Donald W. MacGlashan

Prostaglandin-Induced Activation of Nociceptive Neurons via Direct Interaction with Transient Receptor Potential A1 (TRPA1)

Inflammation contributes to pain hypersensitivity through multiple mechanisms. Among the most well characterized of these is the sensitization of primary nociceptive neurons by arachidonic acid metabolites such as prostaglandins through G protein-coupled receptors. However, in light of the recent discovery that the nociceptor-specific ion channel transient receptor potential A1 (TRPA1) can be activated by exogenous electrophilic irritants through direct covalent modification, we reasoned that electrophilic carbon-containing A- and J-series prostaglandins, metabolites of prostaglandins (PG) E2 and D2, respectively, would excite nociceptive neurons through direct activation of TRPA1. Consistent with this prediction, the PGD2 metabolite 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) activated heterologously expressed human TRPA1 (hTRPA1-HEK), as well as a subset of chemosensitive mouse trigeminal neurons. The effects of 15dPGJ2 on neurons were blocked by both the nonselective TRP channel blocker ruthenium red and the TRPA1 inhibitor (HC-030031), but unaffected by the TRPV1 blocker iodo-resiniferatoxin. In whole-cell patch-clamp studies on hTRPA1-HEK cells, 15dPGJ2 evoked currents similar to equimolar allyl isothiocyanate (AITC) in the nominal absence of calcium, suggesting a direct mechanism of activation. Consistent with the hypothesis that TRPA1 activation required reactive electrophilic moieties, A- and J-series prostaglandins, and the isoprostane 8-iso-prostaglandin A2-evoked calcium influx in hTRPA1-HEK cells with similar potency and efficacy. It is noteworthy that this effect was not mimicked by their nonelectrophilic precursors, PGE2 and PGD2, or PGB2, which differs from PGA2 only in that its electrophilic carbon is rendered unreactive through steric hindrance. Taken together, these data suggest a novel mechanism through which reactive prostanoids may activate nociceptive neurons independent of prostaglandin receptors.

Histamine: A mediator of inflammation

Histamine and its receptors, including the recently discovered receptors (H(3) and H(4)), novel sources of histamine, and the place of histamine in mediator networks continue to be areas of great interest. The 4 major subtypes of histamine receptors, H(1) to H(4), differ in their location, second messengers, and histamine-binding characteristics. In addition, it would appear that different histamine receptor agonists and antagonists bind to different portions of the receptor complex. A fifth receptor subtype, the intracellular H(IC), has only been defined by its location within cell types that are not traditionally associated with histamine. In airway tissue, most cells express at least 1 subtype of histamine receptor; however, blockade of these receptors does not completely abolish the inflammatory response. In addition, some H(1)-antihistamines might also exert anti-inflammatory effects by pathways independent of H(1)-receptor binding. Studies of selected second-generation H(1)-antihistamines have shown that these agents inhibit the release of certain cytokines from basophils, acting at a point downstream from the calcium signaling pathway. It has not yet been determined whether this action represents a class effect.

Releasability of human basophils: cellular sensitivity and maximal histamine release are independent variables.

Within the atopic and nonatopic population, basophils display a wide variation in the extent of histamine release when challenged with an optimal concentration of polyclonal anti-IgE antibody. We have tested the hypothesis that the maximum percent histamine release obtainable with this stimulus is an indicator of basophil sensitivity. We have defined basophil sensitivity, in the context of IgE-mediated release, as the number of cell-surface IgE molecules required to obtain a half-maximal IgE-mediated response, and we have examined this parameter in the basophils of 39 donors. Sensitizing basophils with various densities of antigen-specific IgE, and measuring histamine release at these measured densities, allowed a determination of basophil sensitivity in 38 of the donors. Basophil sensitivities ranged from 300 to 40,000 molecules per basophil (point of 50% maximal response), although most donors fell in the 800 to 8000 range, with 1900 molecules per basophil as the median response. The maximal response, after challenge with an optimal concentration of either polyclonal or monoclonal anti-IgE antibody, ranged from 8% to 94% (median, 50%). There was no correlation between basophil sensitivity and the maximum histamine release obtained by a challenge with anti-IgE antibody. Therefore two independent variables appeared to control the IgE-mediated response in basophils, similar to the parameters of efficacy and potency in drug studies. In the IgE-mediated response one parameter is the maximum histamine release obtainable through IgE-mediated mechanisms, and the second parameter is basophil sensitivity as defined above. As a byproduct of these studies, we also found that the density of unoccupied high-affinity receptors for IgE antibody was uncorrelated with the total receptor density (range, 4200 to 572,000 receptors per basophil). Indeed, the density of unoccupied receptors was remarkably constant across this large range of total receptor densities.

IgE antibody up-regulates high affinity IgE binding on murine bone marrow-derived mast cells

We have examined 3-week-old alcian blue positive cells (putatively mast cells) derived from mouse bone marrow for their expression of Fc epsilon RI. Using an indirect method of sensitizing the cells with immunoglobulin E (IgE) antibody (anti-DNP IgE) and detecting the level of bound IgE antibody by flow cytometry, we found that prolonged culture (1-5 days) with IgE, but not IgG, increased the total receptor density 6 +/- 1.9 fold. During the same period, histamine release in response to antigen (DNP-HSA) increased approximately 6-fold while the cells response to either thrombin or ionomycin remained constant. The greatest up-regulation occurred in the first 2 days of culture. Using 2.4G2 to detect Fc epsilon RII RIII, we could not detect any up-regulation of this receptor. Culturing the cells for 1 h after sensitization did not result in any loss of cell surface IgE, suggesting a reasonably high affinity binding similar to that expected for Fc epsilon RI. This up-regulation was completely inhibited by co-culture with 2 micrograms/ml cycloheximide. These data suggest that IgE is capable of inducing a significant, protein synthesis, dependent up-regulation of its own high affinity receptor on mast cells/basophils.

Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes

Background Activation of human basophils results in the release of many different mediators and the expression of new cell surface proteins. The markers CD63 and CD203c have been used in recent years to assess basophil activation but there have been many studies that demonstrate that expression of these markers can be dissociated from histamine release.

Effects of omalizumab on basophil and mast cell responses using an intranasal cat allergen challenge

BACKGROUND Omalizumab treatment suppresses FcepsilonRI expression faster on blood basophils than skin mast cells. OBJECTIVE We used omalizumab to elucidate the relative contributions of basophil versus mast cell FcepsilonRI activation in a nasal allergen challenge (NAC) model. METHODS Eighteen subjects with cat allergy were enrolled in a 3.5-month, double-blind, randomized (3.5:1), placebo-controlled trial of omalizumab using standard dosing. At baseline, subjects underwent NAC with lavage for prostaglandin D(2) measurement, skin prick test titration (SPTT), and blood sampling for basophil histamine release (BHR) and basophil IgE/FcepsilonRI measurements. Basophil studies were repeated at day 3 and then weekly until cat allergen-induced BHR was <20% of baseline or until day 45. Baseline visit procedures were repeated after the BHR reduction (midstudy NAC) and at the treatment periods completion (final NAC). RESULTS Subjects treated with omalizumab who completed all NACs (n = 12) demonstrated significant mean reduction in BHR to an optimal dose of cat allergen by midstudy NAC compared with baseline (74% decrease; P = .001). In addition, these subjects demonstrated significant decreases in mean combined nasal symptom scores (50% decrease; P = .007) and total sneeze counts (59% decrease; P = .01) by midstudy NAC relative to baseline NAC. In contrast, measures of mast cell response (SPTT and nasal lavage prostaglandin D(2)) were only significantly reduced by the final NAC. Subjects on placebo (n = 4) did not experience a shift in basophil, NAC symptom, or mast cell measures. CONCLUSION Reduction in nasal symptom scores occurred when the basophil, but not mast cell, response was reduced on omalizumab, implicating a role for basophils in the acute NAC response.

IgE antibody-specific activity in human allergic disease

IgE antibody concentration, affinity, clonality and specific activity (also known as the allergen-specific IgE to total IgE ratio) influence the translation of IgE responses into clinically evident allergic symptoms following allergen exposure. Reported IgE-specific activity levels >3–4% place allergic individuals undergoing anti-IgE (Omalizumab®) therapy at a disadvantage for poor resolution of their allergy symptoms following manufacturer’s recommended dosing schemes. We investigated the hypothesis that the specific activity of the IgE antibody response is highly variable with respect to age, allergen specificity and an individual’s total serum IgE level. Second, we investigated whether the IgE-specific activity level influences the extent and rate of loss of effector cell mediator release. IgE-specific activity distributions were plotted against age, allergen specificity and total serum IgE using 18,950 paired total IgE and allergen-specific IgE antibody data obtained from the analysis of sera from 3,614 allergic subjects and covering 182 allergen specificities. The fraction of specific IgE antibody of the total serum IgE was dependent on age of the individual, epitope specificity (clonality) and total serum IgE. The youngest group of allergic individuals with the lowest total serum IgE levels tended to have the highest allergen-specific IgE to total IgE ratios. Hymenoptera venom (54%), peanut (33%) and milk (27%) were the three allergen specificities that elicited the highest frequency of IgE-specific activities >4% among sensitized individuals. A prospective double-blind, placebo-controlled clinical study involving anti-IgE treatment of cat-allergic subjects showed IgE-specific activity was remarkably constant over the 16-week course of treatment, despite the up to 8-fold rise in total serum IgE following repetitive Omalizumab® administration. Changes in specific and total IgE levels paralleled each other in patients receiving anti-IgE therapy. The fastest rate of reduction in cat allergen-induced basophil histamine release following anti-IgE therapy was observed when the cat-specific IgE to total IgE ratio was <2.5%. This reflected the more rapid loss of surface cat-specific IgE antibody with anti-IgE therapy in allergic individuals who displayed a more diverse IgE antibody repertoire. We conclude that IgE-specific activity is an age-, IgE heterogeneity- and total serum IgE-dependent variable that influences the magnitude of effector cell mediator release, and by inference, ultimate allergic symptom induction.

IgE-regulated loss, not IgE-regulated synthesis, controls expression of FcεRI in human basophils

Expression of the high‐affinity receptor on basophils and mast cells is modulated by immunoglobulin E (IgE) antibody. Recent studies have shown that modulation occurs through interaction of IgE with the receptor itself, but the mechanisms underlying this control are not understood. Taking both a theoretical and experimental approach, we examined several competing models that focus on whether there is IgE‐regulated loss, IgE‐regulated synthesis, or both regulated loss and synthesis of the Fc receptor for IgE (FcɛRI). We report that removing IgE from occupied FcɛRI resulted in an accelerated loss only in the unoccupied receptor, with no loss of occupied receptors and no loss of total receptors when all receptors were occupied. Together with previous studies, these results establish that there was IgE‐regulated loss of receptors. An examination of synthetic rates of FcɛRIα using pulse‐labeling with 35S‐methionine indicated no difference in synthetic rates in the presence or absence of IgE. Similarly, the presence or absence of IgE had no influence on the levels of mRNA for either α, β, or γ subunits of FcɛRI. Using model simulations, we found that regulated‐synthesis models could be distinguished from regulated‐loss/constant‐synthesis models on the basis of the relationship between starting FcɛRI densities and changes in density after culture for 1 week in the absence of IgE. Experimental data from this type of study fit a regulated‐loss model that did not include regulation of synthesis. Taken together, these results suggest that IgE regulates cell surface expression of FcɛRI only by regulating the rate that receptor is lost from the cell surface.

Kinetics of mast cell, basophil, and oral food challenge responses in omalizumab-treated adults with peanut allergy.

BACKGROUND Monoclonal antibodies directed at IgE demonstrate clinical efficacy in subjects with peanut allergy, but previous studies have not addressed the kinetics of the clinical response or the role of mast cells and basophils in the food-induced allergic response. OBJECTIVE We sought to determine the kinetics of the clinical response to omalizumab and whether clinical improvement is associated with either mast cell or basophil suppression. METHODS Subjects with peanut allergy were treated with omalizumab for 6 months and assessed for clinical and cellular responses. At baseline, subjects had a double-blind, placebo-controlled oral food challenge (OFC), skin prick test titration (SPTT), and basophil histamine release (BHR) to peanut. BHR was repeated at week 2 and then weekly until it decreased to less than 20% of baseline values. The OFCs and SPTTs were repeated after the BHR reduction (or at week 8 if BHR did not decrease) and again at 6 months. RESULTS Fourteen subjects enrolled in the study. At the second food challenge, there was a significant increase in the threshold dose of peanut inducing allergic symptoms (80 to 6500 mg, P < .01). Peanut-induced BHR was either completely suppressed (n = 5) or 10-fold more allergen was required to induce maximal BHR (n = 9), and SPTT responses were not significantly changed from baseline. After 6 months of omalizumab, further changes in the OFC threshold dose or BHR were not observed, but a significant suppression in SPTTs was identified. CONCLUSIONS The clinical response to omalizumab occurs early in treatment when the basophil, but not the mast cell, is suppressed, supporting a role for the basophil in acute food reactions.

Upregulation of FcϵRI on human basophils by IgE antibody is mediated by interaction of IgE with FcϵRI

Abstract Background: IgE is now known to upregulate the expression of FcϵRI on human basophils. It is not known which receptor on basophils mediates this process of upregulation. Objective: We sought to determine whether galectin-3, FcϵRII (CD23), or FcϵRI were involved in the upregulation of FcϵRI by IgE. Methods: The role of galectin-3 was examined by measuring the influence of α-lactose on upregulation. Basophils were examined for expression of FcϵRII (CD23) by flow cytometry and messenger (m)RNA expression. Functional discrimination between binding to FcϵRII or FcϵRI was examined through the use of mutant IgE-Fc fragments or anti-FcϵRII antibody. Results: Upregulation of FcϵRI on basophils in the presence of IgE was not altered by coincubation with α-lactose, eliminating a role for galectin-3. Basophils were not found to express FcϵRII, as determined by flow cytometry with enriched basophil preparations or RT-PCR with highly purified basophil preparations. A mutant of the Fc fragment of IgE (IgE-Fc), which binds to FcϵRI with a greater than 10-fold lower affinity than IgE or wild-type IgE-Fc but exhibits no change in affinity for FcϵRII, allowed us to distinguish between the functions of the two Fc receptors. The mutant (R334S; Henry et al 1997) was required at about 30-fold higher concentration than the wild-type IgE-Fc for the same stimulation of FcϵRI expression on basophils, thus excluding a role for FcϵRII in the response. In addition, treatment of basophils with anti-FcϵRII antibody (MHM6), which is known to be competitive with IgE, had no effect on the expression of FcϵRI or the ability of IgE to upregulate expression of FcϵRI. Conclusion: Collectively, these data indicate that IgE interacts with FcϵRI to upregulate its expression on human basophils. (J Allergy Clin Immunol 1999;104:492-8.)