The Study On The Correlation Between Helicobacter Pylori Infection And Chronic Spontaneous Urticaria Risk And Its Possible Mechanisms

Background: Chronic urticaria(CU) is a common dermatological disease manifested by recurrent wheals with itching for more than six weeks. Usually, its etiological factor is complicated as well as insidious. The majority of CU cases(up to 80%) without identified exogenous cause is defined as chronic spontaneous urticaria(CSU). Recently, many data have shown that CSU patients had a high helicobacter pylori(H. pylori) infection rate and a symptom remission following H. pylori eradication, which suggests H. pylori infection is closely related to CSU risk.It is generally known the activation of mast cells is a crucial event in the pathogenesis of CU, in which mast cells are mainly triggered by a cross-linking of Ig E and its high affinity receptors(FcεRI) on cell surface. The sera specific immunoglobulin E(Ig E) antibodies against H. pylori also have been detected in some individual CU cases. However, H. pylori infection primarily results in a T-helper type 1(Th1)-polarized response rather than a Th2-polarized response, and the specific anti- H. pylori Ig E might be at a low level and not be enough to stimulate mast cells effectively.Considering that there are many positive receptors on the membrane of mast cells, besides its high affinity FcεRI receptors, the low affinity Ig G receptor, circulating immune complexes receptors, complement receptors as well as toll like receptors(TLR) on the surface could also be bound specifically to activate itself. Some studies have pointed out, the CSU patients with H. pylori infection can be detected high levels of specific anti-H. pylori bacteria or anti-its lpp20 Ig A and Ig G antibodies in the sera, but the sera specific antibody levels against H. pylori bacteria or its protein components in CSU patients are still lack of a large sample epidemiological investigation, and the possible mechanism about the specific anti- H. pylori antibodies involved in the process of CSU needs to be further verified and clarified.Some studies have indicated H. pylori could not activate rat mast cells directly, but the activation effect of H. pylori on human mast cell has not been assessed yet. Although some other studies have demonstrated that a few of H. pylori proteins such as vacuolating cytotoxin A(Vac A) and neutrophil-activating protein(NAP) can activate mast cells directly, which probably suggest H. pylori could participate in the process of CSU through stimulating mast cells in a direct manner, there is still lack of systematic analysis between H. pylori infection and CSU attack.Objective:1. To analysis the correlation between the H. pylori infection and CSU attack;2. To detect the sera specific Ig E, Ig G, Ig A antibodies against H. pylori and Lpp20 protein respectively in CSU group and controls, and further explore the possible correlation and the underlying mechanism involved in the pathogenesis of CSU;3. To study whether there is a direct activation effect of H. pylori bacteria preparations or its protein components on human LAD2 mast cell line in vitro;4. To screen out the most dominant protein components with mast cell activation effect from whole H. pylori bacteria, evaluate its specific ingredients and specific activation effects.Materials and Methods:1. To confirm the correlation between H. pylori infection and CSU risk, the standard ELISA kit was used to determine the sera anti-H. pylori Ig G antibody positive rate in CSU patients group and normal control group;2. To detect the sera specific Ig E, Ig G, Ig A antibodies against Lpp20 protein and H. pylori respectively in CSU group and controls, recombinant Lpp20 protein which was successfully expressed, purified as well as identified, and prepared urea-lysed H. pylori were pre-coated on microplate respectively. Then all of sera samples were tested by ELISA assay;3. To evaluate the direct activation effect of H. pylori preparations on human LAD2 mast cells, three kinds of bacteria preparations were prepared(urea-lysed H. pylori, soluble H. pylori sonicate and formalin-killed H. pylori) and incubated with LAD2 mast cells in vitro respectively, and then mast cell activation in each group was assessed by β-hexosaminidase release assay and toluidine blue staining;4. To detect different mast cell activation effects of various H. pylori protein components, molecular sieve was used to divide urea-lysed H. pylori into 30 kinds of mixed protein components(PCs) with different molecular weight ranges, and then their LAD2 mast cell activation effects were evaluated with β-hexosaminidase release rate in order to screen out the most dominant mixed protein component;5. To investigate the specific activation effects of the most dominant protein component PC17 on LAD2 mast cells, its does-dependent effects on β-hexosaminidase, histamine, TNF-α release, and its time-dependent effects on IL-3, IL-4, IL-17 A, IFN-γ, LTB4 release after co-cultivation were evaluated by ELISA assay;6. To define the specific protein ingredients of PC17, the band of PC17 in SDS-PAGE gel were incised and analyzed by using liquid chromatography-mass spectrometry(LC-MS/MS).Results:1. The positive rate of anti-H. pylori Ig G was significantly higher in CSU patients(73.46%, 155/211) than that in normal controls(51.82%, 71/137)(χ2 = 17.08, P < 0.001);2. The sera specific anti-H. pylori Ig E, Ig G, Ig A antibody levels in CSU patients with H. pylori infection(Ig E 0.082±0.002, Ig G 1.053±0.022, Ig A 0.660±0.022) were not higher than that in normal controls with H. pylori infection(Ig E 0.078±0.003, Ig G 1.222±0.034, Ig A 0.849±0.037);3. The sera specific anti-lpp20 Ig E, Ig G, Ig A antibody levels in CSU patients with H. pylori infection(Ig E 0.085±0.003, Ig G 1.459±0.012, Ig A 1.229±0.020) were not higher than that in normal controls with H. pylori infection(Ig E 0.082±0.005, Ig G 1.494±0.016, Ig A 1.251±0.033);4. β-Hexosaminidase release assay showed urea-lysed H. pylori, soluble H. pylori sonicate and formalin-killed H. pylori can effectively activate LAD2 cells respectively in vitro(The β-Hexosaminidase release rate was compared to that in negative treatment group, P < 0.05), while the activation effects improved as their concentrations increasing;Toluidine blue staining showed the number of degranulated mast cell increased with the concentration of H. pylori preparations, and the change of degranulation rate in each treatment group was roughly consistent with that of their β-hexosaminidase release rate;5. Urea-lysed H. pylori divide into 30 kinds of mixed protein components(PCs) with different molecular weight ranges by molecular sieve chromatography, and their mast cell activation effects evaluated by β-hexosaminidase release rate showed, PC3, PC9, PC10, PC17, PC27 and PC28 had superior activation abilities than other mixed protein components( versus negative treatment group, P < 0.05, β-hexosaminidase release rate > 15%).Meanwhile, PC3, PC9, PC10, PC17, PC27 and PC28 all had a positive dose-response effect on LAD2 cells and PC17 showed a superior activation effect compared to the other five components;6. PC17 and urea-lysed H. pylori all can stimulate LAD2 cellsto release β-hexosaminidase, histamine as well as TNF-α,(versus negative treatment group,P < 0.05) and the release effects increased with stimulation concentration. The three inflammatory mediators release effects induced by 4μg/ml of PC17 were roughly equivalent to those induced by 150 μg/ml of urea-lysed H. pylori(P > 0.05);7. PC17 and H. pylori all can stimulate LAD2 cells to release IL-3, IFN-γ, LTB4,(versus negative treatment group,P < 0.05) and the release levels of the three inflammatory mediators increased with time. Moreover, PC17 showed more prominent stimulation effects on IL-3 and IFN-γ release than urea-lysed H. pylori(P < 0.05), and showed comparable stimulation effect on LTB4 release to urea-lysed H. pylori. The IL-4 and IL-17 A release effects detected at each time point were all at a low level and showed no statistical difference(versus negative control, P > 0.05);8. Twenty-three kinds of proteins were confirmed in PC17 by using liquid chromatography-mass spectrometry(LC-MS/MS), and their molecular weight ranged from 21 to 35 k Da.Conclusions:1. There was an intimate relationship between H. pylori infection and CSU risk, which suggested H. pylori could play a role in the pathogenesis of CSU;2.In the pathogenesis of CSU associated with H. pylori infection, sera specific antibodies against bacteria induced by humoral immunity were not an important factor to trigger and/or exacerbate urticaria;3. Three kinds of H. pylori bacteria preparations, urea-lysed H. pylori, soluble H. pylori sonicate and formalin-killed H. pylori, could activate human LAD2 mast cells directly in vitro, which suggested H. pylori might participate in the pathogenesis of CSU through direct effect rather than antibody-mediated pathway in human mast cell activation;4. PC17 was the most dominant protein component in whole H. pylori bacteria with the mast cell activation ability, and can efficiently activate human LAD2 mast cells to secrete high levels of histamine, TNF-α, IL-3, IFN-γ and LTB4;5. PC17 contained 23 kinds of protein ingredients and their molecular weight ranged from 21 to 35 k Da;6. PC17 should be regarded as the most promising pathogenic factor contributing to the CSU associated with H. pylori infection.