R. Moqbel

Immunohistology of the nasal mucosa in seasonal allergic rhinitis: Increases in activated eosinophils and epithelial mast cells☆

The immunohistology of the nasal mucosa was examined in 13 grass pollen-sensitive patients and in seven normal nonatopic control subjects before and during the pollen season. Cryostat sections (6 microns) of biopsy specimens from the inferior turbinate were immunostained with the alkaline-phosphatase antialkaline-phosphatase method and a panel of monoclonal antibodies. Mast cell subtypes were measured with a double sequential immunostaining method. Within the submucosa, seasonal increases in total (MBP+, p less than 0.01) and activated (EG2+, p less than 0.01) eosinophils were observed for the patients, which were significant when these counts were compared with counts for those of control subjects (MBP+ p less than 0.01; EG2+ p less than 0.001). Within the nasal epithelium, seasonal increases in total (p less than 0.05) and activated (p less than 0.02) eosinophils were also observed. Mast cell counts revealed seasonal increases in tryptase-only positive mast cell (MCT) (p less than 0.02) but not chymase plus tryptase-positive mast cells (MCTC) within the epithelium that were significant when counts were compared with those of control subjects (p less than 0.03). No significant changes were observed within the submucosa or epithelium for total leukocytes (CD45+ cells) or T-lymphocytes (CD3+, CD4+, CD8+, and CD 25+ cells) for either group. Similarly, no significant changes were observed for neutrophils (antielastase), macrophages (CD68+), nor HLA-DR+ cells. In the subjects with rhinitis, seasonal submucosal CD3+ counts correlated with MBP+ eosinophils (r = 0.56; p less than 0.05) and MCTS (r = 0.65; p less than 0.02). Similarly, seasonal epithelial EG2+ eosinophil counts correlated with MCTs (r = 0.56; p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Eosinophils: biology and role in disease.

Publisher Summary The chapter reviews recent findings on the biology of the eosinophil, particularly distinctive eosinophil features, and discusses the possible role of the eosinophil in the pathogenesis of certain diseases with which the cell is associated. There is evidence that eosinophils slow the rate of progression of solid tumors through tumoricidal mechanisms. There is no experiment of nature in that cell, or its progenitors are selectively depleted; and at the present time no pharmacological or biological inhibitor is available for laboratory-based or clinical studies. However, in the past few years, evidence has shifted away from the cell having anti-allergic properties, and even its role in adaptive immunity to helminth infections is seriously questioned. It is important to bear in mind that the eosinophil is an inflammatory cell and that the purpose of inflammation is to restore, repair, and remodel injured tissue. The role of the eosinophil in wound healing has recently received considerable attention, and it seems reasonable to expect that this will be a growth area for eosinophil biologists. Intriguingly, there is now some firm data to implicate the eosinophil in immunological responses against cancer cells. The recent findings on the potential of eosinophils in cancer immunity may stimulate a better understanding of the role of this cell type. It can be stated with reasonable assurity that the eosinophil probably has some homeostatic function in certain physiological situations and that this needs to be considered further.

TNFα mRNA expression in allergic inflammation

Using the technique of in situ hybridization, we have attempted to identify messenger RNA for tumour necrosis factor‐alpha (TNFα) in cells infiltrating allergen‐induced late phase reaetion (LPR) of the skin and the nose ofatopic subjects. We have also compared the number of TNFα mRNA positive cells in bronchoalveolar lavage (BAL) from atopic asthmatics and normal controls. Twenty‐four hours after local allergen challenge, 12/14 skin biopsies and 9/10 nasal biopsies had positive hybridization signals for TNFα mRNA whereas only 4/14 and 2/10 biopsies were positive in the relevant diluent controls. Compared with diluent sites significantly increased numbers of cells expressing mRNA for TNFα were observed in the LPR of skin (P <0.004) and nose(P <0.006). All BAL from asthmatics (n= 10) and from normal volunteers (n= 10) had cells showing positive hybridization signals for TNFα mRN A but these were at increased frequency in asthmatics (P< 0.001). These results suggest that TNFα may be an important cytokine in atopic allergic inflammation.

Interleukin‐8 is a chemo‐attractant for eosinophils purified from subjects with a blood eosinophilia but not from normal healthy subjects

Interleukin‐8 (IL‐8), a pro‐inflammatory cytokine with potent neutrophil chemotactic activity, was studied for its effect on eosinophil migration responses, in vitro. Normal density eosinophils were isolated from healthy, non‐atopic subjects <0.35 × 109 eosinophils/1) and individuals with various diseases associated with a blood eosinophilia (range 0.56 × 109‐12.2 × 109 eosinophils/1). IL‐8 prod need a dose‐dependent migrational response for eosinophils from subjects with an eosinophilia, optimal at 10−8m (P < 0.01) and the major component of the migrationsl response was chemokinesis. On a molar basis, FL‐8 (EC50∼ 10−10m) was 100‐fold more potent than platelet activating factor (PAF), although a comparison of the migrational responses showed that at optimal concentrations IL‐8 (10−8m) produced only 30% maximal responses stimulated by PAF (10−6m). In contrast, IL‐8 tested over a wide concentration range had a negligible effect on eosinophils from normal subjects. A direct correlation between the total blood eosinophil counts for all subjects and the absolute magnitude of the migrational response to IL‐8 (r= 0.727,P < 0.01 at 10−8m), PAF (r= 0.551, P < 0.03 at 10−6m) and N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) (r= 0.689. P < 0.02 at 10−8m), suggested that heightened eosinophil migrational responses in inflammatory mediators may arise as a consequence of in vitro priming mechanism(s) associated with the development of an eosinophilia. In this regard, eosinophils derived from human cord blood mononuclear cells cultured in the presence of eosinophilopoietic cytokines IL‐3 and IL‐5, produced migrational responses to IL‐8 and PA F. that were comparable with that of eosinophils from eosinophilic subjects. Furthermore, incubation of eosinophils from normal donors with IL‐5 (optimal concentration 10−9m), significantly enhanced the subsequent migrational responses to both IL‐8 (10−8m, P < 0.01)and PAF (10−8mP < 0.05). Therefore, the increased responsiveness of eosinophils from eosinophilic subjects may reflect in vivo priming by IL‐5 and this phenomenon may contribute partly to the mechanism(s) by which eosinophils preferentially accumulate at sites of allergic inflammation.

Application of monoclonal antibodies against major basic protein (BMK-13) and eosinophil cationic protein (EG1 and EG2) for quantifying eosinophils in bronchial biopsies from atopic asthma.

A monoclonal antibody prepared against the eosinophil major basic protein (MBP) was compared with the anti‐eosinophil cationic protein (ECP) antibodies (Eg1 and EG2) in immunostaining of bronchial biopsies from atopic asthma and controls. Anti‐MBP (designated BMK‐13) did not cross‐react with other eosinophil basic proteins (i.e. ECP, eosinophil peroxidase [EPO] or eosinophil‐derived neurotoxin [EDN]) and stained more than 98% of peripheral blood eosinophils irrespective of their degree of activation. EG2 stained 15% of resting and 75% of activated eosinophils; EG1 recognized 74% and 78% of resting and activated cells, respectively.

Effect of disodium cromoglycate on activation of human eosinophils and neutrophils following reversed (anti‐IgE) anaphylaxis

Immunological release of histamine and lipid mediators is known to occur when basophils, contained in whole blood human leucocytes, are incubated with anti‐IgE (reversed anaphylaxis). In the present study we show that IgE‐dependent stimulation of basophils was associated with activation of bystander eosinophils and neutrophils, as assessed by enhanced complement (C3b) and IgG (Fc) rosettes, and increased cytotoxicity for complement‐coated schistosomula of Schistosoma mansoni. These changes in eosinophil and neutrophil function were totally inhibited in a dose‐dependent fashion by prior incubation with disodium cromoglycate (DSCG). In all in vitro systems examined, complete inhibition of enhancement was observed with concentrations as low as 10‐7 moles/1. In contrast, DSCG had no effect on histamine release, or the percentage of rosettes or cytotoxicity prior to anti‐IgE stimulation. These results suggest that DSCG inhibits activation of inflammatory cells consequent to an IgE‐dependent stimulus.

Modulation of human eosinophil chemotaxis and adhesion by anti-allergic drugs in vitro

A preponderance of eosinophils at sites of inflammation is a hallmark of helminthic infections and allergic-type diseases such as bronchial asthma (1, 2). The role of eosinophils as prime effector cells of the pathophysiology of asthma is supported by the demonstration that in vitro, activated eosinophils release granule-associated basic proteins that are cytotoxic for respiratory epithelial cells (3). In addition, eosinophils synthesize substantial amounts of lipid mediators such as leukotriene C, (LTC,) and platelet-activating factor (PAF), dti novo. These have the potential to induce the characteristic features of airway obstruction in asthma (4-6). Clinical studies in parallel have emphasized the invariable localization of activated eosinophils in bronchial biopsies from asthmatic subjects (7,8), and a direct correlation has been demonstrated between eosinophil numbers in bronchial lavage and the degree of airway hyperreactivity (9). These findings, however, are circumstantial, and further insight into the role of eosinophils in airway disease can be obtained by investigating agents that inhibit or ablate eosinophil recruitment and/or activation at sites of allergic inflammation. We have shown previously that anti-allergic agents such as sodium cromoglycate (SCG) and nedocromil sodium inhibit eosinophil activation in vitro as assessed by down-regulation of enhanced receptor expression and cytotoxicity to opsonized targets (10-12). Cetirizine, a major metabolite of hydroxyzine, is a potent and HI-receptor antagonist with a limited potential for sedation and anticholinergic side effects (13). The ability of cetirizine to attenuate eosinophil infiltration in vivo to cutaneous sites following allergen challenge ( 1416) and administration of PAF (17) suggests an anti-inflammatory mode of action that is distinct from its anti-Hi, activity. Cetirizine inhibits PAFinduced enhancement of eosinophil complement and immunogloblulin G (1gG)-dependent rossette formation and cytotoxicity to complement coated schistosomula of Schistosoma marzsoni (18). However, unlike SCG and nedocromil sodium, which have no significant effect on PAF-induced chemotaxis of eosinophils from asthmatic subjects ( 19), cetirizine inhibits eosinophilotactic responses towards PAF and formyl-methionyl leucyl phenylalanine in vitro (20,21). In addition, cetirizine selectively inhibits PAF-induced eosinophil but not neutrophil hyperadherence to plasma-coated glass (IS), suggesting a mechanism by which this drug is able to inhibit eosinophil recruitment in viivo. The adhesion of leukocytes to vascular endothelial cells represents a crucial early stage in the transvascular migration of leukocytes.

Eosinophils, Cytokines, and Allergic Inflammation

Eosinophils are prominent inflammatory cells associated with allergic disease and inflammatory responses against metazoan helminthic parasites. This association has been most extensively characterized in relation to asthma where large numbers of eosinophils and their granule products are found in and around the airways in asthma deaths.* An inverse correlation between the degree of bronchial hyperreactivity and peripheral blood eosinophilia has been observed in subjects who exhibited dual response following antigen ~ha l l enge .~ Furthermore, using more invasive methods such as fiberoptic bronchoscopy, an increase in the number of activated eosinophils present in the airways was shown to correlate with asthma ~ e v e r i t y . ~ Despite the close association between eosinophils and the pathogenesis of allergic disease and asthma, the evidence for a cause and effect relationship remains largely circumstantial. Eosinophils are nondividing, granule-containing cells that arise principally in the bone marr0w.j They are 8 pm in diameter and their granules avidly take up acidic dyes such as eosin. In laboratory animals, parasite-induced eosinophilia has been shown to be dependent on T lymphocytes.6 This effect was shown to be mediated by soluble factors released from sensitized lymphocytes. Recent advances in human eosinophil research have indicated that eosinophil infiltration into the tissue in allergic-type responses and asthma may be regulated by a complex series of events that involve immunological and inflammatory mechanisms including T cells and ~ y t o k i n e s . ~ , ~

Synthesis and Storage of Regulatory Cytokines In Human Eosinophils

Eosinophils are prominent cells in allergic inflammation, asthma, and immune reactions against parasitic helminths.1,2 The association between eosinophils and asthma has been extensively documented. Large numbers of eosinophils and their granule products have been observed in the airways of patients with asthma,2 and an inverse correlation between the degree of bronchial hyperreactivity and peripheral blood eosinophilia was shown in subjects in whom a late-phase response developed after antigen challenge.3 With use of fiberoptic bronchoscopy it was possible to correlate the number of activated eosinophils with the degree of bronchial hyperresponsiveness.4 However, despite this close association, the evidence of a cause and effect relationship between eosinophil products and tissue damage remains unproven.

157 IgG-dependent generation of platelet-activating factor by normal and ‘Low density’ human eosinophils

We have compared normal and low density human eosinophils for their ability to generate platelet activating factor (PAF) in response to IgG-dependent and nonimmunologic stimulation. After 45 min incubation with IgG-coated Sepharose beads the concentrations of cell-associated PAF recovered from normal density eosinophils were significantly greater than from low-density eosinophils or neutrophils. Moreover, eosinophils stimulated with calcium ionophore A23187 had a considerably greater capacity to generate PAF than had previously been described. Although the quantities of cell-associated PAF recovered from normal and low density eosinophils and neutrophils after A23187 stimulation were similar, the amounts of extracellular PAF recovered from both eosinophil populations were significantly greater than from neutrophils. The amounts of PAF recovered from the low density eosinophils may not reflect the full synthetic capacity of these cells, because PAF-turnover was found to be more rapid than that observed with normal density eosinophils. When exogenous [3H]PAF was added to the two stimulated eosinophil populations subsequent analysis of the [3H]PAF metabolites by DIOL-HPLC revealed that low density eosinophils incorporated PAF into the phosphatidylcholine (PC) pool more rapidly than did normal density eosinophils or neutrophils. Alkaline hydrolysis of the PC fraction from whole cell extracts followed by treatment with acetic anhydride resulted in all the PC-associated radioactivity being converted to [3H]PAF, confirming PAF incorporation to PC via this pathway. These findings suggest that the contribution of eosinophils to inflammatory processes through the generation of PAF may be greater than previously appreciated, and that Ig-mediated stimulation may be important in initiating generation of the mediator. Low density eosinophils, that are presumed to be similar to tissue eosinophils, may have a role in regulating PAF concentrations in tissues through their enhanced rate of metabolism.