Volume 4 Issue 7 - May 30, 2008
Serine protease inhibitors, nafamostat mesilate (FUT) and gabexate mesilate (FOY), attenuate allergen-induced airway inflammation and eosinophilia in murine model of asthma
Jiu-Yao Wang1,*, MD2, DPhil2

1Department of Pediatrics, College of Medicine, National Cheng Kung University, Tainan, Taiwan
2Instructor, Department of Microbiology and Immunology, National Cheng Kung University
Email:a122@mail.ncku.edu.tw

J Allergy Clin Immunol 2006. 118;105-112.

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The allergic status in Taiwan
Allergic diseases are the most common disorders affecting children. Increasing prevalence of allergy of bronchial asthma, allergic rhinitis, and atopic eczema have been reported from many Western countries in the past decades. Studies from Asian countries show a lower prevalence of allergic disease as compared to Western countries. Taiwan has experienced a rapid economic development and urbanization during the past decades, with a concurrent increase in prevalence of allergic disease from 1975 to 1985 (Table). Between 1995 to 1997, a questionnaire study of allergic diseases in childhood (as part of ISSAC, the International Study of Asthma and Allergies in Childhood) was conducted for kindergarten and primary school children in whole island of Taiwan. The total number of children were 11, 400, the response rate was 93.3%. The result is shown in Table [1,2,3]. Although there was slight difference of reported symptoms of atopy in the different parts of Taiwan, but in the whole, the prevalence rate of allergic disease is rising as compared to the previous study at the same area. In our study, we found that bronchial asthma and allergic rhinitis occurred more frequently in boys than in girls, and no significant difference was found between boys and girls in atopic eczema. The cumulative prevalence of allergic rhinitis showed an increasing trend with age, while the prevalence of bronchial asthma and atopic eczema did not differ between the age groups. When the presence of allergic symptoms in a first degree relative was considered as a positive family history, there was statistically significant relationships between positive family history and atopic diseases. There was a strong relationship between parental history of allergic diseases and the prevalence of atopic diseases in children [2,3,4]

The prevalence of allergens responsible for these atopic diseases in Taiwan was studied by our survey of allergic children using skin prick test and in vitro specific IgE using CAP system (Pharmacia, Sweden). House dust mites, Dermatophagoide pteronyssinus, Der p; and Dermatophagoids farinae, Der f  which shown allergic positive rate, 91% and 88%, respectively, are the most common allergens triggering atopic diseases in the Taiwan area. Following are cockroach (32%) and dog dander (8.5%). The sensitivities to molds, such as Candida (22.6%), Aspergillus (16.5%) and Alternaria (23.2%) are not uncommon. The sensitivity to pollens has major difference in different geography area. In the country side, Ragweed (15.3%), Eucalyptus (7.3%) and Bermuda grass (7,5%) are the most frequently sensitive allergens to allergic children, while in urban area, the pollens sensitivity is rare. [5,6,7,8]

Exposure to indoor allergens have been considered as one important risk factor associated with the occurrence of asthma. However, studies examining concurrently the relationship between multi-allergens exposure and the related respiratory illness have not been complete, particular for the Asian countries. We have conducted a study to asses the effect of domestic allergens exposure on the respiratory health of asthmatic children in a subtropical environment. A representative group of 121 asthmatic children, confirmed clinically, was selected. The allergens measured were Der p I, Der pII, Fel d I, and Bla g II from the mattress dust analyzed by ELISA method. The respiratory outcomes included the self-recorded symptom diary and self-measured Peak expiratory Flow (PEF) using Peak Flow Meter. The results showed: The peak of Der p I concentrations appeared in samples from December to January with a value about 4611 ng/g dust, while the Der p II concentrations from the mattress dust stayed within a range over the study period. The same pattern could be applied to the distribution of Fel d 1 concentrations. The symptom scores calculated from the symptom diary showed a significant association with Der p I concentration in May and June (mean Temp. up to 28 oC). The amplitude of PEF was significantly associated with the concentrations of these allergens for these asthmatic children in a multivariate regression model. Therefore, the association between allergen concentrations and PEF variation appeared to vary with time of the year [9,10,11,12].               
Table: The change of prevalence of childhood atopic disease in different part of Taiwan. [1,2]

Study background
There is mounting evidence that proteases are involved in the pathogenesis of asthma.1 The serine protease tryptase, localized in mast cell granules, constitutes up to 25% of the total mast cell protein, 2 is increased in bronchoalveolar lavage (BAL) fluid of patients with asthma after allergen challenge 3. From in vitro studies, tryptase promotes human mast cell degranulation; 4 induces eosinophil and neutrophil migration; 5 amplifies the bronchoconstrictor effects of histamine on lung tissue; 6 and stimulates the growth of airway fibroblasts, smooth muscle cells, and epithelial cells in the process of airway remodeling. 7 Furthermore, many aeroallergens associated with asthma, such as house dust mite allergens and various fungal allergens, are proteases. 8-10 Ino et al. first characterized Der p 1 as a cysteine protease, 11 which can stimulate production of IL-6, IL-8, PGD2 in airway epithelial cells and activate eosinophils. 12 From an in vitro study, after a short phase of cytokine production, Der p 1, like tryptase, caused detachment of the epithelial cells. 13 Group 3 (Der p 3) and 9 (Der p 9) mite allergens are serine proteases which can increase vascular permeability and detach epithelial cells. 14 Although the receptors for these proteases have not yet been characterized, it is highly probable that the primary basis for this response is activation of PARs or similar molecules. 15,16 The stimulation was blocked by cysteine and serine protease inhibitors. 17

Recently, serine and tryptase inhibitors have been shown to reduce antigen-induced airway hyper-reactivity in allergic guinea pigs and sheep. 17,18 Gabexate mesylate (FOY), as well as nafamostat mesilate (6-amidino-2-naphthyl p-guanidinobenzoate dimethane sulfonate, FUT), are non-antigenic synthetic inhibitor of trypsin-like serine proteases, 19,20 which have been therapeutically used in the treatment of pancreatitis, disseminated intravascular coagulation (DIC), and as a regional anticoagulant for hemodialysis. 21,22 Human tryptase inhibition by FOY may account for the reported prevention of inflammation, erosion, ulceration of mucosa, and the symptom of allergic rhinitis. 23,24 FUT inhibited purified lung tryptase and eliminated pulmonary dysfunction induced in rats by a radiographic contrast medium. 25 A purified human urinary trypsin inhibitor (UTI, ulinastatin), also a serine protease inhibitor, has been shown to have various beneficial effects in treating patients with DIC, shock, and pancreatitis by inhibiting the activation of leukocytes. 26,27 It also can improve peripheral microcirculation and bronchospasm associated with systemic anaphylaxis in rabbits in vivo. 28 The precise modes of anti-inflammatory mechanisms of these synthetic and purified serum tryptase inhibitors are still unclear. The purpose of this study was to determine the therapeutic potential of protease inhibitors such as FUT, FOY and UTI on allergy airway inflammation and eosinophil infiltration in a mouse model of asthma.

Our Results and Discussion:
In this study, we evaluated the effect of serine protease inhibitors on the allergen-induced airway inflammation in a murine model of asthma. In the preventive treatment of protocol 1, we found that only FUT and FOY could inhibit eosinophils infiltration in the lung, thus suppressing allergen-specific IgE production. These findings were in agreement with the decreased inflammatory cytokines and Th2 to Th1 cytokine profiles changes in the FUT- and FOY-treated mice. But, only FUT-treated mice showed a significant change of AHR as compared to other protease inhibitors between treated and non-treated sensitized mice, suggesting that instead of anti-inflammatory action by serine protease inhibition, FUT may have other protective pharmacological effects on AHR. In protocol 2, the results of therapeutic effects of FUT and FOY on allergen-induced airway inflammation, such as attenuating pulmonary eosinophilia, inflammatory cytokine production in the BAL, and reducing AHR, were similar to protocol 1, except that there was no change of allergen-specific IgE. However, the ratio of anti-Der p IgG1/IgG2a antibody was markedly increased and the mast cell activation was significantly inhibited in the sensitized mice treated with FUT and FOY after allergen challenge (Protocol 2) as compared to the sensitized mice when FUT and FOY were administrated during sensitization period (protocol 1). We believed that FUT and FOY administrated in the sensitization period could reverse the allergic status of mice, thus decreasing allergen-induced airway inflammation. After allergen challenge, though allergic status persisted, FUT and FOY still had an inhibitory effect on non-IgE mediated inflammatory process, such as mast cell activation and inflammatory cytokines production in the allergic inflammation of the lung. The detail mechanisms exerted by serine protease inhibitors, FOY and FUT, in allergen-induced airway inflammation still need further investigation.

It is interesting to note that UTI (Unilastine), a urine trypsin inhibitor purified and concentrated from human source had no effect on our allergen sensitized murine model of asthma. Previously reports 27,28 showed that by inhibiting the activation of leukocytes via its elastase inhibitor effect, UTI might play certain roles in the protection of neutrophil-activating, endotoxins-induced sepsis. But this protective effect seems not prominent in the airway inflammation in asthmatic patients, who had significant higher serum UTI levels during asthmatic attack.31 Therefore, the administration of UTI in asthmatic subjects as well as in our murine model of asthma may not have beneficial effect for symptoms controls.

Serine protease inhibitors have been shown to have various beneficial effects in anti-inflammatory effects by inhibiting the activation of leukocytes, and fibrogenolytic activity of human tryptase generated during the coagulation cascade and the inflammatory process,19-24 but the mechanism is still not fully elucidated. Previously, it has been reported that FUT and FOY inhibited tryptase released from mast cells,19,20,25 which may partially account for their anti-asthma effect. Although we don’t have direct evidence to show the change of tryptase activity; however, the mast cell activation, measured by mMCP-1 serum level, was significantly decreased in the FUT and FOY treated mice when administrated during sensitization or after allergen challenged (repository figure 3). Moreover, the anti-inflammatory effect of FUT and FOY were not restricted in their inhibition of tryptase activity. Yuksel et al.23 found FOY was able to inhibit LPS-induced TNF-α production in human monocytes via inhibiting activation of both NF-B and AP-1 to target sites as well as through the activation of mitogen-activated protein (MAP) kinase pathways. FUT was able to suppress LPS-induced nitric oxide synthesis and apoptosis in cultured human trophoblasts,32 inducing IL-12, IL-18, and interferon-γ production, and the expression of intercellular adhesion molecules-1, B7.1, B7.2, CD40, and CD40 ligand in human peripheral blood mononuclear cells.33 In this study, we also found that alveolar macrophages in FUT and FOY treated mice, had decreased NO production and CD86 expression as compared to non-treated allergen sensitized mice. Also, the synthesis of inflammatory cytokines such as IL-1α, IL-6, TNF-α as well as eotaxin was decreased, while anti-inflammatory cytokines, such as IL-10 production were increased in AMs from FUT and FOY treated mice. These protective effects on allergen-induced airway inflammation may through FUT-initiaed intracellular signal transduction for promoting anti-inflammatory cytokines production and inhibit NF-kB activation as shown in repository figure 4. Further study on the immuno-pharmacological action of FUT and/or FOY should be carried out before the application of this new regiment of treating allergic asthma.

In conclusion, we found that serine protease inhibitors, FUT and FOY, could exert their therapeutic effect in allergen-induced airway inflammation not only due to the inhibitory action in the early phase of mast cells activation, but also the immunoregulatory function in the late inflammatory phase of decreasing pulmonary eosinophilia, and attenuating Th2 as well as other inflammatory cytokines production in asthma. Such properties of FUT and FOY might be useful in combination, or as an alternative treatment with present anti-asthma medications for patients suffered from asthma.
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