Regulation of interleukin-5-induced beta2-integrin adhesion of human eosinophils by phosphoinositide 3-kinase

We examined the role of phosphoinositide 3-kinase (PI3K) in integrin-mediated eosinophil adhesion. Deltap85, a dominant-negative form of the class IA PI3K adaptor subunit, was fused to an HIV-TAT protein transduction domain (TAT-Deltap85). Recombinant TAT-Deltap85 inhibited interleukin (IL)-5-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. beta(2)-Integrin-dependent adhesion caused by IL-5 to the plated intracellular adhesion molecule-1 surrogate, bovine serum albumin, was inhibited by TAT-Deltap85 in a concentration-dependent manner. Similarly, two PI3K inhibitors, wortmannin and LY294002, blocked eosinophil adhesion to plated bovine serum albumin. By contrast, beta(1)-integrin-mediated eosinophil adhesion to vascular cell adhesion moelcule-1 was not blocked by TAT-Deltap85, wortmannin, or LY294002. Rottlerin, a protein kinase C (PKC)-delta inhibitor, also blocked beta(2)-integrin adhesion of eosinophils caused by IL-5, whereas beta(1) adhesion to vascular cell adhesion molecule-1 was not affected. IL-5 caused translocation of PKCdelta from the cytosol to cell membrane; inhibition of PI3K by wortmannin blocked translocation of PKCdelta. Western blot analysis demonstrated that extracellular signal-regulated kinase phosphorylation, a critical intermediary in adhesion elicited by IL-5, was blocked by inhibition of either PI3K or PKC-delta. These data suggest that extracellular signal-regulated kinase-mediated adhesion of beta(2)-integrin caused by IL-5 is mediated in human eosinophils by a class IA PI3K through activation of a PKCdelta pathway.     

IL-5-induced integrin adhesion of human eosinophils caused by ERK1/2-mediated activation of cPLA2

We examined the mechanism by which interleukin (IL)-5 causes beta(2)-integrin adhesion of human eosinophils. IL-5 caused time-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38alpha in eosinophils as detected by their phosphorylation. Preincubation of eosinophils with U0126, a mitogen-activated protein kinase/ERK kinase inhibitor, suppressed IL-5-induced activation of cytosolic phospholipase A(2) (cPLA(2)) and eosinophil adhesion, and p38 inhibition by SB203580 had neither effect. ERK1/2 phosphorylation and eosinophil adhesion were blocked by inhibition of the src-family tyrosine kinase, Janus tyrosine kinase (JAK)2, or phosphoinositide-3 kinase (PI3K). Coimmunoprecipitation assay demonstrated that Lyn, a src-family tyrosine kinase, was constitutively associated with PI3K. Inhibition of src-tyrosine kinase but not JAK2 suppressed PI3K activation. Our data suggest that IL-5 induces beta(2)-integrin adhesion of human eosinophils by regulation of cPLA(2) activation caused by ERK1/2 phosphorylation. This phosphorylation results from activation of PI3K and protein tyrosine kinases. We also find that src-family tyrosine kinase, possibly Lyn, is the upstream kinase causing PI3K activation.     

Activation of human AML14.3D10 eosinophils by nanoparticles: Modulatory activity on apoptosis and cytokine production

Eosinophilic inflammation is frequently observed in response to nanoparticle (NP) exposure in airway rodent models of allergies where the number of eosinophils is increased in lungs. Despite this, it is surprising that the potential cytotoxic effect of NP, as well as their direct role on eosinophils is poorly documented. The present study investigated how different NP can alter the biology of the human eosinophilic cell line AML14.3D10. It was found that among NP forms of CeO₂, ZnO, TiO₂, and nanosilver of 20?nm (AgNP₂₀) or 70?nm (AgNP₇₀) diameters, only ZnO and AgNP₂₀ induced apoptosis. Caspases-7 and -9 were not activated by the tested NP while caspase-3 was activated by AgNP₂₀ only. However, both ZnO and AgNP₂₀ induced cytoskeletal breakdown as evidenced by the cleavage of lamin B₁. Using an ELISArray approach for the simultaneous detection of several analytes (cytokines/chemokines), it was found that only ZnO and AgNP₂₀ increased the production of different analytes including the potent pro-inflammatory CXCL8 (IL-8) chemokine. From the data here, we conclude that toxic effects of some NP could be observed in human eosinophil-like cells and that this could be related, at least partially, by induction of apoptosis and production of cytokines and chemokines involved in inflammation. The results of this study also indicate that distinct NP do not activate similarly human eosinophils, since ZnO and AgNP₂₀ induce apoptosis and cytokine production while others such as TiO₂, CeO₂, and AgNP₇₀ do not.     

Glucocorticoid-induced surface expression of annexin 1 blocks beta2-integrin adhesion of human eosinophils to intercellular adhesion molecule 1 surrogate protein

BACKGROUND: Glucocorticoids attenuate the population of eosinophils and T lymphocytes in asthmatic airways. The decrease in airway eosinophilia is caused both by accelerated cell death and by induction of blockade of integrin adhesion. In this study, we examined the hypothesis that annexin 1 surface expression, which is upregulated by the glucocorticoid receptor, prevents integrin adhesion essential to cell migration by blocking intracellular translocation of cytosolic group IV phospholipase A2 (cPLA2). OBJECTIVE: To examine the relationship of the glucocorticoid on annexin 1 expression and the effect of blockade of annexin 1 activity on adhesion of human eosinophils in vitro. To determine the relationship between annexin 1surface expression and nuclear membrane translocation of cPLA2. METHODS: Eosinophils isolated from human peripheral blood were pretreated with fluticasone propionate (FP), and beta2-integrin adhesion was measured after stimulation with IL-5 or eotaxin. Effects of FP on cPLA2 expression, phosphorylation, and translocation were determined. The role of annexin 1 was examined by using annexin 1 blocking antibody and/or mimetic peptides. RESULTS: Fluticasone propionate decreased stimulated eosinophil adhesion and caused 4-fold increase in annexin 1 expression on the plasma membrane. Inhibition of adhesion by FP was blocked with annexin 1 blocking antibody. Annexin 1 N-terminal mimetic peptide also blocked beta2-integrin adhesion. Translocation of cPLA2 to the nuclear membrane was significantly blocked by incubation with FP. Blockade was reversed with annexin 1 blocking antibody. CONCLUSION: Blockade of beta2-integrin adhesion by glucocorticoid is regulated by annexin 1, which blocks cPLA2 translocation to nuclear membrane.     

Proline-rich tyrosine kinase 2 regulates spreading and migration of eosinophils after beta2-integrin adhesion

We examined the role of proline-rich tyrosine kinase (Pyk) 2 in the spreading and migration of human blood eosinophils after beta(2)-integrin ligation. Western blot analysis showed that Pyk2 was activated by phosphorylation at Y402 after eosinophil adhesion to BSA-coated plates after activation with IL-5, platelet-activating factor (PAF), formyl-met-leu-phe (fMLP), or Mn(2)(+). To determine the role of Pyk2 in regulating eosinophil migration, we used a transducable dominant-negative inhibitor of Pyk2, TAT-mediated protein transduction of dominant-negative C-terminal Pyk2 (TAT-Pyk2-CT), a fusion protein in which TAT peptide was fused to the C-terminal Pyk2. TAT-Pyk2-CT blocked tyrosine phosphorylation of Pyk2 caused by beta(2)-integrin adhesion, but did not block adhesion of eosinophils to plated BSA. TAT-Pyk2-CT also blocked subsequent spreading and migration of eosinophils caused by IL-5, PAF, or fMLP. Spreading eosinophils stained with FITC-conjugated phalloidin showed elongation and formation of multiple fillopodia and lamellipodia, whereas nonspreading eosinophils were smaller and round. Treatment of eosinophils with TAT-Pyk2-CT had no effect on the initial cell polarization, but blocked the formation of fillopodia and lamellipodia in adherent cells. Migration of eosinophils through Transwell plates caused by IL-5, PAF, or fMLP was blocked significantly after inhibition of Pyk2. These data indicate that Pyk2, although not involved in beta(2)-integrin adhesion, causes eosinophil spreading and regulates subsequent chemotactic migration after beta(2)-integrin ligation to endothelial counter ligands. We conclude that Pyk2 is activated by beta(2)-integrin adhesion and is a required signal for eosinophil spreading and subsequent chemotactic migration.     

The influence of surface roughness of titanium on beta1- and beta3-integrin adhesion and the organization of fibronectin in human osteoblastic cells

Mechanisms of cell adhesion and extracellular matrix formation are primary processes in the interaction with the material surface of an implant which are controlled by integrin receptors. The aim of our study was to find out whether beta1- and beta3-integrins of osteoblastic cells sense the surface topography of titanium, and if structural alterations of integrin adhesions were involved in the organization of fibronectin. Pure titanium surfaces were modified by polishing (P), machining (NT), blasting with glass spheres (GB), and blasting with corundum particles (CB) resulting in increasing roughness. Confocal microscopic investigations revealed fibrillar adhesions of beta1- and alpha5-integrins on P, NT, and GB, but on CB with its sharp edges these integrin subunits did not form fibrillar adhesions. beta3 generally appeared in focal adhesions. We observed aligned fibrillar structures of fibronectin on NT not only on the basal site but interestingly, also on the apical cell surface. In contrast, on CB, fibronectin appeared apically clustered. We suggest that this alignment of fibronectin fibrils depends on the directed actin cytoskeleton and in particular, on the capability of the beta1-integrins to form fibrillar adhesions, which is affected by the surface roughness of titanium.     

Redox regulation of beta2-integrin CD11b/CD18 activation

Although the central role of beta2-integrin CD11b / CD18 in neutrophil functions is well recognized, signaling pathway that regulate integrin activation remain to be elucidated. We analyzed the contribution of oxido-reduction mechanisms in this signaling. Exogenously added H(2)O(2) induced CD11b/CD18-dependent neutrophil adhesion and expression of an integrin activation neoepitope recognized by monoclonal antibody (mAb) clone 24. H(2)O(2)-triggered beta2-integrin activation was inhibited by tyrosine kinase inhibitors and by complexing sulfhydryl groups with phenylarsine oxide (PAO). CD11b/CD18-dependent adhesion and mAb 24 antigen expression triggered by physiological agonists such as TNF-alpha were inhibited by diphenylene iodonium (DPI, an inhibitor of flavoprotein oxidoreductase), by free radical scavengers, by tyrosine kinase inhibitors and by PAO. No inhibition was observed when adhesion was induced by the integrin-activating KIM 185 mAb. Taken together, these results emphasize the importance of an oxidative S-thiolation step(s) in the tyrosine kinase-dependent signaling pathway leading to beta2-integrin activation. H(2)O(2) would directly mediate this oxidative reaction and bypass the initial agonist/receptor pathway to promote integrin-dependent adhesion. The putative oxidase(s) involved in this process is not NADPH oxidase, since adhesion of neutrophils from patients with chronic granulomatous disease was normal and inhibited by scavengers and DPI. These data shed a new light on the regulation of integrin activation required for cell migration into inflamed organs.     

Calcium ionophore upregulation of AUUUA-specific binding protein activity is contemporaneous with granulocyte macrophage colony-stimulating factor messenger RNA stabilization in AML14.3D10 cells.

Eosinophils produce granulocyte macrophage colony-stimulating factor (GM-CSF), which enhances their survival and function. In T cells and fibroblasts, GM-CSF production is controlled predominantly by variable messenger RNA (mRNA) stability involving 3' untranslated region (3' UTR) adenosine-uridine-rich elements (AREs) and sequence-specific mRNA binding proteins. However, the mode of regulation of this critical cytokine remains unknown in eosinophils. Therefore, we measured GM-CSF mRNA decay in an eosinophil-like cell line (AML14.3D10) and, with a radiolabeled GM-CSF RNA probe, asked whether ARE-specific, mRNA binding proteins were present in cytoplasmic lysates of these cells. Human GM-CSF mRNA transfected into unstimulated AML14.3D10 cells decayed with a half-life of 6 min, which increased to 14 min after 1 h, and to 22 min after 2 h, of ionophore-mediated activation. GM-CSF RNA mobility shift assays using cytoplasmic extracts from resting or ionophore-stimulated AML14.3D10 cells revealed multiple RNA-protein complexes of 55, 60, 85, 100, and 125 kD. A 47-kD complex was also detected with an 80-base RNA probe containing four consecutive AUUUA motifs. On the basis of competition studies, all of the observed binding protein activities interacted with the 3' UTR AREs. In addition, binding activity increased 2.5-fold in cytoplasmic lysates from cells stimulated with calcium ionophore for 2 h, contemporaneous with GM-CSF mRNA stabilization. These data provide direct evidence that ionophore stabilizes GM-CSF mRNA in AML14.3D10 cells and simultaneously increases the activity of a series of AUUUA-specific mRNA binding proteins. We conclude that the interaction of AU-specific binding proteins may stabilize GM-CSF mRNA in activated eosinophil-like cell lines.     

Suppression of cytokine-mediated beta2-integrin activation on circulating neutrophils in critically ill patients.

The beta2 integrin CD11b plays a central role in inflammation and the systemic inflammatory response syndrome (SIRS). The CD11b molecule activates in two ways: the density of membrane-bound CD11b up-regulates and the molecule undergoes a conformational change that confers adhesiveness to counter-receptors. We studied the kinetics of CD11b activation in patients with SIRS. We found a significantly diminished CD11b activation in response to tumor necrosis factor alpha (TNF-alpha). This affected all circulating polymorphonuclear neutrophils (PMN) and was an intrinsic property of the cells and not due to antagonism by soluble TNF-alpha receptors or loss of cellular receptors for TNF-alpha. Diminished responsiveness correlated with the severity of organ failure and lasted for months in some patients but had no impact on mortality. We speculate that reduced CD11b responsiveness in SIRS contributes to the high risk of recurrent infection, but that it may also be protective against excessive PMN activation within the vascular space.     

Attenuated translocation of group IVa phospholipase A2 and up-regulated annexin-1 synthesis by glucocorticoid blocks beta 2-integrin adhesion in neutrophils

We examined the effect of glucocorticoid stimulation in blocking beta 2-integrin adhesion of polymorphonuclear leukocytes (PMNs) isolated from human subjects. Surface expression of CD11b and ERK-1/2-mediated gIVaPLA2 phosphorylation, which are required for beta 2-integrin adhesion, were not affected by treatment with < or =10(-6) M fluticasone propionate (FP) for PMNs activated by either 10(-7) M LTB4 or 30 ng/ml TNF-alpha and caused no significant blockade of beta 2-integrin adhesion in vitro. Baseline expression of annexin-1 (ANXA1) synthesis was increased only after 10(-6) M FP for PMNs; by contrast, comparable increase in ANXA1 expression was demonstrated in human eosinophils from the same subjects with 10(-8) M FP. Viability of PMNs was verified by propidium iodide and by the persistence of beta 2-integrin adhesion in treated groups. Exogenous administration of ANXA1 mimetic peptide fragment blocked significantly and comparably the beta 2-integrin adhesion in PMNs activated by LTB4 and TNF-alpha and in eosinophils activated by IL-5. Translocation of gIVaPLA2 from the cytosol to the nucleus also was refractory for activated PMNs treated with > or =10(-7) M FP; by contrast, complete blockade of nuclear translocation of cytosolic gIVaPLA2 was effected by 10(-9) M FP in eosinophils. Our data indicate that the cell surface ANXA1 synthesis is capable of blocking beta 2-integrin adhesion in both PMNs and eosinophils. However, in contrast to eosinophils, FP does not cause either substantial ANXA1 synthesis or nuclear transport of cytosolic gIVaPLA2 in PMNs and thus does not block beta2-integrin adhesion, a necessary step for granulocyte cell migration in vivo.     

Regulation of the adhesion versus cytotoxic functions of the Mac-1/CR3/alphaMbeta2-integrin glycoprotein

Mac-1/CR3 functions as both an adhesion molecule mediating the diapedesis of leukocytes across the endothelium and a receptor for the iC3b fragment of complement responsible for phagocytic/degranulation responses to microorganisms. Mac-1/CR3 has many functional characteristics shared with other integrins, including bidirectional signaling via conformational changes that originate in either the cytoplasmic domain or extracellular region. Another key to its functions is its ability to form membrane complexes with glycosylphosphatidylinositol (GPI)-anchored receptors such as Fc gammaRIIIB (CD16b) or uPAR (CD87), providing a transmembrane signaling mechanism for these outer membrane bound receptors that allows them to mediate cytoskeleton-dependent adhesion or phagocytosis and degranulation. Many functions appear to depend upon a membrane-proximal lectin site responsible for recognition of either microbial surface polysaccharides or GPI-linked signaling partners. Because of the importance of Mac-1/CR3 in promoting neutrophil inflammatory responses, therapeutic strategies to antagonize its functions have shown promise in treating both autoimmune diseases and ischemia/reperfusion injury. Conversely, soluble beta-glucan polysaccharides that bind to its lectin site prime the Mac-1/CR3 of circulating phagocytes and natural killer (NK) cells, permitting cytotoxic degranulation in response to iC3b-opsonized tumor cells that otherwise escape from this mechanism of cell-mediated cytotoxicity.     

Influence of beta(2)-integrin adhesion molecule expression and pulmonary infection with Pasteurella haemolytica on cytokine gene expression in cattle

beta(2)-Integrins are leukocyte adhesion molecules composed of alpha (CD11a, -b, -c, or -d) and beta (CD18) subunit heterodimers. Genetic CD18 deficiency results in impaired neutrophil egress into tissues that varies between conducting airways and alveoli of the lung. In this study, we investigated whether CD18 deficiency in cattle affects proinflammatory cytokine (PIC) expression in pulmonary tissue after respiratory infection with Pasteurella haemolytica. Cattle were infected with P. haemolytica via fiberoptic deposition of organisms into the posterior part of the right cranial lung lobe. Animals were euthanized at 2 or 4 h postinoculation (p.i.), and tissues were collected to assess PIC gene expression using antisense RNA probes specific for bovine interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) along with the beta-actin (beta-Act) housekeeping gene. Expression of PIC was induced at 2 h p.i. in P. haemolytica-infected cattle and continued to 4 h p.i. At 2 h p.i., induction of gene expression and increase of cells that expressed PIC were observed both in CD18(+) and CD18(-) cattle after inoculation of P. haemolytica. The induction of gene expression with P. haemolytica inoculation was more prominent in CD18(-) cattle than in CD18(+) cattle by comparison to pyrogen-free saline (PFS)-inoculated control animals. At 4 h p.i., however, the induction of PIC, especially IL-1alpha, IL-6, and IFN-gamma, in the lungs of CD18(+) cattle inoculated with P. haemolytica was greater than that in lungs of the CD18(-) cattle. IFN-gamma and TNF-alpha genes were not increased in P. haemolytica-inoculated CD18(-) cattle lungs compared to the PFS-inoculated control lungs at 4 h p.i. In PFS-inoculated lungs, we generally observed a higher percentage of cells and higher level of gene expression in the lungs of CD18(-) cattle than in the lungs of CD18(+) cattle, especially at 4 h p.i. The rate of neutrophil infiltration into the lungs of CD18(-) cattle at 2 h p.i. was significantly higher than that of CD18(+) cattle; at 4 h p.i., there was no difference between the two groups. These data suggest that beta(2)-integrins may contribute to the induction of expression of some PIC genes, as a consequence of P. haemolytica infection.     

海藻硫酸多糖对巨噬细胞所致肿瘤细胞凋亡的调节作用

目的 研究海藻硫酸多糖（sulfated polysaccharides from seaweed,SPS)对巨噬细胞（Mψ）所致HepG2细胞凋亡的调节作用。方法 用MTT比色法,检测Mψ产生的TNF，NO（尤其是NO）及SPS对HepG2细胞的细胞毒性作用。将Mψ与HepG2按一定比例共孵育后，检测培养上清中NO2^-/NO3^-及细胞中cGMP的含量；用透射电镜观察HepG2细胞凋亡的形态学改变。结果 SPS对HepG2细胞没有直接的细胞毒性，但可通过促进Mψ产生NO和TNF，增强Mψ对HepG2细胞的细胞毒性，增加共孵育细胞体系中cGMP的含量，并可明显促进HepG2细胞的凋亡。结论 SPS可间接增强Mψ对HepG2细胞的细胞毒性，并可明显促进HepG2细胞的凋亡。     

Selective activation of VH3A10+ rheumatoid factor producing B cells by staphylococcal enterotoxin D

Staphylococcal enterotoxin D (SED) is a T cell superantigenwhich selectively targets ß TCRs bearing particularV^ß elements. A second function of SED relates to thepreferential activation of a B cell subset characterized bya high frequency of rheumatoid factor (RF) producing B cells.To define the molecular basis of the SED-induced B cell repertoireshift, we have analyzed Ig heavy chain genes in B cell clonesexpanded after SED stimulation and compared them with B cellclones established in the presence of anti-CD3 stimulated helpercells. Gene segments of the V_H3 family were most frequentlyutilized under both stimulation conditions (42% anti-CD3; 47%SED). Sequence analysis of V_H3 gene segments demonstrated thatthe repertoire of V_H3 elements in B cell clones from SED drivenand anti-CD3 driven cultures were distinct (P=0.01). RF activitywas closely associated with the expression of selected V_H3 elements.B cell clones stimulated with SED preferentially expressed V_H3A10,whereas V_H26 was the gene segment dominantly used in B cellclones expanded with anti-CD3 stimulated helper cells. The usageof J_H and D_H elements was indistinguishable in SED and anti-CD3driven B cell clones, suggesting that SED targets V_H3⁺ B cellsthrough a V_H-specific mechanism. Comparison of the closely relatedsequences of the SED responsive V_H3A10 and the SED non-responsiveV_H26 element suggested a role of a sequence polymorphism inthe CDR2 reminiscent of B cell reactivity to conventional antigens.In contrast to conventional antigens, SED can induce differentiationof a high frequency of naive B cells. Thus, this staphylococcalenterotoxin combines selective activation of T cells with selectiveactivation of B cells and might be able to direct T cell helpto RF producing B cells.     

Interleukin-10-secreting "regulatory" T cells induced by glucocorticoids and beta2-agonists

Greater clinical benefit in controlling the symptoms of asthma is frequently observed through combining moderate doses of inhaled glucocorticoids together with long-acting beta(2)-agonists, as compared with increasing glucocorticoid dosage alone. To address in vitro whether glucocorticoids plus beta(2)-agonists, compared with glucocorticoids alone, have greater inhibitory activity on CD4+ T cell responses to allergen, peripheral blood CD4+ T cell responses to allergen were compared in the presence or absence of the glucocorticoid fluticasone proprionate and the short- and long-acting beta(2)-agonists salbutamol and salmeterol, respectively. Fluticasone proprionate inhibited interleukin (IL)-5 and IL-13 and enhanced IL-10 synthesis in allergen-stimulated cultures in a concentration-dependent manner. Salmeterol, but not salbutamol, inhibited IL-5 and IL-13 and enhanced IL-10 synthesis in these cultures. When used in combination the two drugs demonstrated an additive effect on this pattern of cytokine production. Allergen-specific T cell lines induced in the presence of salmeterol and fluticasone proprionate inhibited IL-5 and IL-13 production by allergen-specific Th2 cell lines in an IL-10-dependent manner. Thus fluticasone proprionate and salmeterol increased IL-10 and reduced Th2 cytokine synthesis additively in allergen stimulated human CD4+ T cells.     

Regulation of the IL-10 production by human T cells

Interleukin (IL)-10, an immunomodulatory cytokine predominantly produced by monocytes/macrophages and T cells, inhibits several functions of dendritic cells (DC), monocytes and T cells including their cytokine production, but it stimulates B cell immunoglobulin (Ig) production and cytotoxic T lymphocyte (CTL) generation. A precise knowledge of the mechanisms that control the IL-10 production is therefore highly important for understanding the immunoregulation. The IL-10 production was studied in cultures of freshly isolated human T cells. A rise in intracellular calcium as well as the common gamma-chain containing cytokine receptor triggering or CD28 triggering were found to be important signals for IL-10 induction. CD80, CD58, rIL-12 and rIFN-alpha all had efficacious and independent costimulatory activities on the IL-10 production, while PGE2 was inhibitory. Dependence on autocrine IL-2 signalling was shown by the effects of anti-IL-2 and anti-IL-2R monoclonal antibodies (MoAb), but the IL-10 production proceeded partly IL-2-independent when CD80 provided costimulation. Sensitivity to inhibition by CsA was not removed by CD80 or CD58 costimulation and/or by addition of rIL-12 or rIFN-alpha, pointing to the absolute requirement for calcineurin activity. These data reveal important differences in the regulatory pathways between IL-10 (a cytokine-inhibitory interleukin) and IL-2 (a cytokine-inducing interleukin), which can potentially be exploited therapeutically. The fact that CsA blocks the production of IL-10, which itself has important immunosuppressive properties, should be taken into account in defining immunosuppressive treatment schedules which include the use of CsA.     

The recognition of adsorbed and denatured proteins of different topographies by beta2 integrins and effects on leukocyte adhesion and activation

Leukocyte beta2 integrins Mac-1 and p150,95 are promiscuous cell-surface receptors that recognise and mediate cell adhesion to a variety of adsorbed and denatured proteins. We used albumin as a model protein to study whether leukocyte adhesion and activation depended on the nm-scale topography of a protein adlayer. Albumin adsorbed from the native conformation gave rise to different adlayer topographies and different amounts of adsorbed protein on hydrophobic and relatively hydrophilic polystyrene and silanised silicon-wafer surfaces, whereas adsorption of pre-denatured Alb resulted in similar adlayer topographies and similar amounts of adsorbed protein on these surfaces. All three distinct protein-adlayer topographies supported adhesion of in vitro differentiated, macrophage-like U937 and THP-1 cells, but did not support adhesion of their promonocytic precursors. Human monocytes freshly isolated from peripheral blood did not adhere to adsorbed albumin, not even in the presence of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1alpha chemokines. Adhesion of the macrophage-like cells to albumin in any of the three topographies was inhibited by antibodies against beta2 integrins, but not by antibodies against beta1 integrins, and did not induce secretion of the proinflammatory cytokine tumour necrosis factor-alpha.     

Regulation of desmosomal cell adhesion in human tumour cells by polyunsaturated fatty acids

Desmosomes are key structures in cell-cell adhesion. In this study we examined the effect of n-6 essential fatty acids on the expression of desmoglein (Dsg), desmosomal cadherin and the formation of desmosomes in E-cadherin negative human breast, colon and lung cancer cells and melanoma cells. Electron microscopy revealed that cells cultured with gamma linolenic acid (GLA) showed increased cell-cell adhesion together with an increase in the formation of desmoglein-containing desmosomes. Western blotting studies of cellular proteins demonstrated that, following culture with fatty acids, Dsg expression was modified, with the greatest increase seen after GLA treatment. Other fatty acids increased Dsg expression, but to a lesser extent. It is concluded that GLA regulates desmosome-mediated cell-cell adhesion in human cancer cells, particularly in cells without E-cadherin.     

Inhibition of IgE-induced activation of human mast cells by IL-10

BACKGROUND: IL-10 exhibits anti-inflammatory effects on activated rodent mast cells (MC) in vitro and inhibits allergen-induced airway inflammation in vivo in murine models. The effects of IL-10 on the allergic activation of human MC are presently unknown. OBJECTIVE: In light of the well-known heterogeneity of mast cell reactivity between animal species, one cannot readily predict the response of human MC to IL-10. Moreover, the impact of IL-10 on MC-derived proinflammatory mediators is still unknown. Thus, the objective of this study was to investigate the effects of IL-10 on the release of inflammatory mediators by IgE/anti-IgE-challenged human cord blood-derived mast cells (CBMC), used as an in vitro model of MC phenotypically similar to human lung MC. MATERIALS AND METHODS: Highly purified human MC were obtained by a first step of long-term culture of cord blood mononuclear cells in the presence of human recombinant stem cell factor (rhSCF) and of human recombinant IL-6 (rhIL-6), followed by a second step of purification by depletion of contaminating cells with an immunomagnetic METHOD: The cells were treated with human IgE, then challenged with anti-human IgE, in the presence or the absence of recombinant rhIL-10 used at various concentrations. Histamine, tumour necrosis factor-alpha (TNF-alpha), IL-5 and IL-8 were measured in the various supernatants collected at different times after the beginning of the challenge. RESULTS: IL-10 inhibited the release of TNF-alpha and of IL-8, but not of IL-5, by activated CBMC. Interestingly, IL-10 also inhibited the release of histamine by activated CBMC, contrasting with data reported for rodent MC. CONCLUSIONS: These findings suggest that IL-10 might have anti-inflammatory effects on IgE/anti-IgE-challenged human MC by inhibiting their release of TNF-alpha, IL-8 and histamine. These data provide new insights into the control of human mast cell activation and might lead to a better knowledge of the cellular mechanisms controlling allergic reactions.