Effect of pollen-mediated oxidative stress on immediate hypersensitivity reactions and late-phase inflammation in allergic conjunctivitis

BACKGROUND: Allergic eye diseases are complex inflammatory conditions of the conjunctiva that are becoming increasingly prevalent and present an increasing economic burden because of direct and indirect health expenditures. OBJECTIVE: We sought to identify factors that may synergize with antigen-induced allergic inflammation and lead to allergic conjunctivitis. We used a murine model of allergic conjunctivitis to test the effect of oxidative stress generated by pollen oxidases using nicotinamide adenine dinucleotide (reduced) or nicotinamide adenine dinucleotide phosphate (reduced) (NAD[P]H) as an electron donor present in pollen grains. METHODS: Reactive oxygen species (ROS) generation by hydrated Ambrosia artemisiifolia pollen (short ragweed pollen; RWP) grains was determined by using 2'-7'-dihydro-dichlorofluorescein diacetate, nitroblue tetrazolium reduction, and Amplex Red assay. The RWP-induced changes in intracellular ROS levels were examined in A549 cells, human primary bronchial epithelial cells, and murine conjunctiva. RESULTS: Ragweed pollen grains contain NAD(P)H oxidase activity, which is diphenyleneiodonium-sensitive and quinacrine-sensitive and sodium azide-resistant. These NAD(P)H oxidases generate a superoxide anion that can be converted to H2O2 by pollen grain-associated superoxide dismutase. These diffusible oxygen radicals from pollen grains increase intracellular ROS levels in cultured epithelial cells and murine conjunctiva. Similar phenomena were observed in sensitized and naive mice, indicating that the RWP-induced oxidative stress in conjunctival epithelium is independent of adaptive immunity. Inactivation of NAD(P)H oxidase activity in RWP decreases the immediate-type hypersensitivity and inflammatory cell infiltration into the conjunctiva. CONCLUSION: Our data suggest that ROS generated by NAD(P)H oxidases in pollen grains intensify immediate allergic reactions and recruitment of inflammatory cells in murine conjunctiva.     

Role of pollen NAD(P)H oxidase in allergic inflammation

PURPOSE OF REVIEW: Plant pollens are one of the most common outdoor allergens. Pollen grains and subpollen particles can reach lower airways and induce symptoms of seasonal asthma and allergic rhinitis. Plants possess NAD(P)H oxidase activity that generates reactive oxygen species for physiological functions such as root-hair and pollen-tube growth, defense against microbial infections and cell signaling. The presence of NAD(P)H oxidases in pollens and their role in induction of airway inflammation have not been described until recently. RECENT FINDINGS: We discovered the presence of NAD(P)H oxidase in ragweed and other plant pollens. These oxidases induce reactive oxygen species in mucosal cells (signal 1) independent of adaptive immunity. This reactive oxygen species facilitates antigen (signal 2)-induced allergic inflammation. Inhibiting signal 1 by administration of antioxidants attenuated ragweed extract-induced allergic inflammation. Likewise, abrogating signal 2 by antigen challenge in mice lacking T cells failed to induce allergic inflammation. SUMMARY: Reactive oxygen species generated by pollen NAD(P)H oxidase play a major role in pathogenesis of allergic airway inflammation and airway hypersensitivity. Based on our findings, we propose a 'two signal hypothesis of allergic inflammation' in which both signal 1 (reactive oxygen species) and signal 2 (antigen presentation) are required in order to induce full-blown allergic inflammation.     

Lactoferrin decreases pollen antigen-induced allergic airway inflammation in a murine model of asthma

Pollen grains contain reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and in contact with mucosal surfaces generate superoxide anion (O2*-). In the presence of iron, O2*- may be converted to more reactive oxygen radicals, such as to H2O2 and/or *OH, which may augment antigen-induced airway inflammation. The aim of the study was to examine the impact of lactoferrin (LF), an iron-binding protein, on ragweed (Ambrosia artemisiifolia) pollen extract (RWE)-induced cellular oxidative stress levels in cultured bronchial epithelial cells and accumulation of inflammatory and mucin-producing cells in airways in a mouse model of allergic airway inflammation. Results show that LF lowered RWE-induced increase in cellular reactive oxygen species (ROS) levels in bronchial epithelial cells. Most importantly, LF significantly decreased accumulation of eosinophils into airways and subepithelium of intranasally challenged, sensitized mice. LF also prevented development of mucin-producing cells. Amb a 1, the major allergenic ragweed pollen antigen lacking NADPH oxidase activity, induced low-grade airway inflammation. When administered along with glucose oxidase (G-ox), a superoxide-generating enzyme, Amb a 1 induced robust airway inflammation, which was significantly lowered by LF. Surprisingly, LF decreased also inflammation caused by Amb a 1 alone. Iron-saturated hololactoferrin had only a marginal effect on RWE-induced cellular ROS levels and RWE- or Amb a 1 plus G-ox-induced inflammation. We postulate that free iron in the airways chemically reduces O2*- to more reactive species which augment antigen-induced inflammation in a mouse model of asthma. Our results suggest the utility of LF in human allergic inflammatory disorders.     

Inhibiting pollen reduced nicotinamide adenine dinucleotide phosphate oxidase-induced signal by intrapulmonary administration of antioxidants blocks allergic airway inflammation

BACKGROUND: Ragweed extract (RWE) contains NADPH oxidases that induce oxidative stress in the airways independent of adaptive immunity (signal 1) and augment antigen (signal 2)-induced allergic airway inflammation. OBJECTIVE: To test whether inhibiting signal 1 by administering antioxidants inhibits allergic airway inflammation in mice. METHODS: The ability of ascorbic acid (AA), N-acetyl cystenine (NAC), and tocopherol to scavenge pollen NADPH oxidase-generated reactive oxygen species (ROS) was measured. These antioxidants were administered locally to inhibit signal 1 in the airways of RWE-sensitized mice. Recruitment of inflammatory cells, mucin production, calcium-activated chloride channel 3, IL-4, and IL-13 mRNA expression was quantified in the lungs. RESULTS: Antioxidants inhibited ROS generation by pollen NADPH oxidases and intracellular ROS generation in cultured epithelial cells. AA in combination with NAC or Tocopherol decreased RWE-induced ROS levels in cultured bronchial epithelial cells. Coadministration of antioxidants with RWE challenge inhibited 4-hydroxynonenal adduct formation, upregulation of Clca3 and IL-4 in lungs, mucin production, recruitment of eosinophils, and total inflammatory cells into the airways. Administration of antioxidants with a second RWE challenge also inhibited airway inflammation. However, administration of AA+NAC 4 or 24 hours after RWE challenge failed to inhibit allergic inflammation. CONCLUSION: Signal 1 plays a proinflammatory role during repeated exposure to pollen extract. We propose that inhibiting signal 1 by increasing antioxidant potential in the airways may be a novel therapeutic strategy to attenuate pollen-induced allergic airway inflammation. CLINICAL IMPLICATIONS: Administration of antioxidants in the airways may constitute a novel therapeutic strategy to prevent pollen induced allergic airway inflammation.     

Substrate preference profiles of proteases released by allergenic pollens

BACKGROUND: Pollens are important triggers for allergic asthma and seasonal rhinitis. We have recently reported that proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of epithelial integrity by proteases released following deposition of pollens on mucosal surfaces could promote sensitization and induce inflammation. OBJECTIVE: To compare protease activities released by allergenic pollens of various genera. METHODS: We used a rapid microassay which quantifies cleavage of dipeptide ester substrates to characterize the substrate preference profiles of serine proteases in diffusates of the pollens of perennial ryegrass (Lolium perenne), Kentucky blue grass (Poa pratensis), Bermuda grass (Cynodon dactylon), Western ragweed (Ambrosia spp.), white birch (Betula spp.) and Sydney golden wattle (Acacia longifolia). RESULTS: Comparison of the profiles revealed notable differences as well as similarities between serine protease activities released by these pollens. Diffusates of Kentucky blue grass pollen exhibited very high substrate preference for arginine and lysine. For other pollens, cleavage of the cysteine substrate was usually the most rapid and was associated with marked preference for leucine and methionine. There was considerable variation between these pollens in the rates of cleavage of the histidine substrate. In addition, we observed high rates of cleavage of arginine and lysine substrates by Acacia pollen diffusate. CONCLUSION: At least two dominant patterns of substrate preference are identifiable in the mixtures of proteases released by hydrated pollens. Purification of the proteases responsible for these patterns of activity will facilitate investigation of their role in airway epithelial injury and allergic disease.     

Links between pollen, atopy and the asthma epidemic

Pollen allergy has been found in 80-90% of childhood asthmatics and 40-50% of adult-onset asthmatics. Despite the high prevalence of atopy in asthmatics, a causal relationship between the allergic response and asthma has not been clearly established. Pollen grains are too large to penetrate the small airways where asthma occurs. Yet pollen cytoplasmic fragments are respirable and are likely correlated with the asthmatic response in allergic asthmatics. In this review, we outline the mechanism of pollen fragmentation and possible pathophysiology of pollen fragment-induced asthma. Pollen grains rupture within the male flowers and emit cytoplasmic debris when winds or other disturbances disperse the pollen. Peak levels of grass and birch pollen allergens in the atmosphere correlated with the occurrence of moist weather conditions during the flowering period. Thunderstorm asthma epidemics may be triggered by grass pollen rupture in the atmosphere and the entrainment of respirable-sized particles in the outflows of air masses at ground level. Pollen contains nicotinamide adenine dinucleotide phosphate (reduced) oxidases and bioactive lipid mediators which likely contribute to the inflammatory response. Several studies have examined synergistic effects and enhanced immune response from interaction in the atmosphere, or from co-deposition in the airways, of pollen allergens, endogenous pro-inflammatory agents, and the particulate and gaseous fraction of combustion products. Pollen and fungal fragments also contain compounds that can suppress reactive oxidants and quench free radicals. It is important to know more about how these substances interact to potentially enhance, or even ameliorate, allergic asthma.     

Rapidly released allergens from short ragweed pollen: I. Kinetics of release of known allergens in relation to biologic activity

Study of the kinetics of in vitro release of known antigens from short ragweed pollen revealed a slow release of AgE (1.5% to 4% release after 16 min) but a rapid release of other highly allergenic components. The rapidly released allergens in 16-min pollen extracts were found to be mainly highly basic proteins including the well-characterized Ra5 molecule along with several hitherto uncharacterized components. Considering previous data that suggest that particles of a similar size to ragweed pollen grains remain in the nose only about 6 to 8 min following inhalation, it is difficult to explain the apparent anomaly between the high biologic potency of 16-min extracts and their low AgE content. Increase in the pH of the extracting buffer from the physiologically normal nasal pH of 5.5 to 6.5 to about pH 8.0 to 8.5 (characteristically found in rhinitis) increased the proportion of AgE released after 16-min extraction by about 10-fold, suggesting that response to allergens in ragweed and other inhalants may enhance AgE release and thereby aggravate allergic symptoms. Detailed comparison of the allergenic activity of AgE and of 16-min and 4-day ragweed extracts in 38 ragweed-sensitive subjects suggested that allergens other than AgE were together more important than AgE in causing ragweed allergy in most patients, although large patient-to-patient differences in relative response were observed. Our data emphasize the need to reevaluate materials used in both diagnosis and treatment of ragweed allergy. The importance of AgE relative to other ragweed pollen components with respect to induction of allergic symptomatology deserves to be placed in a more balanced perspective.     

Amb a 1-linked CpG oligodeoxynucleotides reverse established airway hyperresponsiveness in a murine model of asthma

BACKGROUND: Recently, it has been demonstrated that immunostimulatory DNA sequences (ISS) containing CpG motifs prevent the development of allergic airway responses in murine models of disease. However, few studies have addressed the issue of whether these agents will reverse established Tm(H)2-driven allergic airway responses. OBJECTIVE: The aim of this study was to determine whether intradermal delivery of an immunogenic protein of ragweed pollen linked to an immunostimulatory DNA sequence could reverse an established allergic response in the mouse lung. METHODS: Mice sensitized and challenged with ragweed pollen extract were treated intradermally twice at 1-week intervals with an ISS chemically linked to Amb a 1 (Amb a 1-ISS). One week after the Amb a 1-ISS treatment, mice were rechallenged intratracheally with ragweed extract, and airway responses were assessed. RESULTS: Amb a 1-ISS treatment of ragweed-sensitized and ragweed-challenged mice significantly reversed allergen-induced airway hyperresponsiveness and suppressed the total number of eosinophils in bronchoalveolar lavage fluid. The inhibitory effect of Amb a 1-ISS was associated with a marked increase in IFN-gamma levels by Amb a 1-stimulated splenocytes and a shift in the antibody profile from a T(H)2-directed IgG1 response to a T(H)1-directed IgG2a response. Interestingly, the inhibitory effect of Amb a 1-ISS on allergen-driven airway hyperresponsiveness was independent of suppression of T(H)2 cytokine production. CONCLUSION: These results demonstrate that intradermal delivery of allergen-specific DNA conjugates can reverse established allergic responses in the murine lung, supporting their potential use in the treatment of human asthma.     

Requirements for allergen-induced airway inflammation and hyperreactivity in CD4-deficient and CD4-sufficient HLA-DQ transgenic mice.

BACKGROUND: Airway inflammation is central to the pathogenesis of allergic asthma, and molecules that mediate this process obviously represent targets for therapy. OBJECTIVE: To study the role of CD4(+) T cells and/or HLA-DQ molecules in allergic asthma, we have generated and characterized models of short ragweed allergen (SRW)-induced inflammation using transgenic mice with HLA-DQ (DQ6 or DQ8), human CD4 (hCD4), or both on a genetic background that lacks mouse MHC II and CD4 (Abeta(0)/mCD4(0)). METHODS: Mice were actively sensitized and later challenged intranasally with SRW allergenic extract. Bronchoalveolar lavage fluid composition, airway inflammation and hyperresponsiveness, blood eosinophil levels, and cell proliferation were examined. RESULTS: In response to SRW treatment, both DQ6 and DQ8 transgenic mice expressing hCD4 developed pulmonary eosinophilia and associated lung tissue damage with increase in eosinophil peroxidase and T(H)2 cytokines in bronchoalveolar lavage fluid, strong airway hyperreactivity, and persistent blood eosinophilia. The response was independent of mast cells/histamine pathway and was mediated by DQ-restricted hCD4(+) T cells. Interestingly, lungs of CD4-deficient DQ6 transgenic mice showed an eosinophilic inflammation without local increase in cytokines and eosinophil peroxidase. The allergic reaction was absent in double-knockout mice and mice expressing either DQ8 or hCD4 alone. CONCLUSIONS: DQ6 molecules are critical to SRW-induced allergy and can operate in the presence or absence of CD4. However, both DQ antigens and CD4 molecules are critical for full manifestation of allergen-induced asthma in transgenic mice.     

Effect of GM-CSF on immune,inflammatory, and clinical responses to ragweed in a novel mouse model of mucosal sensitization

BACKGROUND: Conventional models of allergic airway inflammation involve intraperitoneal administration of ovalbumin in conjunction with a chemical adjuvant (generally aluminum hydroxide) to generate allergic airways inflammation. Here we have investigated the effect of respiratory mucosal exposure to a ragweed extract in the absence of chemical adjuvant on the generation of allergic responses. OBJECTIVES: We sought to develop a mouse model of ragweed-induced allergic airway inflammation through mucosal sensitization and to investigate the role of GM-CSF in this process. METHODS: Ragweed was delivered intranasally to an airway microenvironment enriched with GM-CSF, which was achieved by means of either multiple coadministrations of recombinant GM-CSF or a single delivery of an adenoviral vector carrying the GM-CSF transgene. RESULTS: Administration of a purified ragweed extract leads to T(H)2 sensitization (and not inhalation tolerance) accompanied by mild airway inflammation, modest clinical symptoms, and moderate production of T(H)2 cytokines by splenocytes on ragweed restimulation. The administration of anti-GM-CSF antibodies in conjunction with ragweed diminished T(H)2-associated cytokine production. These responses were amplified by enriching the airway microenvironment with GM-CSF. Under these conditions, all T(H)2-associated immune-inflammatory responses, as well as the clinical responses, were considerably enhanced. To investigate the mechanism underlying these effects, we examined lung mononuclear cells by means of flow cytometry and detected a substantial expansion of antigen-presenting cells, particularly dendritic cells, as well as a substantially increased activation of these antigen-presenting cells, as demonstrated by the expression of B7 molecules, particularly B7.2. CONCLUSION: GM-CSF plays an important role in the generation of allergic immune-inflammatory responses to ragweed.     

Late summer and fall (March-May) pollen allergy and respiratory disease in Northern New South Wales, Australia.

BACKGROUND: Many people in the subtropical Northern Rivers area of New South Wales, Australia, blame the pollen of Tibouchina tree, which flowers at the same time as ragweed, Bahia grass and Bermuda grass, for hayfever and asthma exacerbations during fall between March and May. OBJECTIVES: To determine whether Tibouchina pollen is allergenic. To determine whether airborne ragweed pollen is present in this region for sufficient length of time and concentration to cause fall respiratory symptoms, and to determine if Bahia grass and Bermuda grass are associated with fall respiratory symptoms. METHODS: Pollen and Alternaria spores were monitored using a Burkard 7-day spore trap. Two hundred and six volunteers in the Northern Rivers area filled in questionnaires before skin prick tests (SPT) were performed with a panel of skin testing extracts. RESULTS: One hundred fifty-three (74.3%) subjects were atopic and reacted to one or more aeroallergens. Seventy were SPT positive to ragweed, OR 3.36 (CI 1.03 to 12.15) and 11 to Tibouchina (OR incalculable). Fifty of the 70 ragweed-positive subjects had fall hayfever or exacerbations of hayfever and/or asthma, OR 23.4 (CI 8.90 to 64.00). Eleven subjects were SPT positive to Tibouchina extract. There was a statistical association between Bermuda grass and hayfever, but not asthma OR 13.44 (CI 1.85 to 27.04). CONCLUSIONS: Ragweed pollen was present for a sufficient length of time and concentration to sensitize and provoke fall hayfever and asthma exacerbations. Tibouchina pollen is an aeroallergen causing mild-to-moderate allergic symptoms in a few people. There is an association between Bahia grass and asthma in children, and between Bermuda grass and allergic rhinitis in adults.     

Production of allergenic pollen by ragweed (Ambrosia artemisiifolia L.) is increased in CO2-enriched atmospheres.

BACKGROUND: The potential effects of global climate change on allergenic pollen production are still poorly understood. OBJECTIVE: To study the direct impact of rising atmospheric CO2 concentrations on ragweed (Ambrosia artemisiifolia L.) pollen production and growth. METHODS: In environmentally controlled greenhouses, stands of ragweed plants were grown from seed through flowering stages at both ambient and twice-ambient CO2 levels (350 vs 700 microL L(-1)). Outcome measures included stand-level total pollen production and end-of-season measures of plant mass, height, and seed production. RESULTS: A doubling of the atmospheric CO2 concentration stimulated ragweed-pollen production by 61% (P = 0.005). CONCLUSIONS: These results suggest that there may be significant increases in exposure to allergenic pollen under the present scenarios of global warming. Further studies may enable public health groups to more accurately evaluate the future risks of hay fever and respiratory diseases (eg, asthma) exacerbated by allergenic pollen, and to develop strategies to mitigate them.     

Environmental risk factors and allergic bronchial asthma

The prevalence of allergic respiratory diseases such as bronchial asthma has increased in recent years, especially in industrialized countries. A change in the genetic predisposition is an unlikely cause of the increase in allergic diseases because genetic changes in a population require several generations. Consequently, this increase may be explained by changes in environmental factors, including indoor and outdoor air pollution. Over the past two decades, there has been increasing interest in studies of air pollution and its effects on human health. Although the role played by outdoor pollutants in allergic sensitization of the airways has yet to be clarified, a body of evidence suggests that urbanization, with its high levels of vehicle emissions, and a westernized lifestyle are linked to the rising frequency of respiratory allergic diseases observed in most industrialized countries, and there is considerable evidence that asthmatic persons are at increased risk of developing asthma exacerbations with exposure to ozone, nitrogen dioxide, sulphur dioxide and inhalable particulate matter. However, it is not easy to evaluate the impact of air pollution on the timing of asthma exacerbations and on the prevalence of asthma in general. As concentrations of airborne allergens and air pollutants are frequently increased contemporaneously, an enhanced IgE-mediated response to aeroallergens and enhanced airway inflammation could account for the increasing frequency of allergic respiratory allergy and bronchial asthma. Pollinosis is frequently used to study the interrelationship between air pollution and respiratory allergy. Climatic factors (temperature, wind speed, humidity, thunderstorms, etc) can affect both components (biological and chemical) of this interaction. By attaching to the surface of pollen grains and of plant-derived particles of paucimicronic size, pollutants could modify not only the morphology of these antigen-carrying agents but also their allergenic potential. In addition, by inducing airway inflammation, which increases airway permeability, pollutants overcome the mucosal barrier and could be able to "prime" allergen-induced responses. There are also observations that a thunderstorm occurring during pollen season can induce severe asthma attacks in pollinosis patients. After rupture by thunderstorm, pollen grains may release part of their cytoplasmic content, including inhalable, allergen-carrying paucimicronic particles.     

Grass pollen immunotherapy as an effective therapy for childhood seasonal allergic asthma

BACKGROUND: Few studies have investigated the use of specific immunotherapy (SIT) for childhood seasonal allergic asthma. OBJECTIVE: We sought to examine the efficacy and safety of SIT with Alutard SQ grass pollen (Phleum pratense Alutard SQ; ALK-Abelló, H?rsholm, Denmark) in children with seasonal allergic asthma. METHODS: A randomized, double-blind, placebo-controlled study assessing the efficacy of grass pollen SIT over 2 pollen seasons was performed. Children (3-16 years) with a history of seasonal allergic asthma sensitized to grass pollen (P pratense) and requiring at least 200 microg of inhaled beclomethasone equivalent per day were enrolled. Subjects with symptomatic asthma or rhinoconjunctivitis outside the grass pollen season were excluded. The primary outcome measure was a combined asthma symptom-medication score during the second pollen season. Secondary outcome measures included end-point titration skin prick testing and conjunctival and bronchial provocation testing to allergen, sputum eosinophilia, exhaled nitric oxide, and adverse events. RESULTS: Thirty-nine subjects were enrolled. Thirty-five subjects provided data for analysis. The use of SIT was associated with a substantial reduction in asthma symptom-medication score compared with that after placebo (P = .04). There were also significant reductions in cutaneous (P = .002), conjunctival (P = .02), and bronchial (P = .01) reactivity to allergen after SIT compared with that after placebo. The 2 groups had similar levels of airway inflammation, despite a trend toward less inhaled steroid use in the active group. No serious adverse events were reported, and no subjects withdrew because of adverse events. CONCLUSION: The study has shown that SIT is effective and well tolerated in children with seasonal allergic asthma to grass pollen.     

Traffic-related air pollutants induce the release of allergen-containing cytoplasmic granules from grass pollen

BACKGROUND/AIM: Pollen cytoplasmic granules (PCG) are loaded with allergens. They are released from grass pollen grains following contact with water and can form a respirable allergenic aerosol. On the other hand, the traffic-related air pollutants NO2 and O3 are known to be involved in the current increase in the prevalence of allergic diseases via their adjuvant effects. Our objective was to determine the effects of air pollutants on the release of PCG from Phleum pratense (timothy grass) pollen. METHODS: P. pratense pollen was exposed to several concentrations of NO2 and O3. The induced morphological damages were observed by environmental scanning electron microscopy, and the amount of PCG released from the pollen upon contact with water was measured. RESULTS: The percentages of damaged grain were 6.4% in air-treated controls, 15% after treatment with the highest NO2 dose (50 ppm) and 13.5% after exposure to 0.5 ppm O3. In treated samples, a fraction of the grains spontaneously released their PCG. Upon subsequent contact with water, the remaining intact grains released more PCG than pollen exposed to air only. CONCLUSIONS: Traffic-related pollutants can trigger the release of allergen-containing granules from grass pollen, and increase the bioavailability of airborne pollen allergens. This is a new mechanism by which air pollution concurs with the current increase in the prevalence of allergic diseases.     

Phleum pratense pollen starch granules induce humoral and cell-mediated immune responses in a rat model of allergy

BACKGROUND: Timothy grass (Phleum pratense) pollen allergens are an important cause of allergic symptoms. However, pollen grains are too large to penetrate the deeper airways. Grass pollen is known to release allergen-bearing starch granules (SG) upon contact with water. These granules can create an inhalable allergenic aerosol capable of triggering an early asthmatic response and are implicated in thunderstorm-associated asthma. OBJECTIVE: We studied the humoral (IgE) and bronchial lymph node cells reactivities to SG from timothy grass pollen in pollen-sensitized rats. METHODS: Brown-Norway rats were sensitized (day 0) and challenged (day 21) intratracheally with intact pollen and kept immunized by pollen intranasal instillation by 4 weeks intervals during 3 months. Blood and bronchial lymph nodes were collected 7 days after the last intranasal challenge. SG were purified from fresh timothy grass pollen using 5 microm mesh filters. To determine the humoral response (IgE) to SG, we developed an original ELISA inhibition test, based on competition between pollen allergens and purified SG. The cell-mediated response to SG in the bronchial lymph node cells was determined by measuring the uptake of [3H]thymidine in a proliferation assay. RESULTS: An antibody response to SG was induced, and purified SG were able to inhibit the IgE ELISA absorbance by 45%. Pollen extract and intact pollen gave inhibitions of 55% and 52%, respectively. A cell-mediated response was also found, as pollen extract, intact pollen and SG triggered proliferation of bronchial lymph node cells. CONCLUSIONS: It was confirmed that timothy grass pollen contains allergen-loaded SG, which are released upon contact with water. These granules were shown to be recognized by pollen-sensitized rats sera and to trigger lymph node cell proliferation in these rats. These data provide new arguments supporting the implication of grass pollen SG in allergic asthma.     

Influence of natural exposure to pollens and domestic animals on airway responsiveness and inflammation in sensitized non-asthmatic subjects

BACKGROUND: Atopy may be a risk factor in the development of asthma. Indoor allergens are considered to be more potent asthma inducers than outdoor ones such as pollens. Lower airway inflammation may be present in non-asthmatic subjects during natural exposure to relevant allergens and may eventually lead to the development of asthma. AIMS: To document seasonal variation in lower airway responsiveness and inflammation in sensitized non-asthmatic subjects, during natural exposure to allergens, and to determine whether it is more marked in those exposed to animals to which they are sensitized. METHODS: Twenty-two atopic subjects were seen during and out of the pollen season. All (but the controls) were sensitized to domestic animals, and to trees, grasses or ragweed. Eleven were not exposed to animals at home and 8 were exposed. They were compared with 3 normal controls. A respiratory questionnaire was administered, allergy skin prick tests, spirometry, methacholine challenge, blood and induced sputum with differential cell counts were obtained during the pollen season for all subjects. These tests were repeated out of the pollen season. RESULTS: Throughout the study, none of the subjects had asthma symptoms. Mean PC(20) was significantly lower in subjects exposed to animals compared with unexposed subjects or controls, both during and out of the pollen season. In season, subjects exposed to animals had significantly higher sputum eosinophil numbers than unexposed or normal control subjects. CONCLUSIONS: Non-asthmatic atopic subjects show variable degrees of airway responsiveness and inflammation. However, subjects exposed to animals show higher airway eosinophilia, which may suggest they are at increased risk of developing airway hyperresponsiveness and asthma.     

Inhalation challenge with ragweed pollen in ragweed-sensitive asthmatics

We reexamined the ability of inhaled ragweed pollen to induce bronchoconstriction in ragweed-sensitive asthmatic patients using a turbo-inhaler to administer pollen quantitatively. Adult subjects were selected for study on the basis of fall season asthmatic attacks, positive skin test, histamine release, RAST, and bronchial challenge responses to ragweed extract. Not one of 12 such subjects had any bronchial response to oral inhalation of whole pollen grains even when the dose was increased to 7640 pollen grains (more than the estimated maximum daily exposure in season), whereas nasal challenge by the same method produced brisk hay fever responses without bronchospasm. On the other hand, when the pollen was ground to fragments with a size range of 1 to 8 micrometers, oral inhalation produced a 35% fall in airways conductance in six of seven subjects in doses ranging from 59 to 20,000 pollen grain equivalents. Atropine pretreatment did not modify the response to pollen fragments, making an irritant response unlikely. These data, coupled with earlier observations that no more than a few pollen grains penetrate further than the larynx, raise further questions about the role of whole ragweed pollen in fall asthma in allergic patients. In addition, ragweed-allergic asthmatics appear not to have their symptoms at the time of maximum pollen load in the air. We believe that small-particle allergens other than ragweed pollen should be considered in most cases of fall seasonal asthma.