrophil recruitment.

Origin-Specific Adhesive Interactions of Mesenchymal Stem Cells with Platelets Influence Their Behavior After Infusion.

We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC-2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC-2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC-2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin-induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018;36:1062-1074.

A role for the integrin alpha6beta1 in the differential distribution of CD4 and CD8 T-cell subsets within the rheumatoid synovium.

OBJECTIVE: CD4 and CD8 T-cell subsets accumulate in distinct microdomains within the inflamed rheumatoid synovium. The molecular basis for their differential distribution remains unclear. Since chemokines and adhesion molecules play an important role in the positioning of leucocytes at sites of inflammation, we tested the hypothesis that the differential expression and function of chemokine and/or adhesion molecules explains why CD4(+) T cells accumulate within perivascular cuffs, whereas CD8(+) T cells distribute diffusely within the tissue. METHODS: Expression of an extensive panel of chemokine receptors and adhesion molecules on matched CD4(+) and CD8(+) T cells from peripheral blood (PB) and synovial fluid (SF) was analysed by multicolour flow cytometry. Migration assays and flow-based adhesion assays were used to assess the functional consequences of any differences in the expression of chemokine and adhesion receptors. RESULTS: CD4(+) and CD8(+) T cells from PB and SF expressed unique yet consistent patterns of chemokine and adhesion receptors. SF CD8(+) T cells were much less promiscuous in their expression of chemokine receptors than SF CD4(+) T cells. The alpha(6)beta(1) integrin was highly expressed on PB CD4(+) T cells, but not on PB CD8(+) T cells. Laminin, the ligand for alpha(6)beta(1), retained CD4(+) T cells, but less so CD8(+) T cells, within inflamed synovial tissue. CONCLUSION: Infiltrating PB CD4(+) T cells, but not CD8(+) T cells, express functional levels of the alpha(6)beta(1) integrin. We propose that this leads to their retention within the rheumatoid synovium in perivascular cuffs, which are defined and delineated by the expression of laminin.

Cross-talk between cell adhesion molecules regulates the migration velocity of neutrophils.

BACKGROUND: Although the adhesive mechanisms underlying the capture and immobilization of circulating neutrophils in inflamed blood vessels have been well described, factors controlling the subsequent migration of neutrophils over and through the blood vessel endothelium are poorly understood. Directional rearrangement of the actin cytoskeleton within the neutrophil, along with modulation of integrin-mediated adhesion, are necessary for neutrophil migration. Signals from chemotactic agents and from the adhesive substrate may regulate these processes, but little is known about their relative importance or their mode of integration. RESULTS: We examined the kinetics of neutrophil migration after formyl tripeptide or platelet-activating factor was perfused over neutrophils that were already rolling on the adhesion molecule P-selectin, which was presented either on the surface of immobilized platelets or in purified form coated on glass capillaries. Upon activation, neutrophils stopped rolling, spread and began to migrate; each of these processes was dependent on beta2 integrin (CD11b/CD18). The rate of migration increased over a period of about 8 minutes and was modulated directly by both the P-selectin and the CD31 surface receptors. Antibody blockade of either CD31 or P-selectin on platelets resulted in a reduction in the velocity of migration, and simultaneous blockade of both receptors reduced velocity further. Purified CD31 and P-selectin (but not a control adhesion molecule, ICAM-1) increased migration velocity in a concentration-dependent and additive manner that reconstituted the migratory behaviour observed on platelets. CONCLUSIONS: These studies show that binding of ligands to CD31 and/or P-selectin modifies the rate of integrin-supported neutrophil migration. This novel example of 'cross-talk' between surface receptors suggests that cell adhesion molecules might generally transduce accessory signals between adjacent cells to modify their migratory responses to chemotactic signals.

Comparison of the pro-inflammatory potential of monocytes from healthy adults and those with peripheral arterial disease using an in vitro culture model.

We adapted a monocyte:endothelial cell co-culture model to investigate the pro-inflammatory potential of monocytes from patients with peripheral arterial disease (PAD). Isolated monocytes were cultured with human umbilical vein endothelial cells (HUVEC) for 24h, after which the ability of the HUVEC to recruit flowing neutrophils was tested. Development of a usable protocol required comparisons of primary HUVEC with cells that had been passaged and/or frozen and thawed, evaluation of optimal culture media and comparison of monocytes from freshly drawn and stored blood. We found, for instance, that expansion of HUVEC was assisted by inclusion of hydrocortisone, but this agent was withdrawn before the test phase because it reduced responses of HUVEC. Using the optimal practical protocol, we found great variation in the ability of monocytes from different donors to cause neutrophil adhesion. Slightly more ( approximately 20%) monocytes from patients with PAD adhered to HUVEC than monocytes from healthy controls, and the monocytes from PAD patients induced approximately 70% greater subsequent adhesion of neutrophils. Thus, we developed a functional model of inflammatory potential usable in clinically-related studies and found that patients with PAD had circulating monocytes with greater than normal ability to activate endothelial cells.

Cellular pathology of atherosclerosis: smooth muscle cells prime cocultured endothelial cells for enhanced leukocyte adhesion.

During the development of an atherosclerotic plaque, mononuclear leukocytes infiltrate the artery wall through vascular endothelial cells (ECs). At the same time, arterial smooth muscle cells (SMCs) change from the physiological contractile phenotype to the secretory phenotype and migrate into the plaque. We investigated whether secretory SMCs released cytokines that stimulated ECs in a manner leading to increased leukocyte recruitment and thus might accelerate atheroma formation. SMCs and ECs were established in coculture on the opposite sides of a porous membrane, and the cocultured cells were incorporated into a flow-based assay for studying leukocyte adhesion. We found that coculture primed ECs so that their response to the inflammatory cytokine tumor necrosis factor-alpha was amplified. ECs cocultured with SMCs supported greatly increased adhesion of flowing leukocytes and were sensitized to respond to tumor necrosis factor-alpha at concentrations 10 000 times lower than ECs cultured alone. In addition, coculture altered the endothelial selectin adhesion molecules used for leukocyte capture. EC priming was attributable to the cytokine transforming growth factor-beta(1), which was proteolytically activated to a biologically active form by the serine protease plasmin. These results suggest a new role for secretory SMCs in the development of atheromatous plaque. We propose that paracrine interaction between ECs and SMCs has the potential to amplify leukocyte recruitment to sites of atheroma and exacerbate the inflammatory processes believed to be at the heart of disease progression.

Eicosanoid generation and effects on the aggregation of thrombocytes from the rainbow trout, Oncorhynchus mykiss.

Fish blood lacks anucleate platelets but contains a nucleated cell type termed the thrombocyte that is thought to be functionally analogous. Thrombocytes were purified from the peripheral blood of the rainbow trout, Oncorhynchus mykiss, by a two step gradient centrifugation method. Following this procedure, the recovered thrombocytes were 78-86% pure as defined by immunoreactivity to a panel of monoclonal antibodies and were of variable morphology from round to spindle-shaped. Incubation of thrombocyte suspensions with either calcium ionophore, A23187, platelet-activating factor or a thromboxane (TX) mimetic, U-46619, generated a range of eicosanoids derived from arachidonic acid including 12-hydroxyeicosatetraenoic acid (12-HETE), TXB2, prostaglandin (PG) E2, leukotriene (LT) B4 and lipoxin (LX) A4. The equivalent products derived from eicosapentaenoic acid were also formed. Co-incubation of thrombocytes with either erythrocytes or granulocytes/monocytes in the presence of calcium ionophore did not result in the formation of any further new lipoxygenase products. Incubation of isolated thrombocytes in plasma-free conditions with U-46619 (0.03-10 microM) resulted in a rapid, dose-dependent aggregatory response. This effect was markedly augmented in the presence of mammalian fibrinogen (400 micrograms ml-1). Thrombin (0.1-1.3 units ml-1), like U-46619, was also a potent proaggregatory compound for trout thrombocytes. LXA4 and LTB4 had limited aggregatory potential and then only at high concentrations (10 microM), while 12-HETE and PAD had no significant effect at all concentrations tested. These results demonstrate that some of the eicosanoids released during the activation of trout thrombocytes are involved in the aggregatory behaviour of this cell type.

A novel system for investigating the ability of smooth muscle cells and fibroblasts to regulate adhesion of flowing leukocytes to endothelial cells.

Stromal cells may contribute to the inflammatory processes which lead to the recruitment of circulating leukocytes. Here, we describe a multicellular model in which chosen cellular elements of tissue can be cocultured with endothelial cells (EC). Cocultures can be incorporated into a novel parallel plate flow chamber to determine if stromal cells influence the patterns of leukocyte adhesion to the EC. As an example relevant to the pathology of atherosclerosis, EC were cultured with arterial smooth muscle cells (SMC) of the 'secretory' phenotype. EC and secretory SMC were cultured on the opposite faces of commercially available porous polyethylene terepthalate (PET) culture inserts, which fitted into a parallel plate flow chamber. Binding of flowing purified lymphocytes, labelled with the fluorochrome calcein-AM, to cocultured EC was assessed by fluorescence microscopy. Lymphocyte adhesion was negligible on unstimulated EC cultured alone or cocultured with SMC. However, when tumour necrosis factor-alpha (TNF) was added to cocultures, the EC supported greatly increased levels of lymphocyte adhesion compared to TNF-treated EC cultured alone. Additionally, cocultured EC responded to TNF at concentrations far below those at which EC cultured alone responded. This priming was specific in that skin fibroblasts cocultured with EC did not modify lymphocyte adhesion induced by TNF. Thus, we have developed a coculture model to determine the ability of tissue stromal cells to modify leukocyte recruitment. This may have wide applications in the study of the cellular pathology of inflammation by allowing the contribution of the local microenvironment to be assessed.

Effects of fluorescent dyes on selectin and integrin-mediated stages of adhesion and migration of flowing leukocytes.

Fluorescent dyes assist visualisation of leukocytes for intravital studies of adhesion or for in vitro studies utilising whole blood. We have used in vitro flow-based assays to investigate the effects of three fluorescent dyes (acridine orange, AO, 5-100 microg/ml; calcein-AM, C-AM, 5-20 microg/ml; rhodamine 6G, R6G, 10-100 microg/ml) on adhesion and migration of isolated neutrophils and mononuclear cells. AO had little effect on the number or velocity of neutrophils rolling on P-selectin presented by a surface coated with platelets. However, AO did cause a dose- and time-dependent conversion of rolling to immobilisation. Pretreatment of neutrophils with an antibody against CD18 prevented this conversion to stationary adhesion, indicating that beta(2) integrins were activated by AO. C-AM had little effect on neutrophil behaviour, but tended to cause some immobilisation at the highest concentration. R6G did not affect the number of neutrophils that bound to the platelet monolayer or the percentage rolling, but the rolling velocity of the neutrophils was increased in a dose-dependent manner. None of the dyes impaired the ability of neutrophils to respond to formyl peptide by converting from rolling to stationary adhesion. Neither C-AM nor R-6G reduced the number of flowing neutrophils or mononuclear cells binding to endothelial cells stimulated with tumour necrosis factor. Interestingly, R-6G inhibited transendothelial migration of mononuclear cells but not neutrophils, while C-AM did not affect transmigration of either cell type. The dose-dependent effects of dyes should be taken into consideration when designing any experimental protocol. AO does not appear to be a suitable dye for adhesion studies. R6G and C-AM can be used at approximately 10 microg/ml (a concentration at which cells can be clearly visualised) although R-6G specifically inhibits the migratory response of mononuclear cells.

Neutrophils rolling on immobilised platelets migrate into homotypic aggregates after activation.

Interactions between platelets and leucocytes are implicated in the pathology of thrombotic vascular disease. Using a flow-based adhesion assay we have investigated a novel route for the formation of neutrophil aggregates on the surface of immobilised activated platelets. Neutrophils perfused over a platelet monolayer formed numerous rolling attachments but rapidly stopped and spread after the superfusion of N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor (both at 10(-7) M). Subsequent integrin-mediated migration across the platelet monolayer enabled formation of homotypic neutrophil aggregates, which was significant within 2.5 min of receipt of either stimulus. Aggregates increased in size with time and had an average projected area of approximately 500 microm2 after 10 min. Increasing size was correlated with an increasing tendency for movement downstream and large aggregates sometimes tumbled in that direction. The formation and stability of homotypic aggregates was dependent on several adhesive mechanisms. Antibody blockade demonstrated that interactions involving CD11a/CD18 and ICAM-3, between alpha(v)beta3-integrin and CD31 and between L-selectin and an unidentified counter-ligand were all required for the complete aggregatory response. Furthermore, blockade of L-selectin allowed initial aggregation which then reversed, suggesting that this receptor might regulate the interactions between other adhesion molecules that directly supported cell-cell adhesion. We propose that this novel route for leucocyte aggregation could promote vascular occlusion in thrombotic vessels or at distal sites in the event of embolisation.

Adhesion of tumor cells to matrices and endothelium.

Cell adhesion is a process fundamental to tumor metastasis. Egress of cells from tumors and their entry into secondary tissues requires the regulated adhesion that underlies the process of cell migration. Thus, adhesion molecules must bind and release counter-receptors on the adhesive substrate in a controlled manner that permits locomotion (1-3). These same molecules must also interact in a complex manner with the intracellular actin cytoskeleton that is the motor for migration (1-3). In addition, it is now clear that there are interactions between adhesive counter receptors that do not mediate primary attachment to a substrate or support migration but are used to sample the adhesive microenvironment. Integration of "adhesive" signals with those from growth factors and cytokines may be used to regulate the pathophysiological responses of cells (1,4,5).

Generation and characterisation of monoclonal antibodies against rainbow trout, Oncorhynchus mykiss, leucocytes.

Monoclonal antibodies were raised against head kidney macrophages of the rainbow trout, Oncorhynchus mykiss. Despite the establishment of a significant number of different hybridoma clones, none of these released antibody specific for determinants only found on macrophages. Instead, all the monoclonal antibodies generated reacted with lymphocytes, granulocytes, and monocytes/macrophages, although thrombocytes (the platelet equivalents in fish) and erythrocytes were not recognized by these antibodies. Western blotting of solubilised macrophages revealed that two of the hybridoma lines, designated 21G6 and 21F11, reacted with at least five proteins of 80, 104, 110, 140, and > 170 kDa. Immunocytochemistry was performed on histological sections of trout alimentary canal, gill, liver, spleen, and haemopoietic head kidney using antibodies from several of the hybridoma lines, and all of these showed a similar pattern of reactivity in each tissue. In the alimentary canal, for example, immunoreactive material was found in the eosinophilic granular cells, blood vessel margins, mucus in the lumen, and in the columnar epithelial cells. In the gills, epithelial cells and blood vessels also showed intense immunoreactive products, while in the liver, such reactivity was localised in the sinusoids and adherent macrophages. Both the spleen and head kidney had largely homogenous immunoreactivity.

Monocytes are a rheologically heterogeneous population of cells.

The effect of increased adhesiveness and decreased deformability of leukocytes following activation can have a profound effect on flow through the microcirculation. Measurement of leukocyte deformability is therefore an important tool in the study of the pathology of vascular diseases. Although much work has been done on the rheological properties of lymphocytes and granulocytes, there is little information available on the larger mononuclear cells, the monocytes. To investigate monocyte rheology, attempts were made to purify monocytes by a variety of methods. Purified monocytes were then filtered through 5 microm polycarbonate filters, with flow profiles (change in volume with time) recorded over 300 seconds. The profiles were analysed by least squares fitting to an appropriate mathematical model. Analysis of filtration data demonstrated 3 distinct sub-populations of monocytes with differing rheological properties. Other workers have characterised monocytes into defined subsets on the basis of their size, phagocytic ability or expression of cell surface markers. The definition of monocytes into defined rheological subsets is a new and useful addition to these studies.

Modulation of endothelial responses by the stromal microenvironment: effects on leucocyte recruitment.

Vascular endothelial cells play a pivotal role in regulating leucocyte recruitment during inflammation, and their responses can be modulated by their local environment, including cells of the tissue stroma. We have developed a model system to examine how the communication between endothelial cells and fibroblasts regulates the recruitment of leucocytes and their subsequent subendothelial fate. Here, we describe a novel co-culture filter-based flow assay and highlight the ability of synovial fibroblasts obtained from chronically inflamed tissue to promote leucocyte recruitment to otherwise 'resting' endothelial cells.

Mediation of endothelial cell damage by serine proteases, but not superoxide, released from antineutrophil cytoplasmic antibody-stimulated neutrophils.

OBJECTIVE: To evaluate potential mediators of endothelial cell injury in systemic vasculitis associated with antineutrophil cytoplasmic antibodies (ANCAs), we investigated the factors controlling the neutrophil respiratory burst and endothelial release of von Willebrand factor (vWF) during neutrophil-endothelial cell interactions. METHODS: Superoxide release from neutrophils binding to purified P-selectin or to tumor necrosis factor-activated endothelial cells was measured under flow or static conditions using the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c. Neutrophils were activated with fMLP, normal IgG, or ANCA IgG. Enzyme-linked immunosorbent assay was used to measure vWF. Serine protease activity was measured enzymatically. RESULTS: ANCA IgG or fMLP induced superoxide release when perfused over neutrophils that were rolling over P-selectin, but not those that were binding to endothelial cells. In static assays, endothelial cells inhibited superoxide production by neutrophils. Adenosine inhibited the respiratory burst, and, in cocultures, adenosine deaminase overcame the inhibitory effects of endothelial cells. Serine proteases were released during activated neutrophil-endothelial cell coculture. There was enhanced release of vWF during activated neutrophil-endothelial cell coculture; this was not inhibited by diphenyleneiodonium or by SOD plus catalase, but was inhibited by diisopropylfluorophosphate. CONCLUSION: Endothelial cells inhibit superoxide generation by fMLP and ANCA-activated neutrophils. The release of vWF occurs during coculture and is sensitive to serine protease, but not NADPH oxidase inhibition. Serine proteases may play a more important role than reactive oxygen species as mediators of endothelial injury during ANCA-associated systemic vasculitis.

Endothelial-borne platelet-activating factor and interleukin-8 rapidly immobilize rolling neutrophils.

The kinetics of the response of integrins to activating signal(s) must be rapid to ensure that rolling neutrophils are localized at the sites of inflammation. From video records, we analyzed the adhesion of individual neutrophils in a flow-based in vitro model of endothelial hypoxia and reoxygenation. There were numerous rolling interactions between flowing neutrophils and P-selectin on human umbilical vein endothelial cells after hypoxia, but 90% lasted for < 1 s, with approximately 30% converted to stationary attachment via beta 2-integrin(s). Interleukin-8 (IL-8) and platelet-activating factor (PAF) were responsible for neutrophil activation in this model [G. E Rainger, A. Fisher, C. Shearman, and G. B. Nash. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1398-H1406, 1995]. In the presence of a PAF-receptor antagonist, IL-8 acting alone induced conversion of rolling to stationary adhesion in as little as 80 ms after the initial attachment of a neutrophil, with a median response time of 240 ms. In the presence of a monoclonal antibody that neutralized IL-8 activity, PAF acting alone required a minimum duration of rolling of 560 ms to promote stationary adhesion, with a significantly longer median duration of 720 ms. In a reconstituted model, treatment of endothelial cells with hydrogen peroxide induced short-lived rolling of neutrophils supported by P-selectin. Exogenously added IL-8 and/or PAF bound to the endothelial surface and successfully induced the immobilization of neutrophils. Rapid and distinct kinetics of the conversion to stationary adhesion were observed again for IL-8 or PAF. Thus although endothelial-presented signals differed in their rate of action, neutrophils could be localized within one or two endothelial cell diameters of their initial adhesive contact point.

Differential ability of exogenous chemotactic agents to disrupt transendothelial migration of flowing neutrophils.

Neutrophils migrate through endothelium using an ordered sequence of adhesive interactions and activating signals. To investigate the consequences of disruption of this sequence, we characterized adhesion and migration of neutrophils perfused over HUVEC that had been treated with TNF-alpha for 4 h and evaluated changes caused by exogenously added chemotactic agents. When HUVEC were treated with 2 U/ml TNF, flowing neutrophils adhered, with the majority rolling and relatively few migrating through the monolayer. If fMLP, IL-8, zymosan-activated plasma (a source of activated complement factor C5a), epithelial cell-derived neutrophil-activating peptide (ENA-78), or growth-regulating oncogene, GRO-alpha, was perfused over these neutrophils, they stopped rolling and rapidly migrated over the monolayer, but did not penetrate it. When HUVEC were treated with 100 U/ml TNF, the majority of adherent neutrophils transmigrated. If neutrophils were treated with fMLP, IL-8, C5a, ENA-78, or GRO-alpha just before perfusion over this HUVEC, transmigration, but not adhesion, was abolished. However, when platelet-activating factor was used to activate neutrophils, migration through HUVEC treated with 100 U/ml TNF was not impaired, and migration through HUVEC treated with 2 U/ml TNF was actually increased. Transmigration required ligation of CXC chemokine receptor-2 on neutrophils, and differential desensitization of this receptor (e.g., by fMLP but not platelet-activating factor) may explain the pattern of disruption of migration. Thus, transmigration may require presentation of the correct activators in the correct sequence, and inappropriate activation (e.g., by systemic activators) could cause pathological accumulation of neutrophils in the vessel lumen.

Kinetics of the different steps during neutrophil migration through cultured endothelial monolayers treated with tumour necrosis factor-alpha.

To enable a better understanding of the regulation of neutrophil migration, we investigated the kinetics of adhesion and migration over, through and under endothelial monolayers. Neutrophils were perfused over human umbilical vein endothelial cells (HUVEC) which had been treated with tumour necrosis factor-alpha (TNF; 2-1,000 U/ml) for 4 h. Videomicroscopy showed that transendothelial migration was complete within about 5 min of completion of perfusion of a bolus of neutrophils. Separate populations of adherent cells could then be observed, either rolling, migrating over the surface of the HUVEC or migrating underneath, at different characteristic speeds. Increasing concentration of TNF had little effect on the kinetics of migration, but shifted the balance from rolling adhesion to transendothelial migration. When individual neutrophils were followed from the moment they bound to HUVEC treated with 100 U/ml TNF, we found that approximately 40% immobilised essentially immediately on contact, while approximately 40% immobilised after rolling for varying periods (average 26 s) and approximately 20% rolled continuously. Most of the immobilised cells went on to migrate through the monolayer after spending 20-200 s migrating on top, and took about 60 s to pass through. Overall, the time from first binding to completion of transmigration averaged 152 s (range approximately 60-240 s). Interestingly, neutrophils moved relatively slowly on top of the monolayer (about 8 microm/min) but more rapidly underneath (about 16 microm/min). We suggest that the different stages during neutrophil transmigration have characteristic kinetics with separate control mechanisms, which critically influence the efficiency and rate of clearance from the vasculature.

Adhesion-dependent release of elastase from human neutrophils in a novel, flow-based model: specificity of different chemotactic agents.

Neutrophils must adhere to the vessel wall, migrate, and degranulate in an ordered manner to perform their protective function. Disruption of these processes may be pathogenic. Current knowledge of the degranulation process is derived almost exclusively from studies on neutrophils in suspension, in which priming with the nonphysiological agent cytochalasin B is necessary to obtain elastase release in response to activating agents. To avoid this, we have adopted a different approach. Using a novel flow-based adhesion system, we have been able to quantify the release of elastase from the primary granules of activated neutrophils adherent to immobilized platelets or purified receptors without priming. Comparing stimuli, formyl tripeptide (fMLP), interleukin-8 (IL-8), activated complement fragment C5a, and platelet-activating factor (PAF) all induced rapid conversion to CD11b/CD18 (MAC-1) -mediated stationary adhesion when perfused over neutrophils already rolling on platelet monolayers or purified P-selectin. However, fMLP, C5a, and IL-8, but not PAF, induced release of elastase from the adherent cells in minutes. Neutrophils stimulated in suspension showed little degranulation. Treatment of neutrophils with an inhibitor of 5-lipoxygenase-activating protein (MK886) and thus synthesis of leukotrienes (LTs) or with an antagonist of the LTB4 receptor (LY223982) blocked the release of elastase. This indicated that endogenous synthesis of 5-lipoxygenase products such as LTs and autocrine activation of neutrophils was required for fMLP-driven elastase release. We hypothesize that the differential ability of PAF and fMLP to induce elastase release from surface-adherent neutrophils could arise from differential ability to generate leukotrienes, such as LTB4, and would be an appropriate mechanism for the control of elastase release during inflammation in vivo, where it is important that cytotoxic agents are not released until activated neutrophils have migrated into the extravascular tissues.

Effects of pentoxifylline on the different steps during adhesion and transendothelial migration of flowing neutrophils.

We used a flow system to observe the stepwise adhesion and migration of neutrophils on cultured human umbilical vein endothelial cells (HUVEC) stimulated with tumour necrosis factor-alpha (TNF) for 4 h, and to evaluate the effects of pentoxifylline (PTX) at each step. When HUVEC had been stimulated with 100 U ml(-1) TNF, treatment of neutrophils with PTX did not reduce the number captured from flow but did cause nearly all adherent cells ( > 90%) to roll, whereas most untreated cells became immobilized and approximately 30% transmigrated within minutes. On washout of the PTX, many rolling cells halted and started to migrate. Treatment of the HUVEC with PTX at the same time as 100 U ml(-1) TNF did not affect the number of neutrophils adhering, but there was a significant increase in the percentage of cells rolling even though PTX was no longer present. Thus PTX reduced presentation of activating agents by HUVEC, as well as inhibiting the response by neutrophils to surface-presented activating agent(s). If HUVEC were stimulated with 10 U ml(-1) TNF with PTX, the adhesion of flowing neutrophils was greatly inhibited compared to TNF alone. Surface ELISA indicated that PTX reduced TNF-induced upregulation of E-selectin. This reduction was only sufficient to reduce capture of neutrophils at the low dose of TNF. Thus, by using a flow-based model, we have been able to separate the effects of a multipotent agent such as pentoxifylline, which acts on leucocytes and endothelial cells, at each stage of migration.