Claudine Ferland

Crucial implication of protein kinase C (PKC)‐δ, PKC‐ζ, ERK‐1/2, and p38 MAPK in migration of human asthmatic eosinophils

Asthma is associated with an eosinophil infiltration into the bronchial mucosa. 5‐Oxo‐6,8,11,14(E,Z,Z,Z)‐eicosatetraenoic acid (5‐oxo‐ETE), a potent eosinophil chemotactic factor, activates cell motility, adherence, and proteolysis, notably, by promoting CD11b expression, matrix metalloproteinase (MMP)‐9 secretion, and plasmin generation. We investigated the intracellular signaling pathways implicated in these various steps by using different, selective inhibitors. Human eosinophil migration through a reconstituted basement membrane in response to 5‐oxo‐ETE was greatly inhibited (≥72%) by the protein kinase C (PKC)‐δ, PKC‐ζ, ERK‐1/2, and p38 inhibitors. Our findings indicate that PKC‐δ mediates cell motility, CD11b expression, and MMP‐9 granule release. PKC‐ζ is also largely involved in eosinophil migration, although its specific targets remain undefined. ERK‐1/2 and p38 modulate CD11b expression; ERK‐1/2 is also involved in long‐term MMP‐9 secretion and p38 in the plasmin activation system. We demonstrated the crucial implication of PKC‐δ, PKC‐ζ, ERK‐1/2, and p38 in human blood eosinophil migration through extracellular matrix components. Targeting specific pathways may have therapeutic potential for the treatment of allergic airway inflammation.

Modulation of eosinophil activation in vitro by a nicotinic receptor agonist

Nicotinic receptor agonists decreased the infiltration of eosinophils into the lung and airways in a mouse model of asthma. To better understand the mechanisms implicated in this anti‐inflammatory phenomenon, the expression of nicotinic acetylcholine receptors (nAChRs) and the effect of dimethylphenylpiperazinium (DMPP), a nonselective nAChR agonist, on human blood eosinophils were studied. The expression of α‐3, ‐4, and ‐7 nAChR subunits on human blood eosinophils was measured by cell ELISA and immunocytochemistry. mRNA expression for all three subunits was evaluated by quantitative RT‐PCR. The effect of DMPP on leukotriene C4 (LTC4) and matrix metalloproteinase‐9 (MMP‐9) production, eosinophil migration, and intracellular calcium mobilization was measured. The results show that the α‐3, ‐4, and ‐7 nAChR subunits and mRNAs are expressed by blood eosinophils. In vitro treatment of these cells with various concentrations of DMPP reduced platelet‐activating factor (PAF)‐induced LTC4 production significantly. DMPP (160 μM) decreased eotaxin, and 5‐oxo‐6,8,11,14‐eicosatetranoic acid induced eosinophil migration through Matrigel by 40.9% and 55.5%, respectively. This effect was reversed by the nAChR antagonist mecamylamine. In addition, DMPP reduced MMP‐9 release and the inositol 1,4,5‐triphosphate‐dependent intracellular calcium increase provoked by PAF. Taken together, these results indicate that functional nAChRs are expressed on eosinophils and that nAChR agonists down‐regulate eosinophil function in vitro. These anti‐inflammatory effects could be of interest in the treatment of allergic asthma.

Expression of membrane type-4 matrix metalloproteinase (metalloproteinase-17) by human eosinophils

Circulating eosinophils need proteinases to mediate a spatially limited and orientated digestion of the extracellular matrix and to migrate into tissue. Moreover, proteinases are likely involved in tissue remodeling, a crucial feature of chronic diseases including asthma. Eosinophils express matrix metalloproteinase (MMP)-9, which is increased upon stimulation with TNF-alpha. Other MMPs, the membrane type (MT)-MMPs, likely play a major role in cell invasion and tissue remodeling. MT4-MMP was identified in peripheral blood leukocyte preparations, but it is not known whether eosinophils express MT4-MMP. We investigated the expression of MT4-MMP and its modulation by TNF-alpha in purified human blood eosinophils. The constitutive expression of MT4-MMP mRNA was detected by RT-PCR in unstimulated eosinophils, lymphocytes, and monocytes, but not neutrophils. Stimulation of eosinophils with TNF-alpha increased MT4-MMP mRNA expression. This effect appeared at 4h and reached a maximum at 8h of incubation. MT4-MMP protein was detected in freshly isolated blood eosinophils by Western blotting and immunocytochemistry. TNF-alpha increased expression of the MT4-MMP protein. MT4-MMP protein was also detected in nasal polyp eosinophils by immunohistochemistry. In conclusion, eosinophils constitutively express MT4-MMP, which is increased upon stimulation with TNF-alpha. Consequently, MT4-MMP may be directly involved in the degradation of extracellular matrix components and/or modulate the activity of other proteins implicated in eosinophil migration and tissue remodeling.

The Endocannabinoid 2-Arachidonoyl-Glycerol Activates Human Neutrophils: Critical Role of Its Hydrolysis and De Novo Leukotriene B4 Biosynthesis

Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B4 biosynthesis, or LTB4 receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB4 biosynthesis, and an autocrine activation loop involving LTB4 receptor 1.

IL-16 Activates Plasminogen-Plasmin System and Promotes Human Eosinophil Migration into Extracellular Matrix via CCR3-Chemokine-Mediated Signaling and by Modulating CD4 Eosinophil Expression

Increased eosinophil counts are a major feature of asthmatic airways. Eosinophil recruitment requires migration through epithelium and tissue extracellular matrix by activation of proteases. We assessed the capacity of IL-16, a CD4+ cell chemotactic factor, to induce migration of eosinophils through a reconstituted basement membrane and evaluated the proteases, mediators, and receptors involved in this migration. IL-16 added to lower chambers of Invasion Chambers elicited eosinophil migration through Matrigel. This effect was decreased by inhibition of the plasminogen-plasmin system (Abs against urokinase plasminogen activator receptor or plasminogen depletion), but not by anti-matrix metalloproteinase-9 Abs. Abs against CD4 also inhibited IL-16-induced eosinophil migration. At the baseline level, few eosinophils (4.6% positive cells with a mean fluorescence of 0.9) expressed surface membrane CD4, while most permeabilized eosinophils (68% positive cells with a mean fluorescence of 18) express the CD4 Ag. TNF-pretreatment increased surface membrane CD4+ expression by 6-fold as previously described, and increased IL-16-induced cell migration by 2.2-fold. Incubation of eosinophils with IL-16 also increased surface membrane CD4 expression by 5.4-fold, supporting the role of CD4 as receptor for IL-16. Abs against CCR3, eotaxin, or RANTES blocked IL-16-induced migration. In conclusion, IL-16 promotes eosinophil migration in vitro, by activating the plasminogen-plasmin system and increasing the membrane expression of its receptor. This effect is initiated via CD4 and mediated via the release of CCR3 ligand chemokines. Interestingly, most eosinophils express intracellular CD4. Hence, IL-16 may play an important role in the recruitment of blood eosinophils to the bronchial mucosa of asthmatics.

Eotaxin promotes eosinophil transmigration via the activation of the plasminogen‐plasmin system

The effect of eotaxin, a potent eosinophil chemotactic factor, on eosinophil transmigration through a reconstituted basal membrane (Matrigel®) was evaluated. Eotaxin induced significant eosinophil transmigration in the presence of 10% fetal bovine serum (FBS) and interleukin‐5. Its effect was optimal at 0.01 μM, and it plateaued at 18 h. Eotaxins effect was greater with eosinophils from asthmatic subjects (61.1 ± 3.4%) than with eosinophils from normal subjects (38.7 ± 4.2%) (P < 0.001). Inhibition of metalloproteinases decreased eotaxin‐induced transmigration by ≤10.4%, whereas inhibition of the plasminogen‐plasmin system decreased eotaxins effect by ≤44.4% (P = 0.0002). Moreover, eotaxin‐induced transmigration was largely diminished in medium with low concentrations of serum [0.5% FBS: 6.1 ± 2.4%; 10% FBS: 40.2 ± 5.8% (P = 0.0001)] but returned to its initial level with the addition of plasminogen (2 U/mL) to 0.5% FBS (43.1 ± 6.5%). These data show that eotaxin is an efficient promoter of eosinophil transmigration in vitro, that it is more potent with cells from asthmatics than with normal cells, and that its effect depends predominantly on the activation of the plasminogen‐plasmin system.

Comparison of two modified techniques for purifying blood eosinophils.

As there is much heterogeneity in the morphology and function of blood eosinophils, comparison of their properties between groups of subjects requires recovering the majority of these cells. In two currently used techniques to isolate eosinophils, blood granulocytes are processed either on Percoll gradients after an incubation of granulocytes with 10(-8) M N-formyl-methionyl-leucyl-phenylalanine (fMLP) or on a magnetic cell sorter (MACS). In this study, these techniques were modified to increase the efficiency of eosinophil recovery. With the Percoll gradients, using 1.078 g/ml as the top gradient instead of 1.082 g/ml doubled the eosinophil recovery from 43 +/- 5.3% (mean +/- SEM) to 86.9 +/- 2.9%, without decreasing the purity (96.1 +/- 1.4% versus 96.2 +/- 0.9%). With a MACS, the neutrophils in granulocytes obtained on Ficoll-Paque (1.077 g/ml) instead of on Percoll gradient 1.082-1.094 g/ml, were tagged with anti-CD16 antibodies and eliminated by passing them through a magnetic field. When blood eosinophils of the same subjects were isolated using the two techniques, similar recovery and purity levels were obtained: Percoll gradients, 72.7 +/- 6.8% and 92.5 +/- 2.2%; MACS, 80.2 +/- 5.1% and 90.4 +/- 3.8%. Eosinophils isolated through the two techniques were also compared for their production of superoxide anion and leukotriene (LT) C4, with and without pre-incubation with cytokines interleukin-3, interleukin-5 and granulocyte-macrophage colony stimulating factor. The release of these products was similar between the two eosinophil preparations under all conditions tested except for interleukin-3 where eosinophils isolated with a MACS produced more LTC4. These results show that both techniques efficiently recover pure eosinophils. Furthermore, cell incubation with 10(-8) M fMLP did not enhance superoxide anion and LTC4 production nor modify the response to cytokines. The two modified techniques are therefore suitable for comparative studies of eosinophils from different groups of subjects.

Role and modulation of CD16 expression on eosinophils by cytokines and immune complexes.

Background: Blood eosinophils express CD16 on their surface when stimulated in vitro with platelet-activating factor or IFNγ. Transient expression of CD16 is also observed in vivo following aeroallergen challenge of asthmatic subjects. The present work is aimed at evaluating the possible mechanisms modulating eosinophil expression of CD16 and the biological functions of this receptor. Methods: First, purified blood eosinophils were incubated with IL-1β, IL-2, IL-4, IL-5, IL-9 or IL-16, GM-CSF, IFNγ, eotaxin or 5-oxo-ETE and CD16 expression was measured. Second, the capacity of CD16 to mediate degranulation induced by IgG immune complexes (IC) was evaluated in eosinophils with low and high CD16 expression. Finally, serum allergen-specific IgE and IgG, and total IgE levels were measured at baseline in allergic asthmatics and correlated with changes observed in blood eosinophil CD16 expression (ΔCD16) following allergen challenge. Results: Only IFNγ and IL-2 significantly increased the number of CD16+ eosinophils, respectively, 37 ± 10% (p = 0.0038) and 38 ± 8% (p = 0.0006), compared to control, 7 ± 2%. IgG IC induced degranulation in eosinophils with low and high CD16 expression and monoclonal anti-CD16 and anti-CD32 antibodies inhibited this. IgG IC increased eosinophil CD16 expression (14 ± 6%, p = 0.0008) and this effect was blocked by pretreatment with anti-CD32 antibodies. ΔCD16 following allergen challenge correlated with the specific IgG/total IgE ratio (r2 = 0.41, p = 0.036). Conclusion: These data suggest that formation of IgG IC is associated with surface eosinophil CD16 expression in asthma and that CD16 in cooperation with CD32 mediates IC-induced degranulation.

Migration through basement membrane modulates eosinophil expression of CD44

Background Tissue eosinophils express more membrane receptors and release more mediators than blood eosinophils, suggesting that migration from blood to tissue modulates eosinophil phenotype and functions.

Interleukin-12 Inhibits Eosinophil Degranulation and Migration but Does Not Promote Eosinophil Apoptosis

Background: Animal and human studies demonstrated that interleukin (IL)-12, a Th1 cytokine, reduces blood and bronchial eosinophilia, and airway hyperreactivity. According to current concepts, these effects are mediated through the release of cytokines promoting eosinophil recruitment and activation. However, the presence of IL-12 receptors on eosinophils suggests that IL-12 also acts directly on eosinophils. We postulated that IL-12 directly modulates eosinophil functions and has the capacity to regulate eosinophil degranulation, migration and survival, in vitro. Method: Effects of IL- 12 on purified human blood eosinophils were evaluated for peroxidase (EPO) release, eotaxin-induced migration through a model of basement membrane (Matrigel), and survival. Results: IL-12 inhibited 50% of PAF and secretory IgA-induced EPO release (n = 8, p < 0.001). IL-12 also reduced eotaxin-induced migration through Matrigel by 54 ±6% (n = 6, p < 0.01). These effects were not explained by an IL-12-induced impaired viability or apoptosis. Conclusion: Our results demonstrate that IL-12 directly modulates eosinophil functions without promoting apoptosis and explain, at least in part, the effects of IL-12 on eosinophils observed in in vivo studies.