David Proud

Inflammatory Mediators in Late Antigen-Induced Rhinitis

To investigate the mechanisms responsible for the late-phase response in patients with allergies, we measured four biochemical mediators (histamine, tosyl-L-arginine methyl ester [TAME]-esterase, kinin, and prostaglandin D2) in nasal secretions after nasal challenge with pollen antigen in 12 patients with allergy. Nine patients had an immediate response and a recurrence of symptoms 3 to 11 hours after challenge. The clinical symptoms during recurrence were accompanied by a second increase in levels of histamine, TAME--esterase, and kinin over base-line values, although kinin levels were lower than during the immediate response. In contrast, although the levels of prostaglandin D2 were significantly increased during the immediate response, they did not increase above base line during the late response. Rechallenge with allergen 11 hours after the initial provocation, however, was associated with reappearance of all four biochemical mediators, including prostaglandin D2. We conclude that the late response to nasal challenge with allergen is accompanied by a second increase in the concentrations of histamine and TAME--esterase but differs from the immediate response in the lack of prostaglandin D2 production and in the amount of kinin generated. Since histamine is released only by mast cells and basophils and prostaglandin D2 is not produced by basophils, we suggest that these cells are partly responsible for the late-phase response.

Infection of a human respiratory epithelial cell line with rhinovirus. Induction of cytokine release and modulation of susceptibility to infection by cytokine exposure.

Rhinovirus infections cause over one third of all colds and are a contributing factor to exacerbations of asthma. To gain insights into the early biochemical events that occur in infected epithelial cells, we develop, for the first time, a model in which a pure respiratory epithelial cell population can be routinely infected by rhinovirus. Viral infection was confirmed by demonstrating that viral titers of supernatants and lysates from infected cell increased with time and by PCR. Infection by rhinovirus 14 was inhibited by homotypic antiserum and by antibodies to intercellular adhesion molecule-1 (ICAM-1), the receptor for this virus. Susceptibility of epithelial cells to infection by rhinovirus 14 (but not rhinovirus 2, an ICAM-1 independent strain) can be increased by preexposure of cells to TNF alpha, whereas IFN gamma reduces susceptibility to infection by both rhinovirus strains. Rhinovirus infection per se does not markedly alter ICAM-1 expression on epithelial cells. Finally, we demonstrate that rhinovirus infection induced increased production of IL-8, IL-6, and GM-CSF from epithelial cells. Production of IL-8 correlated with viral replication during the first 24 h after infection. This model should provide useful insights into the pathogenesis of rhinovirus infections.

Kinins are generated in vivo following nasal airway challenge of allergic individuals with allergen.

Using a recently developed model of nasal challenge, we have obtained data that clearly demonstrate, for the first time, kinin generation during a local allergic reaction in vivo. Allergic individuals (n = 8) and matched nonallergic controls (n = 8) were challenged intranasally with the appropriate antigen and nasal washes were taken before and after challenge. Washes were assayed for kinin, histamine, and [3H]-N-alpha-tosyl-L-arginine methyl ester (TAME)-esterase activity. Increased kinin generation was found by radioimmunoassay (RIA) in the nasal washes of all the allergics (5,560 +/- 1,670 pg/ml) but in none of the controls (38 +/- 16 pg/ml). The presence of kinin was highly correlated with that of histamine and TAME-esterase activity and with the onset of clinical symptoms (P less than 0.001). Serial dilutions of nasal washes produced RIA displacement curves that paralleled the standard curve, and recovery of standard kinins that were added to nasal washes was 100 +/- 4% (n = 14). Kinin recovery was identical in both allergics and controls and did not vary significantly with antigen challenge. The immunoreactive kinin in nasal washes was stable to boiling and not precipitated by ethanol, but completely destroyed by carboxypeptidase B. It was evenly distributed between the sol and gel phases of nasal washes. High performance liquid chromatography analysis of the immunoreactive kinin in nasal washes showed it to be a mixture of lysylbradykinin and bradykinin. We conclude that kinins are produced during local allergic reactions in the nose and may contribute to the symptomatology of the allergic response.

Detection of tissue kallikrein in the bronchoalveolar lavage fluid of asthmatic subjects.

Kininogenase activity was detected by cleavage of radiolabeled substrate (125I-high molecular weight kininogen [HMWK]) in 22 of 24 bronchoalveolar lavage (BAL) fluid samples from 17 asthmatics who either responded to aerosolized allergen challenge or had symptoms of active asthma. In contrast, six of seven normal controls lacked enzymatic activity. Levels of free immunoreactive kinin found in BAL fluid correlated with the presence of kininogenase activity (P = 0.002). The cleavage pattern of 125I-HMWK by the BAL fluid kininogenase (a dominant 65,000-mol wt fragment), and synthetic inhibitor profile (phe-phe-arg-CH2Cl and phenylmethylsulfonyl fluoride) were compatible with a tissue kallikrein. Peak kininogenase activity eluted at an apparent molecular weight of 20,000-34,000 by HPLC gel filtration. Its antigenic identity was established by immunoblotting with anti-human urinary kallikrein antibody and its activity was inhibited by this antibody. Lysylbradykinin was generated during incubation of fractionated BAL fluid and purified HMWK, the characteristic cleavage product of the tissue kallikreins. We conclude that elevated amounts of tissue kallikrein and kinin are present in the bronchoalveolar spaces of asthmatic subjects. Kinin generation may contribute to the asthmatic response directly through edema formation and smooth muscle contraction and by augmenting release and/or production of preformed (histamine) and secondary mediators such as leukotrienes and platelet-activating factor.

Epithelial cells as regulators of airway inflammation

The past decade has seen major advances in our understanding of the metabolic and biochemical functions of the epithelial cell. As a result, the traditional view of the epithelium as a relatively passive physical barrier to the internal environment has been superseded by the concept that the epithelial plays a key role in regulating airway inflammation. The epithelial cell experts this modulatory effect in several ways. Epithelial cells can function maintain mucosal integrity and to modulate local immune responses. They can also limit inflammatory processes by degrading, or inhibiting, proinflammatory mediators and proteins. However, the epithelium also responds to a range of stimuli by producing biologically active mediators that can influence airway inflammation. These include, but are not limited to a broad range of cytokines and chemokines that can exert profound effects on inflammatory cells, as well as lipid and peptide mediators. This review will highlight some of these aspects of the role of the epithelium in regulating airway inflammation.

Inhibition of mediator release in allergic rhinitis by pretreatment with topical glucocorticosteroids

Patients with allergic rhinitis often have immediate symptoms after antigen challenge (the early-phase response), followed several hours later by a recurrence of symptoms (the late-phase response). Systemic glucocorticosteroids are known to inhibit the late-phase but not the early-phase response. We studied the effect of one week of pretreatment with topical (rather than systemic) glucocorticosteroids on the response to nasal challenge with antigen in a double-blind, randomized, placebo-controlled crossover study of 13 allergic patients who had previously had a dual response to nasal challenge. The patients were challenged with three 10-fold increments of allergen, producing an early response, and were then followed for 11 hours, encompassing the late response, before they were rechallenged with the lowest dose of allergen. We monitored their responses by means of symptom scores and measurements of the levels of histamine, tosyl-L-arginine methyl ester (TAME)-esterase activity, and kinins in nasal lavages. Topical glucocorticosteroids significantly reduced both the symptoms and the levels of histamine, TAME-esterase activity, and kinins in the early, late, and rechallenge allergic reactions. The fact that, in contrast to treatment with systemic glucocorticosteroids, prolonged pretreatment with topical glucocorticosteroids inhibited the early-phase response to antigen suggests that the route and duration of administration affect the mechanisms of action of the steroids. We conclude that inhibition of the early-phase as well as the late-phase response by topical glucocorticosteroids may provide an advantage over treatment with systemic glucocorticosteroids in patients with allergic rhinitis.

Nasal challenge with cold, dry air results in release of inflammatory mediators. Possible mast cell involvement.

The purpose of our study was to assess the effect of cold, dry air (CDA) on the nasal mucosa of selected individuals in relation to the release of inflammatory mediators associated with mast cells. 12 subjects with a history of nasal symptoms of rhinorrhea and congestion upon cold or dry environmental exposure were challenged by nasal breathing of CDA and warm, moist air (WMA). Each subject was tested on two occasions with the order of the challenges reversed. Symptom scores were recorded, and the levels of histamine, prostaglandin (PG) D2, kinins, and [3H]-N-alpha-tosyl-L-arginine methyl ester (TAME)-esterase activity in nasal lavage fluids were measured. CDA caused a significant increase in mediator levels and in symptom scores as compared to baseline or to WMA. No significant increase in symptom scores or mediators was noted after WMA challenge, with the exception of a marginal increase in kinins. The response to CDA was similar, regardless of challenge order. Changes in mediators correlated with one another, and symptom scores correlated significantly with the levels of histamine, kinins, and PGD2. Five subjects without a history of nasal symptoms on cold air exposure had no change in mediators or symptom scores after CDA or WMA challenge. We conclude that CDA causes the release of inflammatory mediators possibly associated with mast cells and speculate that such a mechanism may be involved in the bronchospasm induced by CDA in asthmatics.

Nasal challenge with ragweed pollen in hay fever patients. Effect of immunotherapy.

Challenge of the nasal mucosa of allergic subjects with specific allergen induces not only the expected sneezing and rhinorrhea, but also the appearance in nasal secretions of mediators commonly associated with activation of mast cells or basophils: histamine, leukotrienes, prostaglandin D2 (PGD2), kinins, and TAME ([3H]-N-alpha-tosyl-L-arginine methyl ester)-esterase. To determine whether specific immunotherapy alters mediator release in vivo, nasal pollen challenge was used to compare 27 untreated highly sensitive ragweed (RW)-allergic subjects with 12 similarly sensitive patients receiving long-term immunotherapy (3-5 yr) with RW extract (median dose, 6 micrograms RW antigen E). The two groups were equally sensitive based on skin tests and basophil histamine release. The immunized group had a diminished response as demonstrated by (a) the treated group required higher pollen doses to excite sneezing or mediator release; (b) significantly fewer subjects in the treated group released mediators at any dose (TAME-esterase [P = 0.005], PGD2 [P = 0.04]), and (c) the treated group released 3-5-fold less mediator (TAME-esterase [P = 0.01], and histamine [P = 0.02]).

Epithelial cells and airway diseases.

Summary:  The airway epithelial cell is the initial cell type impacted both by inhaled environmental factors, such as pathogens, allergens, and pollutants, and inhaled medications for airway diseases. As such, epithelial cells are now recognized to play a central role in the regulation of airway inflammatory status, structure, and function in normal and diseased airways. This article reviews our current knowledge regarding the roles of the epithelial cell in airway inflammation and host defense. The interactions of inhaled environmental factors and pathogens with epithelial cells are also discussed, with an emphasis on epithelial innate immune responses and contributions of epithelial cells to immune regulation. Recent evidence suggesting that epithelial cells play an active role in inducing several of the structural changes, collectively referred to airway remodeling, seen in the airways of asthmatic subjects is reviewed. Finally, the concept that the epithelium is a major target for the actions of a number of classes of inhaled medications is discussed, as are the potential mechanisms by which selected drugs may alter epithelial function.

Inflammatory mediator release on conjunctival provocation of allergic subjects with allergen

To evaluate the role of inflammatory mediators in the pathogenesis of the ocular allergic response, 23 subjects with positive histories of allergies to either cat dander or ragweed pollen and positive skin tests to the appropriate allergen extract were recruited and were subjected to conjunctival provocation. The tear duct of the left eye of each subject was blocked with a collagen plug while the right eye was left unplugged. In all cases, the eye was initially provoked with saline and subsequently with the appropriate allergen extract. Nonallergic subjects, or allergic subjects provoked with nonrelevant allergen, were used as control subjects. After each provocation, symptoms were recorded, and tears were collected with preweighed strips of filter paper (Schirmer strip). Each strip was placed into a tared tube containing fluid appropriate for the optimal preservation of the mediator to be measured. It was therefore possible to calculate the weight of tears collected and to express mediator levels per milliliter of tears. All allergic subjects demonstrated a positive symptomatic response to allergen challenge, whereas the control subjects remained asymptomatic. Blockage of the tear duct did not significantly alter the response. For allergic subjects, the levels of histamine, kinins, prostaglandin D2, albumin, and TAME-esterase activity were all significantly (p less than 0.005 in each case) greater after allergen challenge than after saline challenge. Furthermore, levels of each of these mediators after allergen challenge (expressed as increases above levels after saline provocation) were significantly greater for allergic subjects than for control subjects (p less than 0.005 in each case). Thus, the clinical response to conjunctival provocation with allergen is associated with increases in the levels of inflammatory mediators in tears.