Adhesion-dependent interactions between eosinophils and cholinergic nerves

Eosinophils adhere to airway cholinergic nerves and influence nerve cell function by releasing granule proteins onto inhibitory neuronal M(2) muscarinic receptors. This study investigated the mechanism of eosinophil degranulation by cholinergic nerves. Eosinophils were cocultured with IMR32 cholinergic nerve cells, and eosinophil peroxidase (EPO) or leukotriene C(4) (LTC(4)) release was measured. Coculture of eosinophils with nerves significantly increased EPO and LTC(4) release compared with eosinophils alone. IMR32 cells, like parasympathetic nerves, express the adhesion molecules vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 (ICAM-1). Inhibition of these adhesion molecules alone or in combination significantly inhibited eosinophil degranulation. IMR32 cells also significantly augmented the eosinophil degranulation produced by formyl-Met-Leu-Phe. Eosinophil adhesion to IMR32 cells resulted in an ICAM-1-mediated production of reactive oxygen species via a neuronal NADPH oxidase, inhibition of which significantly inhibited eosinophil degranulation. Additionally, eosinophil adhesion increased the release of ACh from IMR32 cells. These neuroinflammatory cell interactions may be relevant in a variety of inflammatory and neurological conditions.     

Induction and function of eosinophil intercellular adhesion molecule-1 and HLA-DR.

We have previously established that eosinophils studied ex vivo from the sputum of asthmatics express intercellular adhesion molecule-1 (ICAM-1) and HLA-DR, whereas peripheral blood eosinophils do not express these surface proteins. On incubation of highly purified (greater than 99.5% pure) blood eosinophils from normal subjects with T cell supernatants, eosinophil ICAM-1 was induced in 24 h, whereas HLA-DR was maximally induced within 48 h. Recombinant cytokines that enable eosinophil survival (IL-5, IL-3, and granulocyte macrophage-CSF) were found to be unable to induce ICAM-1 or HLA-DR, even when pooled at concentrations individually required for eosinophil survival. However, synergy between these eosinophil survival factors and TNF (-alpha and -beta) was found mainly responsible for ICAM-1 induction, whereas synergy between IL-3 and IFN-gamma occurred for HLA-DR induction. Culture of eosinophils in the presence of cytokines and cycloheximide prevented expression of ICAM-1 and HLA-DR, showing that de novo eosinophil protein synthesis is occurring. At a functional level we demonstrate that ICAM-1-bearing eosinophils have increased adhesion capacity for autologous T cells. In contrast, HLA-DR-expressing eosinophils mediated Ag-specific proliferation of an autologous HLA-DR-restricted T cell clone that was inhibitable by anti-HLA-DR and anti-ICAM-1 mAb. Since eosinophil-mediated Ag presentation was inhibitable by treatment of eosinophils with glutaraldehyde or chloroquine, this suggests that eosinophils participate in Ag uptake, processing, and presentation and have accessory functions. Thus, through the induction of ICAM-1 and HLA-DR on tissue eosinophils, eosinophils have the capacity to interact with leukocytes and present Ag to T cells.     

Eosinophil adhesion to cholinergic nerves via ICAM-1 and VCAM-1 and associated eosinophil degranulation

In vivo, eosinophils localize to airway cholinergic nerves in antigen-challenged animals, and inhibition of this localization prevents antigen-induced hyperreactivity. In this study, the mechanism of eosinophil localization to nerves was investigated by examining adhesion molecule expression by cholinergic nerves. Immunohistochemical and functional studies demonstrated that primary cultures of parasympathetic nerves express vascular cell adhesion molecule-1 (VCAM-1) and after cytokine pretreatment with tumor necrosis factor-alpha and interferon-gamma intercellular adhesion molecule-1 (ICAM-1). Eosinophils adhere to these parasympathetic neurones after cytokine pretreatment via a CD11/18-dependent pathway. Immunohistochemistry and Western blotting showed that a human cholinergic nerve cell line (IMR-32) expressed VCAM-1 and ICAM-1. Inhibitory experiments using monoclonal blocking antibodies to ICAM-1, VCAM-1, or CD11/18 and with the very late antigen-4 peptide inhibitor ZD-7349 showed that eosinophils adhered to IMR-32 cells via these adhesion molecules. The protein kinase C signaling pathway is involved in this process as a specific inhibitor-attenuated adhesion. Eosinophil adhesion to IMR-32 cells was associated with the release of eosinophil peroxidase and leukotriene C(4). Thus eosinophils adhere to cholinergic nerves via specific adhesion molecules, and this leads to eosinophil activation and degranulation; this may be part of the mechanism of eosinophil-induced vagal hyperreactivity.     

Regulation of eosinophil function by phosphatidylinositol-specific PLC and cytosolic PLA(2)

We examined the regulatory role of cytosolic phospholipase A(2) (cPLA(2)) and phosphatidylinositol (PI)-specific phospholipase C (PLC) in the degranulation of human eosinophils and leukotriene (LT) C(4) synthesis. Activation with formyl-Met-Leu-Phe + cytochalasin B (fMLP/B) caused a time-dependent release of eosinophil peroxidase (EPO) and LTC(4), which was inhibited by pertussis toxin. By immunoblotting, eosinophil PLC-beta2 and -gamma2 isoforms were identified, and PLC activation was measured as a function of inositol 1,4,5-trisphosphate concentration. Stimulated release of EPO and intracellular Ca(2+) concentration was inhibited by ET-18-OCH(3), a PI-PLC inhibitor, whereas trifluoromethylketone (TFMK), a cPLA(2) blocker, had no inhibitory effect. Both TFMK and ET-18-OCH(3) attenuated stimulated arachidonate release and LTC(4) secretion, suggesting that activation of both PLC and cPLA(2) is essential for LTC(4) synthesis caused by fMLP/B. The structurally unrelated protein kinase C inhibitors bisindolylmaleimide, Ro-31-8220, and Go-6976 all blocked fMLP/B-induced EPO release but not LTC(4) secretion. 1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethyl ester, an intracellular Ca(2+) chelator, suppressed both EPO release and LTC(4) secretion. We found that fMLP/B-induced LTC(4) secretion from human eosinophils is regulated by PI-PLC through calcium-mediated activation of cPLA(2). However, cPLA(2) does not regulate eosinophil degranulation.     

Eosinophil transendothelial migration induced by cytokines. I. Role of endothelial and eosinophil adhesion molecules in IL-1 beta-induced transendothelial migration.

IL-1 beta promotes adhesiveness in human umbilical vein endothelial cells (HuVEC) for eosinophils through expression of adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). Using an in vitro endothelial monolayer system, we examined whether IL-1 beta or TNF-alpha can promote eosinophil transendothelial migration. We also evaluated the contributions of ICAM-1, E-selectin, VCAM-1, leukocyte adhesion complex (CD11/18), and very late Ag-4 (CD11b/18) (VLA-4) in this process using blocking mAb, and determined the changes in expression of CD11b and L-selectin on eosinophils that had undergone transmigration. IL-1 beta and TNF-alpha treatment of HuVEC (4 h, 5 ng/ml) induced significant transendothelial migration of eosinophils (a 4.1 +/- 0.4-fold (IL-1 beta) and 2.0 +/- 0.9-fold (TNF-alpha) increase from the spontaneous value of 3.2 +/- 0.3%). Increased CD11b expression and shedding of L-selectin were observed on eosinophils following IL-1 beta-induced eosinophil transendothelial migration. Studies with mAb revealed that blockade of either ICAM-1 or CD11/18 inhibited transmigration, while antibodies against VCAM-1 and VLA-4 had no inhibitory effect. Among antibodies which block beta 2 integrins, anti-CD18 mAb had the best inhibitory effect (88% inhibition). The combined inhibitory effect of anti-CD11a mAb and anti-CD11b mAb was roughly equal to that of anti-CD18, although anti-CD11a (31% inhibition) and anti-CD11b (52% inhibition) were less effective individually. Anti-ICAM-1 by itself inhibited IL-1 beta-induced eosinophil transendothelial migration (24% inhibition) whereas neither anti-E-selectin nor anti-VCAM-1 were effective inhibitors. Interestingly, the combination of anti-E-selectin and anti-VCAM-1 with anti-ICAM-1 inhibited IL-1 beta-induced eosinophil transendothelial migration significantly better (53% inhibition) than anti-ICAM-1 alone. These results suggest that although the initial attachment of eosinophils to IL-1 beta-activated endothelial cells involves VCAM-1, E-selectin, and ICAM-1, the subsequent transendothelial migration process relies heavily on ICAM-1 and CD11/18. Finally, the changes that eosinophils have been observed to undergo during infiltration in vivo, namely increased expression of CD11/18 and shedding of L-selectin, appear to take place as a direct result of the interaction between eosinophils and endothelial cells.     

Mechanism of eosinophil induced signaling in cholinergic IMR-32 cells

Eosinophils interact with nerve cells, leading to changes in neurotransmitter release, altered nerve growth, and protection from cytokine-induced apoptosis. In part, these interactions occur as a result of activation of neural nuclear factor (NF)-kappaB, which is activated by adhesion of eosinophils to neural intercellular adhesion molecule-1 (ICAM-1). The mechanism and consequence of signaling after eosinophil adhesion to nerve cells were investigated. Eosinophil membranes, which contain eosinophil adhesion molecules but not other eosinophil products, were coincubated with IMR-32 cholinergic nerve cells. The studies showed that there were two mechanisms of activation of NF-kappaB, one of which was dependent on reactive oxygen species, since it was inhibited with diphenyleneiodonium. This occurred at least 30 min after coculture of eosinophils and nerves. An earlier phase of NF-kappaB activation occurred within 2 min of eosinophil adhesion and was mediated by tyrosine kinase-dependent phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1). Coimmunoprecipitation experiments showed that both extracellular signal-regulated kinase 1/2 and IRAK-1 were recruited to ICAM-1 rapidly after coculture with eosinophil membranes. This was accompanied by an induction of ICAM-1, which was mediated by an IRAK-1-dependent pathway. These data indicate that adhesion of eosinophils to IMR-32 nerves via ICAM-1 leads to important signaling events, mediated via IRAK-1, and these in turn lead to expression of adhesion molecules.     

Transcellular secretion of group V phospholipase A2 from epithelium induces beta 2-integrin-mediated adhesion and synthesis of leukotriene C4 in eosinophils

We examined the mechanism by which secretory group V phospholipase A(2) (gVPLA(2)) secreted from stimulated epithelial cells activates eosinophil adhesion to ICAM-1 surrogate protein and secretion of leukotriene (LT)C(4). Exogenous human group V PLA(2) (hVPLA(2)) caused an increase in surface CD11b expression and focal clustering of this integrin, which corresponded to increased beta(2) integrin-mediated adhesion. Human IIaPLA(2), a close homolog of hVPLA(2), or W31A, an inactive mutant of hVPLA(2), did not affect these responses. Exogenous lysophosphatidylcholine but not arachidonic acid mimicked the beta(2) integrin-mediated adhesion caused by hVPLA(2) activation. Inhibition of hVPLA(2) with MCL-3G1, a mAb against gVPLA(2), or with LY311727, a global secretory phospholipase A(2) (PLA(2)) inhibitor, attenuated the activity of hVPLA(2); trifluoromethylketone, an inhibitor of cytosolic group IVA PLA(2) (gIVA-PLA(2)), had no inhibitory effect on hVPLA(2)-mediated adhesion. Activation of beta(2) integrin-dependent adhesion by hVPLA(2) did not cause ERK1/2 activation and was independent of gIVA-PLA(2) phosphorylation. In other studies, eosinophils cocultured with epithelial cells were stimulated with FMLP/cytochalasin B (FMLP/B) and/or endothelin-1 (ET-1) before LTC(4) assay. FMLP/B alone caused release of LTC(4) from eosinophils, which was augmented by coculture with epithelial cells activated with ET-1. Addition of MCL-3G1 to cocultured cells caused approximately 50% inhibition of LTC(4) secretion elicited by ET-1, which was blocked further by trifluoromethylketone. Our data indicate that hVPLA(2) causes focal clustering of CD11b and beta(2) integrin adhesion by a novel mechanism that is independent of arachidonic acid synthesis and gIVA-PLA(2) activation. We also demonstrate that gVPLA(2), endogenously secreted from activated epithelial cells, promotes secretion of LTC(4) in cocultured eosinophils.     

Structural determinants of blockade of eosinophil activation, adhesion and secretion by synthetic analogs of phomactin

We examined the structural determinants of phomactin analogs to assess their efficacy as antagonist of PAF. Six analogs of phomactin were synthesized to determine their inhibitory effects on adhesion, superoxide release, leukotriene C4 (LTC4) synthesis and [3H]PAF binding in human eosinophils. Phomactin analogs inhibited both PAF- and IL-5-induced eosinophil adhesion. Analog A, which bears an alkene moiety between C-1 and C-14, a ketone at the C-2 position, and an alkyne moiety between C-3 and C-4, had the greatest anti-adhesive effect. Change of the alkene between C-1 and C-14 to an alkane (analog I) decreased the anti-adhesive effect by 2.5-4 fold, while substitution of ketone by hydroxyl (analog G) at the C-2 position caused an 11-fold decrease in the anti-adhesive effect. Substitution of the alkyne moiety between C-3 and C-4 by an alkene (B and E) or alkane (D) blocked completely the anti-adhesive effect. Analogs A and I completely blocked superoxide release from eosinophils caused by phorbol-12-myristate-13-acetate or PAF and LTC4-release caused by fMLP plus cytochalasin B. Change of the alkyne moiety between C-3 and C-4 to an alkene (B and E) or alkane (D) blocked completely these inhibitory effects of phomactin. Analog A decreased the maximal binding of [3H]PAF binding to eosinophils without change of the apparent dissociation constant. We conclude that phomactin analogs are specific non-competitive PAF antagonists and have exceptional efficacy in inhibiting adhesion, metabolic activity and leukotriene secretion in human eosinophils. We further define the structural alterations in the phomactin molecule that regulate its inhibitory functions.     

Regulation of eosinophil trafficking by SWAP-70 and its role in allergic airway inflammation

Eosinophils are the predominant inflammatory cells recruited to allergic airways. In this article, we show that human and murine eosinophils express SWAP-70, an intracellular RAC-binding signaling protein, and examine its role in mediating eosinophil trafficking and pulmonary recruitment in a murine model of allergic airway inflammation. Compared with wild-type eosinophils, SWAP-70-deficient (Swap-70(-/-)) eosinophils revealed altered adhesive interactions within inflamed postcapillary venules under conditions of blood flow by intravital microscopy, exhibiting enhanced slow rolling but decreased firm adhesion. In static adhesion assays, Swap-70(-/-) eosinophils adhered poorly to VCAM-1 and ICAM-1 and exhibited inefficient leading edge and uropod formation. Adherent Swap-70(-/-) eosinophils failed to translocate RAC1 to leading edges and displayed aberrant cell surface localization/distribution of α4 and Mac-1. Chemokine-induced migration of Swap-70(-/-) eosinophils was significantly decreased, correlating with reduced intracellular calcium levels, defective actin polymerization/depolymerization, and altered cytoskeletal rearrangement. In vivo, recruitment of eosinophils to the lungs of allergen-challenged Swap-70(-/-) mice, compared with wild-type mice, was significantly reduced, along with considerable attenuation of airway inflammation, indicated by diminished IL-5, IL-13, and TNF-α levels; reduced mucus secretion; and improved airway function. These findings suggest that regulation of eosinophil trafficking and migration by SWAP-70 is important for the development of eosinophilic inflammation after allergen exposure.     

LFA-3 plasmid DNA enhances Ag-specific humoral- and cellular-mediated protective immunity against herpes simplex virus-2 in vivo: involvement of CD4+ T cells in protection

Adhesion molecules are important for cell trafficking and delivery of secondary signals for stimulation of T cells and antigen-presenting cells (APCs) in a variety of immune and inflammatory responses. Adhesion molecules lymphocyte function-associated antigen (LFA)-1 and CD2 on T cells recognize intercellular adhesion molecule (ICAM)-1 and LFA-3 on APCs, respectively. Recent studies have suggested that these molecules might play a regulatory role in antigen-specific immune responses. To investigate specific roles of adhesion molecules in immune induction we coimmunized LFA-3 and ICAM-1 cDNAs with a gD plasmid vaccine and then analyzed immune modulatory effects and protection against lethal herpes simplex virus (HSV)-2 challenge. We observed that gD-specific IgG production was enhanced by LFA-3 coinjection. However, little change in IgG production was observed by ICAM-1 coinjection. Furthermore, both Th1 and Th2 IgG isotype production was driven by LFA-3. LFA-3 also enhanced Th cell proliferative responses and production of interleukin (IL)-2, interferon-gamma, IL-4, and IL-10 from splenocytes. In contrast, ICAM-1 showed slightly increasing effects on T-cell proliferation responses and cytokine production. beta-Chemokine production (RANTES, MIP-1alpha, and MCP-1) was also influenced by LFA-3 or ICAM-1. When animals were challenged with a lethal dose of HSV-2, LFA-3-coimmunized animals exhibited an enhanced survival rate, as compared to animals given ICAM-1 or gD DNA vaccine alone. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vitro and in vivo T-cell subset deletion. These studies demonstrate that adhesion molecule LFA-3 can play an important role in generating protective antigen-specific immunity in the HSV model system through increased induction of CD4+ Th1 T-cell subset.     

Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.     

Cross-talk between ICAM-1 and granulocyte-macrophage colony-stimulating factor receptor signaling modulates eosinophil survival and activation

Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of asthma pathogenesis. In this regard, GM-CSF is a primary candidate cytokine regulating eosinophil activation and survival in the lung; however, its molecular mechanism of propagation and maintenance of stimulated eosinophil activation is not well understood. In this study, we elucidate those late interactions occurring between the GM-CSF receptor and activated eosinophil signaling molecules. Using coimmunoprecipitation with GM-CSF-stimulated eosinophils, we have identified that the GM-CSF receptor beta-chain (GMRbeta) interacted with ICAM-1 and Shp2 phosphatase, as well as Slp76 and ADAP adaptor proteins. Separate experiments using affinity binding with a tyrosine-phosphorylated peptide containing an ITIM (ICAM-1 residues 480-488) showed binding to Shp2 phosphatase and GMRbeta. However, the interaction of GMRbeta with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophil lysates, suggesting that the interaction of GMRbeta with ICAM-1 required phosphorylated Shp2 and/or phosphorylated GMRbeta. Importantly, we found that inhibition of ICAM-1 in activated eosinophils blocked GM-CSF-induced expression of c-fos, c-myc, IL-8, and TNF-alpha. Moreover, inhibition of ICAM-1 expression with either antisense oligonucleotide or an ICAM-1-blocking Ab effectively inhibited ERK activation and eosinophil survival. We concluded that the interaction between ICAM-1 and the GM-CSF receptor was essential for GM-CSF-induced eosinophil activation and survival. Taken together, these results provide novel mechanistic insights defining the interaction between ICAM-1 and the GM-CSF receptor and highlight the importance of targeting ICAM-1 and GM-CSF/IL-5/IL-3 receptor systems as a therapeutic strategy to counter eosinophilia in asthma.     

The dependence for leukocyte function-associated antigen-1/ICAM-1 interactions in T cell activation cannot be overcome by expression of high density TCR ligand

The leukocyte-specific integrin, LFA-1, can enhance T cell activation. However, it is unclear whether the binding of LFA-1 to its ligand, ICAM-1, functions through intercellular adhesion alone, resulting in an augmentation of the TCR signal, or involves an additional LFA-1-mediated cellular signal transduction pathway. We have previously shown that naive CD4+ lymph node T cells, isolated from DO11.10 TCR transgenic mice, are activated by increasing doses of exogenous OVA peptide presented by transfectants expressing both class II and ICAM-1, but not by cells expressing class II alone. To determine whether LFA-1/ICAM-1 interactions were simply enhancing the presentation of low concentrations of specific MHC/peptide complexes generated from exogenously added peptide, we transfected cells with class II that is covalently coupled to peptide, alone or in combination with ICAM-1. These cells express 100-fold more specific class II/peptide complexes than can be loaded onto class II-positive cells at maximum concentrations of exogenous peptide. Despite this high density of TCR ligand, activation of naive CD4+ T cells still requires the coexpression of ICAM-1. LFA-1/ICAM-1 interactions are not required for effective conjugate formation and TCR engagement because presentation of class II/peptide complexes in the absence of ICAM-1 does induce up-regulation of CD25 and CD69. Thus, high numbers of engaged TCR cannot compensate for the lack of LFA-1/ICAM-1 interactions in the activation of naive CD4+ T cells.     

Cytosolic phospholipase A2 activation is essential for beta 1 and beta 2 integrin-dependent adhesion of human eosinophils.

We examined the role of cytosolic phospholipase A2 (cPLA2) during human eosinophil adherence to ICAM-1- or VCAM-1-coated plates. IL-5-stimulated eosinophils adhered to ICAM-1 through the beta 2 integrin CD11b/CD18, while nonstimulated eosinophils did not. By contrast, nonstimulated eosinophils adhered to VCAM-1 through the beta 1-integrin VLA-4/CD29. Both IL-5-induced adhesion to ICAM-1 and spontaneous adhesion to VCAM-1 corresponded temporally to cPLA2 phosphorylation, which accompanied enhanced catalytic activity of cPLA2. The structurally unrelated cPLA2 inhibitors, arachidonyl trifluoromethylketone and surfactin, significantly inhibited eosinophil adhesion to ICAM-1 and VCAM-1 in a concentration-dependent manner. Inhibition of secretory PLA2, 5-lipoxygenase, or cyclooxygenase did not affect eosinophil adhesion. Addition of arachidonic acid to eosinophils after cPLA2 inhibition with arachidonyl trifluoromethylketone or surfactin did not reverse the blockade of adhesion to ICAM-1 or VCAM-1. However, CV-6209, a receptor-specific antagonist of platelet-activating factor, inhibited all integrin-mediated adhesion. The activated conformation of CD11b as identified by the mAb, CBRM1/5, as well as quantitative surface CD11b expression were up-regulated after IL-5 stimulation. However, cPLA2 inhibition neither prevented CBRM1/5 expression nor blocked surface Mac-1 up-regulation caused by IL-5. Our data suggest that cPLA2 activation and its catalytic product platelet-activating factor play an essential role in regulating beta 1 and beta 2 integrin-dependent adhesion of eosinophils. This blockade occurs even in the presence of up-regulated eosinophil surface integrin.     

Differential regulatory effects of intercellular adhesion molecule-1 on costimulation by the CD28 counter-receptor B7.

Although ligation of the CD3/TCR complex initiates an activation signal in T cells, additional costimulatory signals generated during cell-to-cell interactions with APC transduced via ligation of CD11a/CD18 and CD28 by their specific counter-receptor intercellular adhesion molecule (ICAM)-1 and B7, respectively, are required for optimal T cell proliferation and cytokine synthesis. Using soluble IgC gamma 1 fusion proteins of these costimulatory counter-receptors, we have recently shown that unactivated resting CD4+ T cells and Ag-primed CD4+ T cells differ in their response to the costimulation by ICAM-1 and B7. Preferential proliferative responses of resting T and Ag-primed T cells to ICAM-1 and B7, respectively, prompted us to speculate that ICAM-1-induced signals may regulate coupling of the CD28 signaling pathway. Furthermore, both B7 and ICAM-1 are co-expressed on APC and thus, may co-regulate activation-driven maturation of T cells. In this study, we have examined regulatory effects of IgC gamma 1 fusion proteins of B7, ICAM-1, and ICAM-2 (a homologue of ICAM-1) on each other's costimulation. We first demonstrate that TCR-directed costimulation of resting CD4+ T cells with ICAM-1 (ICAM-1 priming) but not ICAM-2 induces increased responsiveness to B7. Priming of CD4+ T cells with ICAM-1 induced higher expression of both CD18 and CD28 than that with either B7 or ICAM-2. Cross-linking of CD28 induced faster and significantly higher cytoplasmic free calcium mobilization response in ICAM-1-primed CD4+ T cells than in resting, B7-primed, or ICAM-2-primed CD4+ T cells. B7 synergized with ICAM-1 but not ICAM-2 to augment proliferative responses of not only resting CD4+ T cells but also those that had been primed with either ICAM. Unlike resting or ICAM-2-primed CD4+ T cells, ICAM-1-primed CD4+ T cells efficiently proliferated in response to the synergistic costimulation of B7 and ICAM-2. In contrast, both ICAM-1 and ICAM-2 inhibit B7-driven proliferation of Ag-primed CD4+ T cells. Thus, B7 and ICAM-1 exert contrasting regulatory effects on the proliferation of CD4+ T cells depending on their state of activation-induced maturation.     

Immune restoration in head and neck cancer patients after in vivo COX-2 inhibition

Purpose To determine the immunomodulatory effects of in vivo COX-2 inhibition on leukocyte infiltration and function in patients with head and neck cancer. Experimental design Patients with squamous cell carcinoma of the head and neck preoperatively received a specific COX-2 inhibitor (rofecoxib, 25 mg daily) orally for 3 weeks. Serum and tumor specimens were collected at the start of COX-2 inhibition (day 0) and again on the day of surgery (day 21). Adhesion to peripheral blood monocytes to ICAM-1 was examined. Percentages of tumor-infiltrating monocytes (CD68, CCR5) and lymphocytes (CCR5, CD4, CD8 and CD25) were determined by immunohistochemistry. Results Monocytes obtained from untreated cancer patients showed lower binding to ICAM-1 compared to monocytes of healthy donors but significantly regained adhesion affinity following incubation in sera of healthy donors. Conversely, sera of cancer patients inhibited adhesion of healthy donors’ monocytes. Tumor monocyte adhesion to ICAM-1 was increased (P < 0.001) after 21 days of COX-2 inhibition, and concomitant increases in tumor infiltrating monocytes (CD68+), lymphocytes (CD68− CCR5+, CD4+ and CD8+) and activated (CD25+) T cells were observed. Conclusions Short-term administration of a COX2 inhibitor restored monocyte binding to ICAM-1 and increased infiltration into the tumor of monocytes and Th1 and CD25+ activated lymphocytes. Thus, in vivo inhibition of the COX-2 pathway may be useful in potentiating specific active immunotherapy of cancer.     

Enhancement of human eosinophil cytotoxicity and leukotriene synthesis by biosynthetic (recombinant) granulocyte-macrophage colony-stimulating factor

Culture medium conditioned by activated human T lymphocytes enhances the in vitro cytotoxicity of purified human eosinophils toward Schistosoma mansoni larvae, suggesting the existence of a mechanism for T lymphocyte regulation of eosinophil function. Here we show that purified biosynthetic (recombinant) human T lymphocyte granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced markedly two eosinophil functions: cytotoxicity toward schistosomula by a mean of 676%, and calcium ionophore A23187-induced generation of leukotriene C4 (LTC4) by a mean of 135%. Augmentation of each eosinophil function by GM-CSF was time- and dose-dependent, with a dose-response relationship at concentrations between 1 and 20 pM. Tumor necrosis factor (TNF) enhanced eosinophil cytotoxicity with slower kinetics, a different dose-dependence relationship, and to a lower maximum, as compared with GM-CSF. There was no detectable effect of TNF on calcium ionophore A23187-induced generation of LTC4. The effect of GM-CSF on arachidonic acid metabolism to LTC4 reached a plateau with 60 min of incubation before stimulation with ionophore, and was characterized by an initial augmentation of the intracellular level of LTC4 and a subsequent increment in extracellular LTC4. Thus, GM-CSF can serve as a mediator for T lymphocyte regulation of functions of mature eosinophils. It is also the first defined macromolecule known to enhance metabolism of membrane-derived arachidonic acid via the 5-lipoxygenase pathway.     

Augmentation of LTC(4) synthesis in human eosinophils caused by CD3-stimulated Th2-like cells in vitro

We assessed the effect of anti-CD3-stimulated secretion of cultured human Th1- and Th2-like cells on leukotriene C(4) (LTC(4)) secretion in isolated human eosinophils. T helper (Th) cell subsets were generated from human naive CD4(+) T cells cocultured with irradiated human transformed B cells and either recombinant human interleukin (rhIL)-1beta plus rhIL-6 plus rhIL-12 for Th1-like cells or rhIL-1beta plus rhIL-6 plus rhIL-4 for Th2-like cells. Coincubation of eosinophils with 1:5 dilution of Th2-supernatant (Sup) caused an increase in LTC(4) secretion caused by 0.1 microM formyl-Met-Leu-Phe and 5 microg/ml cytochalasin B from 921 +/- 238 to 3,067 +/- 1,462 pg/10(6) eosinophils (P < 0.01). Th1-Sup at the same dilution had no augmenting effect on stimulated secretion of LTC(4) in eosinophils despite substantial concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the supernatant. Dilution of Th1-Sup caused increased LTC(4) that returned to baseline after immunoabsorption of GM-CSF, suggesting the presence of a possible inhibitory factor. We demonstrate that pretreatment of eosinophils with 1:5 dilution of Th2-Sup but not of Th1-Sup causes substantial augmentation of LTC(4) secretion in vitro and establishes that human Th2 cells cause direct augmentation of LTC(4) secretion within 15-30 min of exposure.     

The role of CD11a/CD18-CD54 interactions in human T cell-dependent B cell activation.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in human T cell and B cell collaboration was examined by studying the effect of mAb to these determinants on B cell proliferation and differentiation stimulated by culturing resting B cells with CD4+ T cells activated with immobilized mAb to the CD3 molecular complex. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) inhibited B cell responses significantly. The mAb did not directly inhibit B cell function, inasmuch as T cell-independent activation induced by formalinized Staphylococcus aureus and IL-2 was not suppressed. Moreover, DNA synthesis and IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells were not suppressed by the mAb to LFA-1 or ICAM-1. Although the mAb to LFA-1 inhibited enhancement of IL-2 production by co-culture of immobilized anti-CD3-stimulated CD4+ T cells with B cells, addition of exogenous IL-2 or supernatants of mitogen-activated T cells could not abrogate the inhibitory effects of the mAb to LFA-1 or ICAM-1 on B cell responses. Inhibition was most marked when the mAb were present during the initial 24 h in culture. Immobilized anti-CD3-stimulated LFA-1-negative CD4+ T cell clones from a child with leukocyte adhesion deficiency could induce B cell responses, which were inhibited by mAb to LFA-1 or ICAM-1. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the collaboration between activated CD4+ T cells and B cells necessary for the induction of B cell proliferation and differentiation, and for enhancement of IL-2 production by CD4+ T cells. Moreover, the data are consistent with a model of T cell-B cell collaboration in which interactions between LFA-1 on resting B cells and ICAM-1 on activated CD4+ T cells play a critical role in initial T cell-dependent B cell activation.     

T cell subsets and their soluble products regulate eosinophilia in allergic and nonallergic asthma.

Lymphokines derived from activated T cells regulate the proliferation and postmitotic differentiation of eosinophils in vitro. We investigated whether peripheral blood eosinophilia, which is a characteristic feature of both allergic and nonallergic asthma, correlates with T cell activation and lymphokine production in asthmatic patients. Flow cytometric analysis of T cell activation markers revealed that asthmatic individuals are characterized by increased numbers of IL-2R (CD25)-bearing T cell subsets. The absolute number of IL-2R+ T cells correlated with the eosinophilia observed in the asthmatic patients. Purified CD4+ and CD8+ T cells from allergic and nonallergic asthmatic individuals spontaneously secreted factors that extend the lifespan of eosinophils in vitro. T cells from normal donors displayed this effect only after polyclonal stimulation with anti-CD3 antibody. The eosinophil lifespan-extending factors were also found in sera of asthmatic patients. Identification of these factors was performed by using neutralizing antibodies against IL-3, IL-5, and granulocyte-macrophage CSF. In sera, mainly IL-5 and granulocyte-macrophage CSF were responsible for prolonged eosinophil survival, whereas granulocyte-macrophage CSF was dominant in T cell supernatants. These results indicate that T cells and secretion of lymphokines play an important regulatory function toward eosinophils, which are thought to represent major proinflammatory effector cells in certain types of asthma.