Hemolysis in the spleen drives erythrocyte turnover.

Red pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear. In this study, we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPMs, we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By using in vivo imaging and transfusion experiments, we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. In addition, we showed that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule-driven retention of senescent erythrocytes under low shear conditions was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by RPMs. These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated.

Differential interaction between DARC and SDF-1 on erythrocytes and their precursors.

The Duffy Antigen Receptor for Chemokines (DARC) is expressed on erythrocytes and on endothelium of postcapillary venules and splenic sinusoids. Absence of DARC on erythrocytes, but not on endothelium, is referred to as the Duffy negative phenotype and is associated with neutropenia. Here we provide evidence that stromal cell-derived factor 1 (SDF-1), the chemokine that restricts neutrophil precursors to the bone marrow, binds to erythrocyte progenitors in a DARC-dependent manner. Furthermore, we show that SDF-1 binding to DARC is dependent on the conformation of DARC, which gradually changes during erythroid development, resulting in the absence of SDF-1 binding to mature erythrocytes. However, SDF-1 binding to erythrocytes was found to be inducible by pre-treating erythrocytes with IL-8 or with antibodies recognizing specific epitopes on DARC. Taken together, these novel findings identify DARC on erythrocyte precursors as a receptor for SDF-1, which may be of interest in beginning to understand the development of neutropenia in situations where DARC expression is limited.

In vitro detection of cholangiocarcinoma cells using a fluorescent protein-expressing oncolytic herpes virus.

Pathological confirmation is desired prior to high-risk surgery for suspected perihilar cholangiocarcinoma (PHC), but preoperative tissue diagnosis is limited by poor sensitivity of available techniques. This study aimed to validate whether a tumor-specific enhanced green fluorescent protein (eGFP)-expressing oncolytic virus could be used for cholangiocarcinoma (CC) cell detection. Extrahepatic CC cell lines SK-ChA-1, EGI-1, TFK-1 and control cells (primary human liver cells) were exposed to the oncolytic herpes simplex type 1 virus NV1066 for up to 24 h in adherent culture. The technique was validated for cells in suspension and cultured cells that had been exposed to crude patient bile. Optimal incubation time of the CC cells with NV1066 at a multiplicity of infection of 0.1 was determined at 6-8 h, yielding 15% eGFP-expressing cells, as measured by flow cytometry. Cells were able to survive 2-h crude bile exposure and remained capable of producing eGFP following NV1066 infection. Detection of malignant cells was possible at the highest dilution tested (10 CC cells among 2 × 105 control cells), though hampered by non-target cell autofluorescence. The technique was not applicable to cells in suspension due to insufficient eGFP production. Accordingly, as yet the technique is not suitable for standardized clinical diagnostics in PHC.

ICAM-3 activation modulates cell-cell contacts of human bone marrow endothelial cells.

The Ig-like cell adhesion molecule ICAM-3 is mainly expressed on human leukocytes and is involved in cell-cell interactions. Its expression on endothelium is observed during disorders such as Crohn's disease and in solid tumors. We found low but detectable expression of ICAM-3 on VE-cadherin-expressing cells from primary human bone marrow aspirates, i.e. endothelial cells, and on primary human endothelial cells from cord blood. Also, immortalized human umbilical cord endothelial cells and human bone marrow endothelial cells showed ICAM-3 expression. However, its function on human endothelium is not known. Surprisingly, activation of endothelial ICAM-3 by crosslinking with specific antibodies resulted in a drop in the electrical resistance of bone marrow endothelial monolayers. In line with this, immunocytochemical analysis showed a loss of endothelial cell-cell contacts after ICAM-3 crosslinking in HBMEC. Detailed biochemical analysis showed an association of moesin and in a later stage ezrin with ICAM-3 upon crosslinking in HBMEC. Moreover, ICAM-3 crosslinking induced the production of reactive oxygen species (ROS), which are known to be involved in the control of endothelial cell-cell contacts. In conclusion, we showed that ICAM-3 is expressed on human bone marrow endothelial cells and controls endothelial integrity via ROS-dependent signaling.

Regulation and kinetics of platelet-activating factor and leukotriene C4 synthesis by activated human basophils.

BACKGROUND: Allergic disease is the result of an interplay of many different cell types, including basophils and mast cells, in combination with various inflammatory lipid mediators, such as platelet-activating factor (PAF) and leukotrienes (LT). LTC4 synthesis by human basophils has been studied quite extensively. However, not much is known about the synthesis of PAF by human basophils. OBJECTIVE: In this study, we have made a comprehensive comparison between the kinetics of PAF and LTC4 synthesis, in highly purified basophils, activated with different stimuli or with combinations of stimuli. METHODS: Synthesis of PAF and LTC4 by human basophils was determined with commercially available assay kits. The basophils were activated with C5a, fMLP, PMA, allergen or anti-IgE, in the absence and presence of IL-3 and/or in combination with elevation of cytosolic free Ca2+ by the sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. RESULTS: Most stimuli were found to induce both PAF and LTC4 synthesis. PAF synthesis and LTC4 release were enhanced by preincubation of the basophils with IL-3 or by elevation of cytosolic free Ca2+ by thapsigargin. Incubation of human basophils with IL-3 alone or thapsigargin alone did not result in detectable synthesis of PAF and LTC4, whereas the combination of the two resulted in high amounts of PAF and LTC4 synthesis. Depending on the stimulus used, LTC4 release was 5-100-fold higher than PAF synthesis. In addition, PAF, but not LTC4, was transiently detected, probably due to PAF degradation. LTC4 and PAF synthesis was strongly blocked by inhibitors of cytosolic phospholipase A2, indicating that this enzyme is involved in PAF and LTC4 synthesis by activated human basophils. CONCLUSION: This study provides a first comprehensive comparison of PAF and LTC4 synthesis in highly purified human basophils, stimulated with a variety of stimuli.

Beta1 integrin activation on human neutrophils promotes beta2 integrin-mediated adhesion to fibronectin.

Although the importance of beta1 integrin-mediated binding to adhesion molecules and extracellular matrix (ECM) molecules is well established for most types of leukocytes, the expression patterns and functional importance of beta1 integrins on neutrophils have remained controversial. Using flow cytometry, we found that human neutrophils express the alpha4, alpha5, alpha9 and beta1 integrin subunits. To examine whether the integrins VLA-4 (alpha4/beta1) and VLA-5 (alpha5/beta1) have a functional role on neutrophils, we studied adhesion to their ligand fibronectin. Treatment of neutrophils with antibody 8A2, which specifically binds and activates beta1 integrins, resulted in increased binding to fibronectin. However, addition of blocking mAb revealed that 8A2-induced adhesion did not depend on beta1 integrins, but on the beta2 integrin CD11b/CD18. Similarly, activation of beta1 integrins by 8A2 resulted in CD11b-dependent binding of neutrophils to fibrinogen. 8A2 treatment increased expression of an activation epitope of CD11b/CD18, which depended on phosphoinositide 3-OH kinase activity and an adequate concentration of intracellular free Ca2+. These data suggest that engagement of beta1 integrins on neutrophils results in a cross-talk signal that leads to activation of the beta2 integrin CD11b/CD18, followed by CD11b-mediated adhesion. As transmigrated neutrophils are surrounded by both beta1 and beta2 ligands in the ECM, this integrin cross-talk could play a role in modifying migration and cellular activation in inflamed tissues.

Neutrophils enhance eosinophil migration across monolayers of lung epithelial cells.

During the late-phase asthmatic response eosinophils and neutrophils infiltrate the lungs and cause severe damage. In this study, we investigated in vitro the migration of eosinophils, in the absence and presence of neutrophils, across a monolayer of lung H292 epithelial cells. The migration of eosinophils towards the complement fragment 5a (C5a) was increased when neutrophils were added to the upper compartment of the Transwells, and decreased when neutrophils were added to the lower compartment. Moreover, neutrophils exclusively stimulated eosinophil migration towards C5a, and not towards other chemoattractants such as RANTES, IL-8 or PAF. Neutrophils and eosinophils differed in that neutrophils, but not eosinophils, rapidly inactivated C5a, suggesting that neutrophils in the upper compartment remove part of the active C5a that has diffused into the upper compartment. Indeed, we found that the addition of other C5a-degrading agents, such as human serum or carboxypeptidase B, also enhanced eosinophil migration when added to the upper compartment and decreased migration when added to the lower compartment. Taken together, these results indicate that the presence of neutrophils influences the migratory behaviour of eosinophils in vitro. The neutrophils presumably maintain a proper C5a chemotactic gradient in the transmigration model, which results in enhanced eosinophil chemotaxis.

Sequential migration of neutrophils across monolayers of endothelial and epithelial cells.

In the course of granulocyte-dominated lung inflammation, granulocytes migrate across the endothelium and epithelium of the lung and cause severe tissue damage. To study this process in more detail, we developed a bilayer transmigration model composed of primary human endothelial and lung epithelial cells, simultaneously cultured on opposite sides of Transwell filters. Electron microscopical analysis showed that the morphology of the cells and the expression of junctional proteins remained unaltered and that matrix components were deposited onto the filter. Intriguingly, neutrophil migration was more efficient across the bilayers than across single epithelial monolayers and did not differ from migration across single endothelial monolayers. Coculture experiments showed that endothelial cells stimulated epithelial cells to release IL-6 and that epithelial cells enhanced release of IL-8 from endothelial cells. Together these data reveal bidirectional signaling and enhanced neutrophil migration in a transmigration model of primary human epithelial and endothelial cells.

Influence of bronchial allergen challenge on histamine release by human basophils.

BACKGROUND: Basophils can be primed by cytokines such as interleukin (IL) -3, IL-5 or granulocyte macrophage-colony stimulating factor (GM-CSF). It has been described that the concentrations of these cytokines are enhanced at sites of allergic inflammation as well as systemic in allergic asthma. OBJECTIVE: To investigate the priming status of basophils as detected by thapsigargin-induced histamine release during bronchial allergen challenge. METHODS: Ten subjects allergic to house dust mite were challenged via an aerosol delivery system. Spontaneous leucocyte histamine release as well as histamine release induced by various stimuli was measured in vitro at several time points. In addition, lung function parameters, serum IL-5 and blood eosinophil counts were evaluated. RESULTS: We found no effect of bronchial allergen challenge upon spontaneous leucocyte histamine release, nor upon histamine release induced by anti-immunoglobulin (Ig) E, house dust mite extract, C5a, fMLP, IL-3, PMA+ thapsigargin or IL-3+ thapsigargin. However, the priming status of basophils as measured by thapsigargin-induced histamine release was enhanced at 24 h after bronchial allergen challenge. Analysis of the individual data showed a heterogeneous initial response (30 min, 6 h) followed by a predominant increase at 24 h after allergen challenge. This increase in the thapsigargin-induced histamine release correlated with the increase in serum IL-5 levels at 24 h after allergen challenge. CONCLUSION: The priming status of human basophils as measured by thapsigargin-induced histamine release is enhanced 24 h after allergen challenge.

Basophils from patients with allergic asthma show a primed phenotype.

BACKGROUND: IL-3, IL-5, and GM-CSF are not able to induce histamine release in purified basophils of nonallergic donors. However, we have recently found that preincubation with 2 micromol/L thapsigargin, which induces a rise in intracellular free calcium ions, renders human basophils extremely sensitive for IL-3, IL-5, or GM-CSF, leading to enhanced histamine release. Histamine release was also induced in the reverse order (first cytokine and then thapsigargin). OBJECTIVE: Because these cytokines are supposed to be increased in allergic inflammation, we examined whether basophils of patients with allergic asthma showed an enhanced response to thapsigargin. METHODS: We measured the histamine release induced by thapsigargin in a group of allergic asthmatic subjects (n = 24) and compared this response with those of 3 control groups. The control groups consisted of healthy control subjects (group 1, n = 21); patients with a nonallergic, nonasthmatic lung disease (group 2, n = 22); and patients with nonallergic asthma (group 3, n = 9). RESULTS: There was no difference in spontaneous histamine release. Also, no significant difference in histamine release was found when anti-IgE or formyl-methionyl-leucyl-phenylalanine was used as a stimulus. Histamine release induced by IL-3 alone or a combination of IL-3 and thapsigargin also did not differ. In contrast, basophils from the group with allergic asthma showed a significantly higher percentage of histamine release induced by thapsigargin (38.2% +/- 13.2%) than did basophils from the 3 control groups (healthy control subjects, 22.5% +/- 6.9%; subjects with lung disease, 24.9% +/- 8.9%; subjects with nonallergic asthma 15.0% +/- 3.0%; all mean +/- SD). CONCLUSION: These data indicate that basophils in peripheral blood of subjects with allergic asthma have a primed phenotype and that thapsigargin-induced histamine release is a practical tool to study this phenomenon.

Neutrophil activation in sickle cell disease.

Vascular occlusion is the main cause of the morbidity and mortality observed in patients with sickle cell disease (SCD). Increasing evidence indicates that (activated) neutrophils could play an important role in the initiation and propagation of vaso-occlusive processes in SCD. In this study, the activation state of neutrophils in sickle cell patients was analyzed by determining the level of expression of neutrophil antigens such as CD62L, CD11b, CD66b, CD63, and Fcgamma receptors. We also analyzed plasma levels of lactoferrin, elastase, soluble (s)CD16 (sFcgammaRIII), and serum levels of soluble (s)CD62L (sL-selectin) as neutrophil activation markers in these patients. Significant differences were observed in the activation state of neutrophils in non-symptomatic sickle cell patients compared to healthy HbAA controls as exemplified by significant decrease in L-selectin expression, enhanced expression of CD64, and increased levels of soluble markers like sL-selectin, elastase, and sCD16. During vaso-occlusive crisis the differences were even more pronounced. These results show neutrophils to be activated in sickle cell patients, suggesting a role of importance in the pathophysiology of sickle cell disease.

Vascular-endothelial-cadherin modulates endothelial monolayer permeability.

Vascular endothelial (VE)-cadherin is the endothelium-specific member of the cadherin family of homotypic cell adhesion molecules. VE-cadherin, but not the cell adhesion molecule platelet/endothelial cell adhesion molecule (PECAM-1), markedly colocalizes with actin stress fibers at cell-cell junctions between human umbilical vein endothelial cells. Inhibition of VE-cadherin-mediated, but not PECAM-1-mediated, adhesion induced reorganization of the actin cytoskeleton, loss of junctional VE-cadherin staining and loss of cell-cell adhesion. In functional assays, inhibition of VE-cadherin caused increased monolayer permeability and enhanced neutrophil transendothelial migration. In a complementary set of experiments, modulation of the actin cytoskeleton was found to strongly affect VE-cadherin distribution. Brief stimulation of the beta2-adrenergic receptor with isoproterenol induced a loss of actin stress fibers resulting in a linear, rather than 'jagged', VE-cadherin distribution. The concomitant, isoproterenol-induced, reduction in monolayer permeability was alleviated by a VE-cadherin-blocking antibody. Finally, cytoskeletal reorganization resulting from the inactivation of p21Rho caused a diffuse localization of VE-cadherin, which was accompanied by reduced cell-cell adhesion. Together, these data show that monolayer permeability and neutrophil transendothelial migration are modulated by VE-cadherin-mediated cell-cell adhesion, which is in turn controlled by the dynamics of the actin cytoskeleton.

Triple role of platelet-activating factor in eosinophil migration across monolayers of lung epithelial cells: eosinophil chemoattractant and priming agent and epithelial cell activator.

Infiltration of eosinophils into the lung lumen is a hallmark of allergic asthmatic inflammation. To reach the lung lumen, eosinophils must migrate across the vascular endothelium, through the interstitial matrix, and across the lung epithelium. The regulation of this process is obscure. In this study, we investigated the migration of human eosinophils across confluent monolayers of either human lung H292 epithelial cells or primary human bronchial epithelial cells. Established eosinophil chemoattractants (IL-8, RANTES, platelet-activating factor (PAF), leukotriene B4, and complement fragment 5a (C5a)) or activation of the epithelial cells with IL-1beta induced little eosinophil transmigration (<7% in 2 h). In contrast, addition of PAF in combination with C5a induced extensive (>20%) transepithelial migration of unprimed and IL-5-primed eosinophils. Eosinophil migration assessed in a Boyden chamber assay, i.e., without an epithelial monolayer, was only slightly increased upon addition of PAF and C5a. Preincubation of eosinophils with the PAF receptor antagonist WEB 2086 only inhibited migration of unprimed eosinophils toward PAF and C5a, whereas preincubation of epithelial cells with WEB 2086 abolished migration of both IL-5-primed and unprimed eosinophils. This latter result indicated the presence of PAF receptors on epithelial cells. Indeed, addition of PAF to epithelial cells induced an increase in cytosolic free Ca2+, which was blocked by the PAF receptor antagonists WEB 2086 and TCV-309. Our results show that PAF induces permissive changes in epithelial cells, and that PAF acts as a chemoattractant and priming agent for the eosinophils.

Degranulation of human basophils by picomolar concentrations of IL-3, IL-5, or granulocyte-macrophage colony-stimulating factor.

In most secretory cells, an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) is associated with the exocytosis response. In this study we have evaluated the effect of thapsigargin on histamine release from purified (70% to 97% pure) human basophils of nonallergic donors. Thapsigargin (2 micromol/L), by inhibiting the uptake of Ca2+ in the stores of the endoplasmic reticulum, leads within 1 minute to a gradual increase in [Ca2+]i in human basophils. Incubation of basophils with thapsigargin by itself induced only a very small release of histamine (5.6% +/- 1.8%). However, under suboptimal conditions of stimulation with other agonists, preincubation of basophils with thapsigargin significantly enhanced histamine release. Most strikingly, addition of thapsigargin made basophils extremely sensitive for histamine release induced by IL-3 (maximum histamine release, 71% +/- 7%), IL-5 (maximum histamine release, 43% +/- 8%), or granulocyte-macrophage colony-stimulating factor (GM-CSF) (maximum histamine release, 57% +/- 10%). These cytokines by themselves did not induce histamine release in purified basophils. The effect of thapsigargin was mimicked to a limited extent by addition of platelet-activating factor. We conclude that depletion of the Ca2+ stores may be a critical event in the activation of receptor-mediated histamine release in human basophils.

Macrophages influence gap junctional intercellular communication between smooth muscle cells in a co-culture model.

The present study demonstrates for the first time that cells cultured on pore membrane inserts (macrophages) modulate gap junctional intercellular communication (GJIC) between a second cell type (smooth muscle cells (SMC)) co-cultured in Transwell-COL cell culture chambers. Unstimulated J774A.1 murine macrophages reduced GJIC between human SMC. Stimulation of J774A.1 cells by lipopolysaccharide (LPS) or interferon-γ abrogated this modulation of GJIC. Unstimulated human monocyte-macrophages did not affect GJIC between human SMC. Upon stimulation of these monocyte-macrophages with LPS, a substantial increase in GJIC between co-cultured SMC was observed. Thus, activation of macrophages alters their interaction with co-cultured SMC. Since these results were obtained in an indirect co-culture system in which direct cell-cell contact is prevented, it is hypothesized that soluble factors released by macrophages may be involved in this modulation of GJIC between SMC. The possible nature of the responsible soluble factors is discussed in the context of atherosclerosis.

Transmigration of human neutrophils across airway epithelial cell monolayers is preferentially in the physiologic basolateral-to-apical direction.

To study the mechanisms involved in the movement of neutrophils from the blood stream into the lung airways, we investigated human neutrophil transmigration across a monolayer of human airway epithelial cells, both in the apical-to-basolateral direction and in the more physiologic basolateral-to-apical direction. Migration of human neutrophils across monolayers of human airway epithelial H292 cell-line cells and primary bronchial epithelial cells occured most efficiently in the basolateral-to-apical direction, both after the addition of chemoattractants to resting epithelial cells and across interleukin-1beta (IL-1beta)-stimulated epithelial cells. Blocking studies with monoclonal antibodies revealed that the migration of neutrophils was mediated by the CR3 adhesion molecule (CD11b/CD18) on the neutrophils. IL-1beta-treated epithelial cells caused neutrophil movement via the secretion of chemoattractants. The most potent chemoattractant released by the epithelial cells was found to be IL-8, because the IL-1beta-induced migration was inhibited for 75 +/- 10% by the addition of an antibody against IL-8. After apical stimulation of the epithelial cells with an optimal concentration of IL-1beta, 27 +/- 4 ng/ml IL-8 was found in the supernatant at the apical side of epithelial cells. Platelet-activating factor (PAF) synthesis by the epithelial cells did not play a role in neutrophil transmigration, as was demonstrated by the lack of inhibition of this process after addition of the PAF-receptor antagonist WEB 2086. We conclude that the movement of neutrophils across airway epithelial cell monolayers occurs preferentially in the physiologic basolateral-to-apical direction, indicating that the polarity of epithelial cells is important for neutrophil transmigration.

Neutrophil transmigration across monolayers of endothelial cells and airway epithelial cells is regulated by different mechanisms.

Neutrophil recruitment from the blood stream into the airways is one of the important features of many inflammatory disorders in the lung. In this process, neutrophils migrate first from the blood across the ECs in the apical-to-basolateral direction into the tissues and then across airway epithelium in the basolateral-to-apical direction into the lung lumen. We investigated and compared the different mechanisms of neutrophil transmigration in vitro across monolayers of these two cell types in both directions. Neutrophil migration induced by chemoattractants was stronger across resting EC than across resting epithelial cells when both cell types were growing on top of the filters (i.e., migration in the apical-to-basolateral direction). Much higher neutrophil transepithelial migration was found in the physiological, basolateral-to-apical direction (cells hanging underneath the filters) than in the opposite direction across either resting or IL-1 beta-activated epithelial cells. In contrast, no significant difference was observed with EC under these conditions. After a 4-h IL-1 beta activation, neutrophil migration across EC was dependent on IL-8 and PAF. However, the migration across epithelial cells relied, to a greater extent, on IL-8 production, and not on PAF. The adhesion molecule ICAM-1 contributed much less to neutrophil migration across epithelium than that across endothelium. Our study provides evidence of different mechanisms of neutrophil transmigration across monolayers of EC and epithelial cells in vitro, indicating that different processes control neutrophil transmigration across EC and epithelial cells in vivo.

The role of basophils in allergic disease.

During allergic disease, leucocytes infiltrate the affected tissues and release their mediators and cytokines. In this way, the local inflammatory process is induced and maintained. Basophilic granulocytes have been demonstrated in lung and sputum of allergic asthmatics, in nasal mucosa and secretion of allergic rhinitis patients, and in skin lesions of atopic dermatitis patients. The number of basophils correlates with the severity of the disease. Analysis of mediator profiles and cellular contents of lavages of nose, skin and lung during allergic late-phase reactions (LPR) have demonstrated histamine, but not tryptase or prostaglandin D2. The histamine-containing cells have been characterized as basophilic granulocytes. This indicates that infiltrating basophils but not mast cells are activated and release their inflammatory contents in the LPR. We are interested in the cellular mechanisms that determine the degranulation of basophils during LPR. Basophil activators, such as allergens and activated complement, are not present at these sites. However, cytokines that prime basophils but do not induce degranulation, such as interleukin-5 (IL-5) and granulocyte/macrophage colony-stimulating factor (GM-CSF), have been detected at sites of LPR. We have now observed that after emptying intracellular Ca2+ stores by means of the Ca2+ adenosine triphosphatase (ATPase) inhibitor, thapsigargin, basophils become extremely sensitive to stimuli that do not affect the Ca2+ stores themselves but that induce degranulation, such as the phorbolester, phorbol myristate acetate (PMA). The most interesting finding was that although both thapsigargin and IL-3, IL-5 or GM-CSF do not induce basophil degranulation by themselves, a 2 min preincubation of basophils with thapsigargin followed by addition of one of these cytokines resulted in extensive histamine release: IL-3 induced 71 +/- 7% histamine release (conc1/2max 6 pM), IL-5 induced 43 +/- 8% histamine release (conc1/2max 41 pM) and GM-CSF induced 57 +/- 10% histamine release (conc1/2max 140 pM). Interestingly, the effect of thapsigargin could be mimicked by platelet-activating factor (PAF) (range 10(-9) to 10(-6) M), although to a lesser extent. Our results indicate that basophil degranulation in tissues during late-phase reactions might be caused by a combination of mediators or cytokines depleting Ca2+ stores, as platelet-activating factor or thapsigargin do, concurrent with activation by interleukin-3, interleukin-5 or granulocyte/macrophage colony-stimulating factor. The response of the basophils towards these cytokines might also be influenced by cell adhesion events, such as binding of basophils via integrins. This is the subject of further study.

The effect of salmeterol and nimesulide on chemotaxis and synthesis of PAF and LTC4 by human eosinophils.

Salmeterol is a long-acting beta 2-adrenoceptor agonist, with several antiasthma properties. Nimesulide is a nonsteroidal anti-inflammatory drug, supposed to act by inhibition of phosphodiesterase type IV. This might indicate that the effects of both drugs are mediated by an increase in cyclic adenosine monophosphate (cAMP). For salmeterol, it has been shown that it inhibits the influx of eosinophils into the lungs of guinea-pigs after platelet-activating factor (PAF) challenge, suggesting an effect of cAMP on eosinophil migration. For neutrophils, it has been shown that PAF synthesis is inhibited by cAMP. In the present study, we have, therefore, measured the effect of salmeterol and nimesulide on two important human eosinophil functions: chemotaxis; and synthesis of PAF and leukotriene C4 (LTC4). Both drugs were found to be inhibitors of the chemotactic responses of human eosinophils. However, at comparable concentrations, only nimesulide was able to inhibit the synthesis of PAF and LTC4 in activated eosinophils. These results indicate that although the effects of both drugs are thought to be mediated by an increase in cyclic adenosine monophosphate, they have differential effects on eosinophil chemotaxis and synthesis of lipid mediators.

Neutrophil adherence to and migration across monolayers of human peritoneal mesothelial cells. The role of mesothelium in the influx of neutrophils during peritonitis.

Increased adherence to and subsequent migration of leukocytes across cultured human peritoneal mesothelial cell monolayers takes place after pretreatment of the mesothelial cells with interleukin-1beta. The contribution of the leukocyte beta2 integrins (CD11/CD18) and the mesothelial adhesion protein intercellular adhesion molecule-1 (ICAM-1) and the role of the cytokines interleukin-8, platelet-activating factor (PAF), and transforming growth factor-beta (TGF-beta) were studied in a three-dimensional model system for neutrophil-mesothelial monolayer interaction. Polymorphonuclear leukocytes (PMNs) showed minimal adherence to and migration across unactivated mesothelial monolayers, despite an extensive amount of ICAM-1 on the mesothelial membrane. Pretreatment of the monolayers with rIL-1beta induced enhanced PMN adherence to the mesothelial monolayer together with a further increase in ICAM-1 expression on the mesothelial membrane. PMN migration was observed across rIL-1beta-activated mesothelial cell (MC) monolayers whenever cytokines secreted by the MCs were present during migration. Monoclonal antibody (mAb) R6.5 against ICAM-1 and mAb CLB-LFA1/1 against CD18 both reduced the migration of PMNs across mesothelial monolayers with a predominant inhibitory effect of CLB-LFA1/1, indicating a significant role of the beta(2) integrins of PMNs in this process. Interleukin-8 was the major cytokine synthesized by the MCs to stimulate the migration of PMNs; both PAF and TGF-beta had a more modest role in our system. Adherence of PMNs to MC monolayers was not dependent on these latter cytokines. Neuraminidase did not have any effect, indicating that selectins were not involved in the adherence process. rIL-1beta-pretreated MCs induced a rapid increase in intracellular Ca2+ in PMNs; actinomycin D blocked this effect and was also able to prevent adhesion of neutrophils to activated MC monolayers. Neutrophil migration across activated cultured MCs is thus a cascade of events in which the MCs are actively involved.