Clinical immunology
Plasma neutrophil elastase in children with recurrent aphthous stomatitis

Abbreviations: RAS – recurrent aphthous stomatitis, N – neutrophils, NE – neutrophil elastase, NE-α1PI – neutrophil elastase complexed with α1-proteinase inhibitor, ELISA – enzyme-linked immunosorbent assay
Introduction
Recurrent aphthous stomatitis (RAS) is a common and very painful ulcerative condition of the mouth. The disease has a chronic course with periodic exacerbations. Pathogenesis of RAS is not fully understood. According to some authors immunological and neutrophil abnormalities may be involved in the etiology and pathophysiology of RAS [1]. RAS may be a big clinical and therapeutic problem. It may be the only sign of the onset stage of many systemic diseases. Several indicators have been evaluated in the diagnosis of inflammatory stage, and include various leukocyte indices and acute- phase proteins. The examination of plasma levels of NE-α1PI can be used as a novel and sensitive indicator of the acute and chronic response and neutrophils activation in different diseases. The aim of the study was to determine the concentration of plasma NE-α1PI in children in the acute period of RAS.
Material and methods

Subjects
For this study the blood of 12 children with the acute stage of minor form RAS was obtained. The subject: 7 girls (58%) and 5 boys (42%) at the age of 1.5 to16 years (median: 12 years) were patients of the Clinic of Immunology, Medical University of Wroclaw. None of these patients had a history of any additional acute or chronic disorders. Medications for patients included only topical nonsteroidal anti-inflammatory drugs. Healthy children (n=15), aged from 1.5-12.5 years (median: 6 years), without chronic illness in the anamnesis were studied as a control group.
Assesment of elastase complexed with α1-proteinase inhibitor
Plasma NE-α1PI was measured by means of reagents manufactured by Merck, Germany. The assessment was carried out by ELISA method.
Statistical analysis
The obtained results were subjected to statistical analysis using the nonparametric Wald-Wolfowitz test. The level of statistical significance was assumed to be p<0.05.
Results
As shown in Figure 1, NE-α1PI was significantly increased (p<0.00001), in plasma of all RAS patients compared to control group. In 10 of ill children NE-α1PI concentration was above 150 µg/L, and in 5 cases above 200 µg/L (RAS: interquartile range (25-75%): 155.06-638.48 µg/L; control: interquartile range (25-75%); 38.08-68.18 µg/L).
Discussion
Many of studies indicate different causes of RAS e.g. haematologic and nutritional deficiencies, food allergies, immune disorders, stress. There is also evidence indicating for certain abnormalities of neutrophils in RAS concerning both their count and function. Neutrophils (N) not only provide a principal means in defense against bacterial or fungal infection, but also have been implicated in the pathogenesis of non-infectious inflammatory diseases. Neutrophilic inflammation is a characteristic feature e.g. in acute and chronic lung diseases, Sweet’s syndrome [2, 3]. The granules of N contain the strong proteolytic enzyme – elastase (NE). During degranulation NE is released into intracellulary space where together with oxygen radical and other enzymes, such as cathepsin G, myeloperoxidase, lactoferrin, inactivates foreign phagocytized substances. After excessive stimulation N release enzymes and oxidizing substances outside the cell. Free NE perpetuates the cycle of inflammation by promoting the generation of chemoattractants particularly interleukin-8 and leukotriene B4, which recruit more N into the tissue. In the extracellular environment free NE is rapidly bound and inactivated mainly by α1-proteinase inhibitor (α1-PI). Degranulation of large numbers of N causes the release of so large amounts of NE that it can’t be neutralized by local inhibitors. The extent of tissue injury depends on the balance between the enzymes and its inhibitor. NE can destroy e.g. elastin, collagen, immunoglobulins, clotting factors and proteinase inhibitors [2]. NE may degrade cadherins – components of endothelial basement membrane [4] and contribute to microvascular injury, which facilitates the migration of not only the cells taking part in the inflammation but also some harmful factors. The value of free NE and NE-α1PI may be useful as an indicator of local and systemic inflammation. The concentration of the NE-α1PI correlates with the released free elastase. Schroeder et al. [5] demonstrated in the biopsy specimens neutrophil infiltrates in the ulcers margins. The function of N performed by Sistig et al. [1] show that RAS patients had significantly lower spontaneous migration and ingestion values in the acute period. Also ingestion ability of salivary N in patients with acute RAS was reduced [6]. These disorders may contribute to the recurrence of the aphths. Another study demonstrated that in patients with minor RAS no significant differences in chemotaxis and spontaneous migration of N in blood were found [7]. On the other hand the increased reactive oxygen intermediates and increased expression of CD11b on resting and fMLP-stimulated N in RAS patient [8], and increased adherence of stimulated N were demonstrated [9]. The excessive activation of N and action of NE, as shown in the present study, may initiate immunopathological changes that lead to RAS.
Conclusions
Our data suggest that the stimulation status of N in inflamed tissue in RAS patients can be estimated by measuring the plasma concentration of NE-α1PI. These observations could have implications for development of new therapeutic strategies in the future.
References
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