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Although mediators, such as lipids, cytokines, and chemokines, are related to the appearance of an IPR, there has been no reliable indicator to predict conditions for the appearance of an IPR.
In this study, we adopted a proteomic approach to investigate the pathogenesis at the level of the plasma proteins and to develop plasma markers to predict the appearance of an IPR following an inhalation challenge with Dermatophagoides pteronyssinus (D.p.). Sixteen mild asthmatics were recruited. Plasma was obtained before challenge and when a decline in forced expiratory volume in 1 s (FEV1) values greater than 20% from the phosphate-buffered saline value was achieved during D.p. allergen challenge (positive responders), or at 60 min after the highest concentration of D.p. allergen was inhaled (negative responders). After comparing normalized volumes of the spots in the two groups, differentially expressed spots were identified using intra-gel digestion and mass spectrometric analysis. Before D.p. antigen challenge, four spots of gamma fibrinogen and its isoforms were significantly decreased and two spots of complement C3 fragments were significantly increased in the positive responders compared to the negative responders. After D.p. antigen challenge, complement C3 fragment was persistently higher, while gamma fibrinogen was lower in the positive responders than in the negative responders. A validation study using Western blotting showed that gamma fibrinogen expression in the IPR-positive asthmatics was significantly decreased compared to the average of the IPR-negative asthmatic control group. These results indicate that alterations in the complement cascade and fibrinogen may predispose patients to the appearance of an immediate response to D.p. allergen challenge and may provide plasma markers to predict the appearance of an IPR.
Allergic asthma is characterized by immunoglobulin E (IgE)-mediated sensitization following allergen exposure, leading to CD4+ lymphocyte responses and subsequent eosinophilic inflammation of the airways, resulting in enhanced bronchial reactivity and reversible airflow obstruction (1). Allergen inhalation in sensitized asthmatic patients results in an immediate-phase reaction (IPR), occurring within minutes and resolving in 30–60 min. A substantial number of patients also present with late-phase reactions (LPRs) that typically begin 3–4 h after allergen inhalation (2). Mechanisms behind airway narrowing in the IPR are primarily secondary to mast cell activation, while LPR is thought to depend on the infiltration of inflammatory cells, including eosinophils and T cells, into the bronchial wall (3, 4). However, even in subjects strongly sensitized to inhalant allergens, some are IPR-negative (5). Why some subjects do not react to allergen inhalation, even though they are sensitized to the allergen, is unclear. Several physiological and immunological factors have been studied in relation to IPRs. Among them, nonspecific bronchial responsiveness is mostly related to the appearance of IPR, although the degree of antigen sensitization, indicated by total and specific IgE, skin test reactions, and the ability to cause basophil histamine release, are less related to the appearance of an IPR (6, 7).
The IPR is initiated by mast cells releasing preformed mediators from their granules, such as histamine, tryptase, and chymase (8). In addition, activated mast cells generate leukotriene C4, prostaglandins, thromboxane, cytokines, and chemokines when stimulated with specific allergens. The changes in these mediators in the peripheral blood, bronchoalveolar lavage (BAL), and urine parallel the appearance of the IPR (9–12). Additionally, mediators, such as thrombin, and lung tissue kallikrein, from activated resident and recruited inflammatory cells, including eosinophils, platelets, alveolar macrophages, and neutrophils, have been demonstrated in the peripheral blood, airway secretions, and urine in IPR following specific allergen challenge (13, 14). However, these mediators do not differ in amount between the IPR-positive and -negative groups before a specific allergen challenge. Thus, to our knowledge there is no reliable indicator in the peripheral blood or urine to predict conditions for the appearance of an IPR.
In this study, we adopted a proteomics approach to investigate the pathogenesis at the level of the plasma proteins and to develop plasma markers to predict the appearance of an IPR following Dermatophagoides pteronyssinus (D.p.) inhalation challenge, because house dust mites are globally the most important sources of indoor allergens responsible for sensitization and the development of asthma.
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- Material and methods
Although sensitization to an allergen is not equivalent to the development of asthma, allergic asthma requires systemic or local sensitization to the allergen and the subsequent development of bronchial hyper-responsiveness (5). Approximately 50% of allergic children and young adults who are exposed to relevant allergens in their houses do not wheeze (19). Thus, identification of factors predisposing patients to bronchospasm in connection with inhaled allergens would be of great help in understanding the pathogenesis behind allergen-induced bronchospasm. Furthermore, proteins in plasma that are related to a predisposition to bronchospasm would be useful biomarkers for predicting the development of bronchospasm in allergen-sensitized persons. For this purpose, we adopted a proteomics approach. As an initial step, we screened the target proteins on a 2-D gel to differentiate IPR-positive from -negative asthmatics. At baseline, the expression levels of two complement fragments (spots 5 and 6) were higher in the IPR-positive than in the -negative group. In contrast, the expression levels of gamma fibrinogen (spots 1, 2, 3, 4) were lower in IPR-positive responders. Gamma fibrinogen (spot 4) and complement C3d (spot 6) were differentially expressed at baseline and after D.p. challenge. This suggests that these differences between IPR-positive and -negative responders after D.p. challenge were caused by differences at baseline, rather than by the D.p. challenge. Additionally, IPR-positive asthmatics, but not IPR-negative asthmatics, showed decreased C3 (spot 5) and increased C3d fragment (spot 6) and C4a fragment (spot 7) following D.p. antigen challenges. This indicates that alterations in the complement cascade may predispose patients to the appearance of immediate responses to D.p. allergen challenge.
The complement cascade is associated with triggers evoking the development of asthma. Segmental allergen challenge of allergic asthmatics elevates C3a and C5a in BAL levels (20). In addition to allergens, ozone (21), RSV (Respiratory syncytial virus) infection (22), smoking (23), and particles (24) can result in activation of the complement cascade. We previously reported that plasma levels of C3a and C4a were higher in aspirin-sensitive individuals, and these levels correlated with changes in FEV1 in aspirin-sensitive individuals after aspirin challenge (25). These data suggest that alterations in the complement cascade may not be limited to D.p. antigen challenge, but may be a general predisposing factor in the development of bronchospasm.
As an explanation for the mechanism behind C3 and C4 complement activation following the D.p. antigen provocation, proteases such as trypsin, thrombin, and elastase released from mast cells may cleave C5 and C3 in the airway wall via activation of IgE-mediated processes. Second, some macromolecular structures from dust mites may be recognized by the alternative pathway and fix the complement directly. Lastly, exogenous proteases derived from allergens, such as Der p3 and Der f3, directly cleave C3 and C5 into their active fragments (26). However, the process involved remains unknown at present.
Another interesting finding is that the gamma fibrinogen chain and its isoforms were significantly decreased in IPR-positive responders, compared to IPR-negative responders (Fig. 4). The differences in the gamma fibrinogen chain in plasma in the two groups were confirmed by Western blot analysis. The expression level of the gamma fibrinogen chain in IPR-positive asthmatics was less than in the -negative group, while fibrinogen alpha and beta chain levels were the same in both groups (Fig. 5).
Fibrinogen is the six-chain protein precursor to the main clot structural protein, fibrin. Fibrinogen is a dimer of three polypeptide chains termed α (or Aα), β (or Bβ), and γ (27). While all three chains are essential for the normal function of fibrinogen, due to their intertwined three-dimensional structure (28), the gamma chain contains a number of sites that interact with other fibrin(ogen) molecules, clotting factors, growth factors, and integrins (29). The gamma chain can bind to factor XIII zymogen and to thrombin. Fibrinogen interactions with integrins modulate the processes of platelet aggregation, inflammation, and wound healing. Interestingly, many of these processes involve the fibrinogen gamma chain (27). Fibrinogen gamma participates in inflammation by binding to leukocyte integrin αMβ2 (CD11b/CD18, Mac-1, CR3). This binding interaction mediates the recruitment of phagocytes during inflammation (30). In addition to adhesion molecules, fibrinogen contains binding sites for several different growth factors and cytokines, including vascular endothelial growth factor (31), fibroblast growth factor-2 (32), and interleukin-1β (33). Thus, these binding sites may serve as storage depots to concentrate growth factors and cytokines in inflammatory and repair processes. The γ chain arises from an alternative mRNA processing event (34), and differs only in its carboxyl terminus from the gamma A isoform. This isoform is often referred to as gamma A/γ fibrinogen, and constitutes approximately 10% of an individual’s fibrinogen, although the ratio can vary from 3% to 40% among individuals (35). The fibrinolysis rate of recombinant γ′/γ′ fibrinogen is about 10-fold slower than recombinant γA/γA fibrinogen, in the presence of factor XIIIa (36). This suggests that the function of fibrinogen may be dependent on the ratio of these isoforms. In the present study, spots 1, 2, 3, and 4 were identified as fibrinogen gamma. Spots 3 and 4 are likely isoforms of spots 1 and 2, respectively, because spots 3 and 4 had molecular weights of 48.1 kDa and spots 1 and 2 had molecular weights of 49.4 kDa (Fig. 2). Spots 2 and 4 are likely post-translational modifications of spots 1 and 3, respectively, because spots 2 and 4 had pIs of 5.56 and spots 1 and 3 had pIs of 5.44. All the spots were significantly decreased in the IPR-positive group compared to the IPR-negative group. The sum of these four isoforms in IPR-positive asthmatics was also lower than in the IPR-negative asthmatic controls (41.7%vs 100%, P < 0.005).
The role of platelet activation and fibrinolytic activity has been demonstrated in asthma. Activation of fibrinogen is the initial and essential step in platelet aggregation and fibrin clot formation (37). Platelets are activated during antigen-induced airway reactions in asthmatic subjects (38). Symptomatic asthmatics have increased concentrations of beta-thromboglobulin and platelet factor-4 in plasma and BAL fluid (39, 40). In experimental asthma models, platelet activation within the circulation is necessary for transmigration of eosinophils and lymphocytes into the airways (41). In our study, we did not evaluate changes in fibrinogen gamma in the LPR. However, intravascular platelet activation after bronchial allergen exposure occurs during the IPR independent of the pattern of asthmatic response, and the degree of platelet activation during the IPR may affect the development of prolonged inflammation during the LPR (42). It is unknown why IPR was associated with a decreased amount of fibrinogen gamma in our study. A possible mechanism is that the released mediators from mast cells may activate fibrinogen gamma in IPR positive subjects. Recent data suggest that mast cells and their products such as heparin and proteases are involved in the regulation of coagulation and fibrino(geno)lysis (43). IgE-mediated immediate hypersensitivity reactions have been known to result in local extravasation of fibrinogen and deposition of fibrin (44). IgE-mediated activation of mast cells may inactivate the fibrinogen gamma in IPR positive subjects subclinically before specific antigen challenge. In IPR-negative asthmatics, lung mast cells are not activated. As the results, relatively high concentration of plasma fibrinogen gamma blocks the tissue injury by blood clotting and inflammation by competing local fibrinogen. However, in IPR-positive patients, fibrinogen gamma does not protect because its lower levels in plasma. However, the exact mechanism is remained unsolved to date. In addition, our result of the specific antigen challenge should be cautiously extended to the natural exacerbation of allergic asthma on exposure to allergens.
In conclusion, our study is the first to show differences in protein expression in the plasma of IPR-positive and -negative asthma patients at baseline and after D.p. antigen challenge. As a result, levels of fibrinogen gamma and complement components may be determinants for the IPR following D.p. antigen inhalation, possibly providing plasma markers to predict the likelihood of IPR.