Tyrosine kinase inhibition is an important factor for gene expression of CRTH2 in human eosinophils and lymphocytes: A novel mechanism for explaining eosinophils recruitment by the neuro-immune axis in allergic rhinitis.

We recently shown a novel neuro-immune competition between vasoactive intestinal peptide (VIP) and PGD2 for CRTH2 receptor, and that genistein augmented VIP and PGD2-induced eosinophil chemotaxis. However, there are neither studies on the CRTH2 gene expression in allergic rhinitis (AR) nor in the effect of tyrosine kinase inhibitors in CRTH2 gene regulation. Our Objectives were to study the gene expression modulation of CRTH2 receptor in AR patients and the effect of tyrosine kinase inhibitors (TKIs) on CRTH2 gene modulation. Nasal provocation tests, ELISA, qRT-PCR, western blot, flow cytometry and chemotaxis assays in modified micro-Boyden chambers, were all used, to achieve our objectives. Herein we show that AR patients increased the amounts of VIP and PGD2 in their nasal secretions in the early phase reaction, however CRTH2 gene expression from leukocytes recovered in their nasal secretions was upregulated only during the late phase reaction. The TKIs; Genistein, Erbstatin and Herbimycin A, induced the gene expression of CRTH2 and increased the protein content of CRTH2 in both human lymphocytes and eosinophils. This was functional as PGD2/VIP-induced eosinophil chemotaxis was augmented by the TKIs and inhibited by pervanadate, the tyrosine phosphatase inhibitor. These results open channels for therapeutic modalities targeting CRTH2 molecules in AR.

Novel cooperation between CX3CL1 and CCL26 inducing NK cell chemotaxis via CX3CR1: a possible mechanism for NK cell infiltration of the allergic nasal tissue.

BACKGROUND: Recent data indicated that natural killer (NK) cells and chemokines could play a pivotal role in nasal inflammation. CX3CR1, the only receptor for fractalkine/CX3CL1, is abundantly expressed by NK cells, and was recently shown to also be a receptor for eotaxin-3/CCL26. However, no reports explored the NK cells-CX3CL1-CCL26 axis via CX3CR1 in allergy. OBJECTIVE: Our goals were first to determine specifically NK cell recruitment pattern in nasal tissue of allergic chronic rhinosinusitis (ACRS) and non-allergic chronic rhinosinusitis (NACRS) patients in comparison with healthy controls, and secondly, to investigate the function of CX3CR1 in NK cell migration. METHODS: Immunohistochemistry, microchemotaxis chambers, flow cytometry and confocal microscopy were used in this study. RESULTS: Herein, we showed that NK cells infiltrated the epithelial layers of nasal tissue only in ACRS patients and not in NACRS patients or controls. NK cells were also more numerous in the stroma of the nasal tissue from ACRS patients compared with NACRS patients or controls. This migration could be mediated by both CX3CL1 and CCL26, as these two chemokines induced NK cell migration. Moreover, both molecules also stimulated cytoskeleton changes and F-actin reorganisation in NK cells. Chemotaxis and cytoskeleton changes were sensitive to genistein, a tyrosine kinase inhibitor. By flow cytometry, we demonstrated that a single antigen nasal provocation challenge increased the expression of CX3CR1 on NK cells in allergic rhinitis (AR) patients. The function of this receptor was associated with a significant augmentation of NK cell chemotaxis against the optimal doses of CX3CL1 and CCL26. CONCLUSIONS AND CLINICAL RELEVANCE: Our results highlight a novel role for CX3CR1 in NK cell migration that may contribute to the NK cell trafficking to the allergic upper airway. This could be mediated largely by CX3CL1 and CCL26 stimulation of the tyrosine kinase pathway.

Anatomical variations of the lateral nasal wall: The secondary and accessory middle turbinates.

The aim of the current anatomical and clinical study was to audit our cases of patients who presented with secondary and/or accessory middle turbinates during a two-year period. We investigated the incidence and the clinical impact of these variations. Twenty-eight patients, 19 males and 9 females with a mean age of 41.5 years, representing different ethnic origins, were diagnosed with double middle turbinates based on endoscopic examination. Of those, 92.8% had a main symptom of refractory frontal headache. A secondary nasal symptom was sensation of blocked nose. Patients who underwent endoscopic surgery (n = 13) for reduction of the extra turbinate, reported significant symptom scores improvement (P < 0.0001) of frontal headache and blocked nose, from means of 9.07 ± 0.26 and 8.57 ± 1.39 to 1 ± 0.31, and 1.42 ± 0.35, respectively. Our results indicate that double middle turbinates may be encountered in rhinology practice (2%). Clinically they may present with refractory headache and blocked nose. Endoscopic surgical approach seems to be an effective way of improving the symptoms.

IFN-γ and TNF-α potentiate prostaglandin D2-induced human eosinophil chemotaxis through up-regulation of CRTH2 surface receptor.

Prostaglandin D2 (PGD2) receptor CRTH2, is a pro-inflammatory molecule involved in eosinophil recruitment to the allergic airway. We investigated the expression of CRTH2 in eosinophil from allergic rhinitis patients (AR) and tested the modulatory role of several TH1 and TH2 cytokines closely related to the allergic immunological response, on the expression of CRTH2 receptor, utilizing human eosinophil cell line (Eol-1).The expression of CRTH2 was tested by immunohistochemistry and flow cytometry (FACS). Chemotaxis was performed in micro-chemotaxis chambers. It is shown that the expression of CRTH2 by eosinophils was significantly higher in the nasal tissue and peripheral blood of AR patients, when compared to control subjects. PGD2 exhibited a typical bell shape dose response in attracting eosinophil from AR patients with optimal activity at 10(-7) M. Eol-1 cell surface expression of CRTH2 was significantly up-regulated by 10 ng/ml IFN-γ and TNF-α. The percentage of Eol-1 cells expressing the receptor increased by IFN-γ and TNF-α from 12.74%±2.66 to 55%±8 and 33.8%±9.4, respectively. PGD2-induced Eol-1 chemotaxis was not blocked by SB203580, H-89 Dihydrochloride, Bisindo-lylmaleimide, or Genistein. PGD2-induced Eol-1 chemotaxis was potentiated by IFN-γ and TNF-α without changing the signal transduction pathway. Correlation of our results to peripheral blood eosinophils from allergic rhinitis patients confirmed that 3 hour pretreatment of eosinophils by 10 ng/ml IFN-γ and TNF-α, increased the mean fluorescence intensity (MFI) of CRTH2 from 8.23 to 9.68 and 9.38, respectively, and potentiated PGD2-induced eosinophil chemotaxis. Our results demonstrate a novel synergism between PGD2, IFN-γ and TNF-α, in eosinophil chemotaxis.

Modulation of NK cell autocrine-induced eosinophil chemotaxis by interleukin-15 and vitamin D(3): a possible NK-eosinophil crosstalk via IL-8 in the pathophysiology of allergic rhinitis.

Natural killer cells (NK) secrete eosinophilotactic cytokines, however, whether they contribute to eosinophil chemotaxis by secreting IL-8 is not known. We investigated the ability of CD56+CD3-ve (NK cells) to induce chemotaxis of peripheral blood eosinophils from allergic rhinitis (AR) patients, through IL-8 secretion, and the effects of IL-15, the NK cell proactivating cytokine, and calcitriol: 1α, 25-dihydroxy Vitamin D(3) (vitamin D(3)), the immunomodulator agent, in this scenario. Herein, it is shown that supernatants from unstimulated NK cells exhibited chemotactic activity against eosinophil. This effect was significantly augmented by IL-15 (1 ng/mL) treatment, resulting in an increase in the chemotactic index of approximately 3 folds and was abrogated by neutralizing antibody (Ab) to IL-8 in a dose-dependent fashion. The amount of IL-8 secreted by NK cells was increased by IL-15 treatment from levels of 88.64 ± 21.5 to 178.9 ± 23.6 Pg/mL and was significantly reduced by 10(-6) M vitamin D(3) to levels of 59.2 ± 16.3 Pg/mL. Our results indicate a novel inflammatory crosstalk between NK cells and eosinophils via IL-15/IL-8 axis that can be modulated by vitamin D(3).

2B4 (CD244) is involved in eosinophil adhesion and chemotaxis, and its surface expression is increased in allergic rhinitis after challenge.

A role for the subtypes of CD2 Ig superfamily receptors has been recently demonstrated in eosinophilic inflammation in experimental asthma and atopic asthmatics. We investigated the functions of 2B4 (CD244) molecules in eosinophil adhesion and chemotaxis, and correlated the results to the pathophysiology of allergic rhinitis (AR). Herein, we show that agonistic stimulation of 2B4 by C1.7, the anti-human 2B4 functional grade purified antibody, resulted in significant increase of eosinophils and eosinophil cell line (Eol-1 cells) adhesion to collagen type IV, and random migration. These functions were associated with tyrosine kinase phosphorylation of several protein residues of low molecular weight. Flow cytometry (FACS) experiments demonstrated that Eol-1 cells, normal peripheral blood eosinophils and eosinophils from AR patients, express surface 2B4 molecules. In vitro AR model demonstrated that the CC-chemokine receptor CCR3 stimulation by eotaxin induced significant increase in the expression of surface 2B4 in eosinophils and Eol-1 cells. Immunofluorescence confocal microscopy images showed that eotaxin induces also redistribution of 2B4 molecules towards the pseudopods in eosinophils and Eol-1 cells, changing their shape. Blocking of 2B4 molecules by the corresponding neutralizing antibody inhibited eotaxin induced Eol-1-adhesion, chemotaxis and the cytoskeleton changes. Pretreatment of Eol-1 cells with 1 microM genistein blocked eotaxin-induced Eol-1 adhesion, chemotaxis and 2B4 up-regulated expression. In vivo correlation demonstrated the expression of 2B4 molecules in eosinophils from AR patients to be significantly increased, after nasal provocation challenge. These results identify a novel role for 2B4 molecules in eosinophil functional migratory response and may point to a novel tyrosine kinase-mediated ligation between CCR3 receptor and 2B4 co-receptor in eosinophil chemotaxis. If so, then 2B4 molecules would be a novel target for therapeutic modalities in diseases characterized by eosinophilia such as AR.

Hepatitis C virus induces nasal epithelial erosion and sub-epithelial rhinitis.

BACKGROUND: Hepatitis C virus (HCV) affects extra-hepatic organs, but its effect on the nose is poorly defined. OBJECTIVES: To investigate the histological changes in nasal tissue induced by HCV and whether the nasal mucosa harbors the virus for extrahepatic replication. STUDY DESIGN: We investigated nasal biopsies from 20 patients with HCV infection, and from 10 control subjects. All biopsies were subjected to real time polymerase chain reaction (RT-PCR) as well as histology. RESULTS: Our analyses showed that 60% of HCV positive samples showed nasal epithelial erosion, 95% showed subepithelial non-specific inflammation and/or fibrosis, while only 5% showed normal histology. However, none of the twenty PCR samples showed the presence of HCV nucleic acids sequences in the nasal tissues. On the other hand, all control subjects had normal histology and the absence of the viral m-RNA in the PCR (100%). CONCLUSIONS: HCV induces histopathological rhinitis with nasal epithelial erosion. However, it does not seem that the nasal tissue harbors the virus.

Mechanisms involved in activation of human eosinophil exocytosis by substance P: an in vitro model of sensory neuroimmunomodulation.

Substance P (SP), a tachykinin with a wide range of biological activities including a priming effect on human eosinophil chemotaxis, was investigated for its influence on eosinophil cytotoxic function measured as degranulation of eosinophil-derived neurotoxin (EDN). Peripheral blood was obtained from healthy volunteers and the degranulation assays were performed using radioimmunoassay (RIA). SP and its C-terminal elicited EDN release in a time-dependent mode at a narrow range of doses with optimal activity of 10(-6) M. FK888 (NK-1 receptor antagonist) inhibited EDN release stimulated by SP in dose dependency, also a complete inhibition was observed when eosinophils were preincubated with 1000 ng/ml pertussis toxin (PTX). Pre-exposure of eosinophils to staurosporine resulted in blockage of SP-induced EDN release in a dose-dependent mode. On the other hand, SP at 10(-7) M and 10(-8) M primed eosinophils to suboptimal dose (10(-8) M) of Platelet activating factor (PAF) resulting into significant enhancement of EDN release. SP(4-11) fragment showed a similar activity while SP(1-4) fragment was not active. SP priming of eosinophils was not affected by Ca2+ depletion, however, it caused a change in the pattern of the intracellular calcium influx against the suboptimal dose of PAF. These results suggest that SP i) may induced human eosinophil matrix protein degranulation through a receptor mediated mechanism coupled to PTX sensitive G protein(s) with the probability of linkage to phospholipase C activation, and, ii) primes human eosinophils for an exalted inflammatory response through a Ca2+ independent pathway.

Inhibition of human eosinophil chemotaxis in vitro by the anti-allergic agent emedastine difumarate.

Emedastine difumarate (emedastine), an anti-allergic agent with anti-histaminic properties, was studied for its effect on human eosinophil chemotaxis induced by platelet activating factor (PAF). Peripheral blood eosinophils (98% purity) were obtained from healthy donors and chemotaxis assay were performed in microchemotaxis chambers. Emedastine showed a significant inhibitory effect on 10(-6) M PAF-induced eosinophil chemotaxis, in dose dependent fashion, at concentrations from 10(-6) to 10(-8) M. Conversely, no inhibitory effect was observed on human neutrophil chemotaxis. Pretreatment of eosinophils with Pyrilamine did not affect PAF-induced eosinophil chemotaxis. Thus emedastine appears to possess a potent and selective inhibitory effect on eosinophils chemotaxis, an action which is probably unrelated to its anti-histamine properties.

Immunoregulatory effect of substance P in human eosinophil migratory function.

Substance P (SP) is a tachykinin involved in the regulation of inflammatory processes. To investigate a modulatory role of the neuropeptide SP in allergic inflammation, we studied its priming effect on human eosinophil chemotaxis and kinetic responses towards platelet activating factor (PAF) and recombinant human interleukin 5 (rhIL-5). Blood was obtained from normal subjects and eosinophils were separated by Percoll discontinuous density gradient centrifugation. High purification was obtained by negative selection procedure (CD16-beads) and the experiments were performed in a 48-well microchemotaxis Boyden chamber. In the present study we demonstrate a potent synergistic effect of 1OOnM dose of SP on the migratory function of human eosinophils stimulated by PAF and rhIL- 5. This synergism was chemotaxis specific and was abolished by NK-1 receptor antagonist (FK888). The results suggest that neurogenic stimuli may play a significant role in eosinophil infiltration via its priming effect on the cell.