Inhibition of human eosinophil chemotaxis and of the IgE-dependent stimulation of human blood platelets by cetirizine

Cetirizine is a new anti-allergic compound with a potent, long-acting, and specific antihistaminic property. Strongly active in the therapy of urticaria and seasonal or perennial rhinitis, it has been shown to inhibit the in vivo eosinophil attraction at skin sites challenged with allergen in atopic patients. In the present work, we confirmed that, at a therapeutical concentration, this molecule had a potent inhibitory action in vitro on eosinophil chemotaxis induced either by N-formyl-Met-Leu-Phe or platelet-activating factor and also on the IgE-dependent stimulation of platelets. These observations appear in favour of a possible role for cetirizine in the modulation of inflammatory cell interactions in allergic processes.     

Inhibition of human eosinophil chemotaxis by IgA paraproteins

Sera from patients with IgA myeloma inhibit normal human eosinophil chemotaxis. No correlation was noted between inhibition and the absolute concentration of IgA or - light-chain type. Eosinophil chemotactic inhibitory activity was associated with isolated IgA paraproteins and was found to be cell directed and stable at 56° C. Pepsin digestion of IgA paraproteins resulted in loss of both IgA Fc fragment and eosinophil chemotactic inhibitory activity. Polymeric IgA accounted for most of the inhibitory activity as evidenced by sucrose density gradient centrifugation studies and a loss of inhibitory activity following dithiothrietol reduction and iodoacetamide alkylation which converted polymeric IgA to monomeric IgA. Comparative studies with neutrophils showed that both neutrophil and eosinophil chemotaxis and chemokinesis were effectively inhibited by IgA paraproteins. The mechanisms of suppression of eosinophil and neutrophil chemotaxis by IgA paraproteins appear to be similar and possibly may involve a membrane receptor for IgA.     

Pharmacologic profile of a new antiallergic compound PRD-92-Ea.

PRD-92-Ea [5,5-Dimethyl-11-oxo-5H, 11H-(2) benzopyrano (4,3-g) (1) benzopyran-9-carboxylic acid ethanolamine], was an active antiallergic compound in rat and monkey experimental models of immediate hypersensitivity. It inhibited, in a dose-dependent manner, the rat PCA reaction after both intravenous and oral administration. It also inhibited the degranulation of rat peritoneal mast cells after antigenic challenge. PRD-92-Ea was also active in preventing bronchoconstriction in Ascaris-sensitive Rhesus monkeys after intravenous, topical and oral administration. Using chopped monkey tissues, it was found that PRD-92-Ea prevented histamine release from the respiratory mast cells, but not from the cutaneous mast cells. No reason for this dichotomy of effect is known. PRD-92-Ea showed antagonistic activity against the allergic mediators released from mast cells. In order of decreasing potency it was active against SRS-A (monkey lung), PGF2alpha, PGE2, serotonin, bradykinin and histamine. Apart from its antiallergic effects PRD-92-Ea had no other significant pharmacological activity.     

Modulation of eosinophil chemotaxis and eosinophil chemotactic factor release by concanavalin A

Concanavalin A (Con A) is not a chemotactic factor for guinea pig eosinophil leukocytes. It does, however, modulate the response of cells to neutrophil-derived eosinophil chemotactic factor (ECF) or to C5a. This effect occurs by a direct interaction of Con A with the target cell and is reversible by appropriate doses of alpha-methyl-D-mannoside (alpha-MM). The inhibition of chemotaxis at high doses of Con A is due to agglutination of the cells on top of the filter. The mechanism of enhancement at low doses is not understood, but several possible explanations are explored. Careful definition of the effect of Con A on eosinophil chemotaxis makes it possible to exclude an effect of Con A on ECF secretion by neutrophils.     

Modulation of eosinophil chemotaxis by interleukin-5.

Eosinophilia and eosinophil function are regulated by cytokines such as granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5). We have investigated the modulatory role of IL-5 on N-formyl-methionyl-leucyl-phenylalanine (FMLP), neutrophil-activating factor (NAF/IL-8), platelet factor 4 (PF4), and cytokine-induced chemotaxis of eosinophils from normal individuals. These eosinophils show a small chemotactic response toward PF4 but not to NAF/IL-8 and FMLP. Preincubation of eosinophils with low concentrations of IL-5 caused significantly increased responses toward PF4 and induced a significant chemotactic response toward FMLP and NAF/IL-8. In marked contrast, IL-5 (or IL-3) priming of eosinophils from normal donors resulted in a strong inhibition of GM-CSF-induced chemotaxis. A similar decrease in the chemotactic response toward GM-CSF was observed in eosinophils derived from allergic asthmatic individuals. This finding suggests that the latter eosinophils may have had a prior exposure to IL-5 (or IL-3). Washing of the cells after priming did not abrogate the inhibition of the GM-CSF response. Our data indicate that at low concentrations IL-5 is an important modulator of eosinophil chemotaxis, causing selective upregulation or downregulation of chemotactic responses toward different agents.     

Enhancement of eosinophil leukocyte chemotaxis by a factor in normal human serum (SEF).

An activity in normal human serum is described which enhances the migration of eosinophils towards neutrophil-derived eosinophil chemotactic factor (ECF). The serum-enhancing factor (SEF), in contrast to the inhibitors in serum, is expressed primarily during weak chemotaxis, acts in a cell-directed fashion, increases the chemokinesis of eosinophils and, in the presence of ECF, enhances chemotaxis in a synergistic fashion. SEF is found in sera of several mammalian species, affects human and guinea pig eosinophils, and it is heat-labile (56 degrees C). On Sephadex column chromatography, SEF has a molecular weight of approximately 800,000 daltons. In some sera, a second activity with a molecular weight of approximately 200,000 daltons is eluted. SEF does not enhance eosinophil migration once the cells have been deactivated in vitro by their chemotactic factor. Because of its wide distribution, SEF may play an important modulating role both in in vitro assay systems and in vivo.     

CMRF35-like molecule 1 (CLM-1) regulates eosinophil homeostasis by suppressing cellular chemotaxis

Eosinophil accumulation in health and disease is a hallmark characteristic of mucosal immunity and type 2 helper T cell (Th2) inflammation. Eotaxin-induced CCR3 (chemokine (C-C motif) receptor 3) signaling has a critical role in eosinophil chemotactic responses. Nevertheless, the expressions of immunoreceptor tyrosine-based inhibitory motif-bearing receptors such as CMRF35-like molecule-1 (CLM-1) and their ability to govern eosinophil migration are largely unknown. We now report that CLM-1 (but not CLM-8) is highly and distinctly expressed by colonic and adipose tissue eosinophils. Furthermore, Clm1^−/− mice display elevated baseline tissue eosinophilia. CLM-1 negatively regulated eotaxin-induced eosinophil responses including eosinophil chemotaxis, actin polymerization, calcium influx, and extracellular signal-regulated kinase (ERK)-1/2, but not p38 phosphorylation. Addition of CLM-1 ligand (e.g., phosphatidylserine) rendered wild-type eosinophils hypochemotactic in vitro and blockade of CLM-1/ligand interactions rendered wild-type eosinophils hyperchemotactic in vitro and in vivo in a model of allergic airway disease. Interestingly, suppression of cellular recruitment via CLM-1 was specific to eosinophils and eotaxin, as leukotriene B₄ (LTB₄)- and macrophage inflammatory protein-1α (MIP-1α)-induced eosinophil and neutrophil migration were not negatively regulated by CLM-1. Finally, peripheral blood eosinophils obtained from allergic rhinitis patients displayed elevated CLM-1/CD300f levels. These data highlight CLM-1 as a novel regulator of eosinophil homeostasis and demonstrate that eosinophil accumulation is constantly governed by CLM-1, which negatively regulates eotaxin-induced eosinophil responses.     

Inhibition of polymorphonuclear leukocyte chemotaxis by streptokinase-streptodornase

Leukotaxis of circulating polymorphonuclear leukocytes (PMN's) appears to be a critical step in the generation of local inflammatory reactions. The present studies demonstrate a reversible, dose-dependent inhibition of PMN chemotaxis by streptokinase-streptodornase (SK-SD). This inhibition does not appear to depend upon the presence of lymphocytes or the release of chemokinetic humoral factors and phagocytic and bactericidal capacities of PMN's are unaffected by the doses of SK-SD employed.     

Endothelial protein C receptor-dependent inhibition of human eosinophil chemotaxis by protein C

BACKGROUND: Eosinophil infiltration is a characteristic feature of allergic inflammation. Allergic responses are associated with local activation of the coagulation pathway and accumulation of fibrin. OBJECTIVE: We tested whether protein C and activated protein C (APC), which are endogenous anti-inflammatory coagulation inhibitors, affect eosinophil function. METHODS: Eosinophils were from venous blood of healthy donors. Cell migration and apoptosis were studied by using micropore filter assays and fluorometry, respectively. Receptor expression was investigated by means of RT-PCR and SDS-PAGE of immunoprecipitated protein. RESULTS: Protein C and APC had no significant chemotactic effects on eosinophils. Eosinophils pretreated with protein C or APC showed significantly reduced migration toward chemoattractants. No effect of either protein C preparation was seen in eosinophil apoptosis assays. The inhibiting effect on migration was reversed by an antibody against the endothelial protein C receptor (EPCR). Synthesis of EPCR by eosinophils is suggested by demonstration of receptor mRNA expression and detection of metabolically labeled receptor protein. CONCLUSIONS: Data suggest that an EPCR is expressed by eosinophils whose activation with protein C or APC arrests directed migration. Protein C-affected eosinophil chemotaxis is a novel thrombin-independent component of the protein C pathway.     

Inhibitory effect of cetirizine 2HCl on eosinophil migration in vivo

The effect of a potent antihistamine, cetirizine, was studied on allergic patients and normal subjects by means of an in-vivo 'skin window' technique. All subjects showed significant inhibition of skin-test responses to grass pollen, compound 48/80, histamine and methacholine, after administration of a single dose (10 mg) of cetirizine. Compared to placebo, cetirizine significantly decreased the eosinophils attraction at skin sites challenged with grass pollen and compound 48/80. In allergic patients no change in eosinophil migration pattern was noted with histamine and methacholine skin-tested sites. In normal subjects, compound 48/80 and histamine did not induce eosinophil accumulation and cetirizine did not modify cellular patterns as compared to placebo. These results suggest that cetirizine acts on eosinophil migration by inhibiting the release of mast cell mediators or inhibiting the eosinophilotactic mediators themselves.     

Effects of cetirizine on human eosinophil and neutrophil activation in vitro.

The ability of cetirizine, a novel antihistamine agent, to inhibit the in vivo activation of human eosinophils, neutrophils and monocytes has been investigated using C3b- and IgG-dependent rosette formation, cytotoxicity against opsonised parasitic larvae and adherence to plasma-coated glass (PCG). The drug inhibited platelet-activating factor (PAF)-induced enhancement of eosinophil and neutrophil IgG (Fc) and complement (C3b) rosettes with an IC50 of 2 x 10(-5) M. There was also comparable inhibition of PAF-dependent enhancement of eosinophil cytotoxicity (for complement-coated schistosomula of Schistosoma mansoni). Cetirizine inhibited PAF-induced eosinophil, but not neutrophil, hyperadherence to PCG. These data support the view that cetirizine may exert some of its anti-allergic effects by inhibiting the activation of human granulocytes and that it may also selectively inhibit PAF-induced eosinophil hyperadherence.     

Inhibition of passive cutaneous anaphylaxis (PCA) by azelastine: dissociation of its antiallergic activities from antihistaminic and antiserotonin properties

Azelastine and methysergide injected i.v. 5 min prior to antigen challenge and disodium cromoglycate (DSCG) injected i.v. immediately before antigen challenge produced dose-dependent inhibition of IgE-mediated 72 h passive cutaneous anaphylaxis (PCA) responses with ID50S of 0.3, 0.2 and 1.0 mg/kg, respectively. Thus, azelastine is about three times as effective as DSCG (a mast cell stabilizing agent) and somewhat less active than methysergide (a specific serotonin "D" receptor antagonist). Oral administration of azelastine and other drugs 2 h prior to antigen challenge produced strong inhibitory effects on PCA. The ID50S (mg/kg) were as follows: azelastine = 1.4; astemizole = 1.6; ketotifen = 2.0; aminophylline = 4.6; and diphenhydramine = 10.9. After 4 h of oral administration, azelastine and other drugs inhibited PCA responses with the following ID50S (mg/kg): azelastine = 1.8; astemizole = 2.3; ketotifen = 2.3; and aminophylline = 12.5. Azelastine administered orally 24 h before antigen challenge was still capable of exerting significant anti-PCA activity with an ID50 of 2.6 mg/kg, whereas none of the other drugs tested produced any significant inhibitory effects on PCA. In subsequent experiments, it was established that the antiallergic and antihistaminic activities of azelastine are inseparable 2 h after oral administration (ID50 of azelastine mg/kg, p.o., 2 h: PCA = 2.6 and histamine = 3.1). However, the persistence of the oral antiallergic (anti-PCA) effects of azelastine for 24 h (ID50 = 3.7 mg/kg) does not seem to be associated with its antihistaminic or antiserotonin activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by diethylcarbamazine (hetrazan) of the eosinophil response which trichina extract elicits in guinea-pig lymph nodes

The injection of Trichina extract into guinea-pig footpads led to eosinophilia in the draining lymph nodes. Primary injections resulted in para-follicular accumulations within 24 hr in about 15% of animals tested; multiple injections resulted in diffuse eosinophilia in all animals, and, after it was allowed to dissipate, a brisk anamnestic response was elicited by a single challenge. Diethylcarbamazine eliminated the primary response, but had no effect on the secondary response. The drug was effective only when given (5 min–3 hr) before the extract; it failed to block when given (5 min or 1 hr) after the extract. The data indicate that the eosinophilia which accompanies Trichina infestation is, at least in part, an immunologic response to larval antigens, and that SRS-A may be one of the mediators.     

Quantitative analysis of eosinophil chemotaxis tracked using a novel optical device -- TAXIScan

We have reported previously the development of an optically accessible, horizontal chemotaxis apparatus, in which migration of cells in the channel from a start line can be traced with time-lapse intervals using a CCD camera (JIM 282, 1–11, 2003). To obtain statistical data of migrating cells, we have developed quantitative methods to calculate various parameters in the process of chemotaxis, employing human eosinophil and CXCL12 as a model cell and a model chemoattractant, respectively. Median values of velocity and directionality of each cell within an experimental period could be calculated from the migratory pathway data obtained from time-lapse images and the data were expressed as Velocity–Directionality (VD) plot. This plot is useful for quantitatively analyzing multiple migrating cells exposed to a certain chemoattractant, and can distinguish chemotaxis from random migration. Moreover precise observation of cell migration revealed that each cell had a different lag period before starting chemotaxis, indicating variation in cell sensitivity to the chemoattractant. Thus lag time of each cell before migration, and time course of increment of the migrating cell ratio at the early stages could be calculated. We also graphed decrement of still moving cell ratio at the later stages by calculating the duration time of cell migration of each cell. These graphs could distinguish different motion patterns of chemotaxis of eosinophils, in response to a range of chemoattractants; PGD₂, fMLP, CCL3, CCL5 and CXCL12. Finally, we compared parameters of eosinophils from normal volunteers, allergy patients and asthma patients and found significant difference in response to PGD₂. The quantitative methods described here could be applicable to image data obtained with any combination of cells and chemoattractants and useful not only for basic studies of chemotaxis but also for diagnosis and for drug screening.     

Inhibition of eosinophil infiltration into the mouse peritoneal cavity by a traditional Chinese medicine, Bu-zhong-yi-qi-tang (Japanese name: Hochu-ekki-to)

Our previous study showed that the serum level of antigen-specific IgE antibodies in primary response was decreased by a traditional Chinese medicine, Bu-zhong-yi-qi-tang (Japanese name; Hochu-ekki-to, HOT). In this study, we examined inhibition of secondary IgE response and of eosinophil infiltration by HOT. BALB/c mice were intraperitoneally immunized with aluminum hydroxide adsorbed with DNP-KLH (DNP-KLH + alum) on day -14 and on day 0. In mice treated with HOT daily from day -14, the serum level of antigen-specific IgE antibodies after the secondary immunization was significantly decreased compared to that in mice not treated with HOT. Eosinophils increased in number after 6 and 24 hr, and CD4+ T cells in the peritoneal cavity increased in number 24 hr after the secondary immunization. HOT suppressed accumulation of eosinophils and CD4+ T cells in the peritoneal cavity. Furthermore, HOT suppressed the numbers of IL-4- and IL-5-producing cells 24 hr after the secondary immunization, but did not inhibit the number of IFN-gamma-producing cells. HOT also suppressed IL-5 mRNA expression. Furthermore, HOT also inhibited antigen-induced late-phase reaction (LPR) in the skin. These results suggested that HOT exhibited anti-allergic effects mainly by inhibiting Th2 cell responses.     

Red blood cells regulate eosinophil chemotaxis by scavenging RANTES secreted from endothelial cells

BACKGROUND: Eosinophils play a critical role in the pathogenesis of allergic diseases. CC chemokines, such as regulated on activation, normal, T cell expressed, and secreted (RANTES), are key regulators of eosinophil locomotion. Although eosinophils migrate from the bloodstream into tissues, mechanisms that generate a chemogradient across the endothelium remain to be fully elucidated. OBJECTIVE: We first examined the polar secretion of RANTES by endothelial cells. We also studied the functional scavenging effect of red blood cells (RBCs) on RANTES secreted into the intravascular side. METHODS and RESULTS: Endothelial cells were cultured in a transwell chamber with a membrane pore size of 0.45, 3.0, and 8.0 microm and stimulated with TNF-alpha, IL-1beta, or IFN-gamma from the apical or basolateral side for 16 h. The measurement of RANTES in the supernatant was performed by ELISA. We did not see any difference in the amount of RANTES secreted from the cytokine-stimulated endothelium between inner (intravascular side) and outer (extravascular side) wells separated by the 8.0-microm membrane, although apical polarization was observed with the 0.45-microm membrane. The addition of RBCs (hemoglobin (Hb): 0.5-15 g/dL) to the apical supernatant of TNF-alpha-stimulated endothelial cells reduced the RANTES level in a concentration-dependent manner. The treatment of supernatant on the intravascular side with RBCs significantly enhanced the migration of eosinophils. CONCLUSION: RBCs possess a scavenging effect on intravascular RANTES, and thereby regulate transendothelial migration of eosinophils. Our findings suggest a new role of RBCs in allergic inflammation.     

Myosin light chain kinase mediates eosinophil chemotaxis in a mitogen-activated protein kinase-dependent manner

BACKGROUND: Eosinophil migration in the tissue is one characteristic feature of allergic diseases. The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Myosin light chain kinase (MLCK) is known to regulate cytoskeletal rearrangement and cell motility by means of phosphorylation of myosin light chain (MLC). OBJECTIVE: We have previously shown that mitogen-activated protein (MAP) kinases are important for eosinophil migration. In the present study we hypothesized that MLCK is downstream of MAP kinases, thereby linking the MAP kinase pathway to the activation of cytoskeletal components required for eosinophil chemotaxis. METHODS: Blood eosinophils were purified by using Percoll and anti-CD16 antibody-coated magnetic beads. We investigated the phosphorylation of MLCK and MLC by using the phosphorous 32-orthophosphates-labeled eosinophils. The kinase activity of MLCK was determined by measuring the phosphotransferase activity for the MLCK-specific peptide substrate. The chemotaxis assay was performed in a 48-well Boyden microchamber. RESULTS: The phosphotransferase activity of MLCK for a substrate peptide was enhanced in eotaxin-stimulated eosinophils. We also found that eotaxin induced phosphorylation of MLCK in vivo in phosphorous 32-orthophosphate-labeled eosinophils. PD98059 (MAP/extracellular signal-regulated kinase inhibitor) or SB202190 (p38 MAP kinase inhibitor) abrogated the eotaxin-induced phosphorylation of MLCK. The phosphorylation of MLC was upregulated by eotaxin. Eosinophil chemotaxis was inhibited by means of pretreatment of the MLCK inhibitor ML-7. CONCLUSION: These results suggest that eotaxin regulates MLCK through both extracellular signal-regulated kinase 1/2 and p38 MAP kinase. MLCK activation is a critical step in the cytoskeletal rearrangements leading to eosinophil migration.     

Inhibition of sperm-zona binding by suramin, a potential 'lead' compound for design of new anti-fertility agents

Progress in developing new contraceptive agents, particularlyfor the male, is extremely slow. Here, we report on a novelproperty of the anti-trypanosomal drug suramin: the abilityto act at the sperm-egg interface and prevent fertilization.Suramin is a polysulphonated compound that inhibits bindingof capacitated mouse spermatozoa to the zona pellucida in vitrowith an IC₅₀ of 12.4 µM. The drug is only effective atthe time of fertilization and is not inhibitory if gametes arepre-treated separately. Autoradiographic localization of suraminbinding sites on mouse, boar and human spermatozoa shows thatthey are intracellular, principally in the head region. Thesperm protein recognized by suramin has been identified as proacrosinwhich is known to interact with sulphate groups on zona glycoproteins.Zone pellucida glycoproteins do not bind suramin, suggestingthat the drug blocks the ability of proacrosin/acrosin, exposedduring the acrosome reaction, to mediate the secondary bindingphase of spermatozoa to the zona during fertilization. Structurebaseddesign studies have the potential to generate safe suramin mimeticsthat would form the basis for a new generation of non-steroidalcontraceptive agents. fertilization/proacrosin/sucramin/zona pellucida