The Effect Of 4.1R Gene Deletion On IgE-mediated Mast Cell Activation

BackgroundMast cells are derived from haematopoietic progenitors in the bone marrow.Developing mast cells subsequently migrate to peripheral tissues,such as the skin,nerves,intestinal,airways and other organs,where they terminate their differentiation.Mast cell is via the receptors to identify relevant pathogens.In the body’s natural immune mast cell against pathogenic microorganism infection and plays an important function.Activated mast cells secrete preformed chemicalmediators,including histamine cytokines and chemokines,which are stored in cytoplasmic secretory granules.This process involved in type I allergy.Activated mast cells can also lead to inflammatory cell infiltration of the late inflammation,tissue reconstruction and fibrosis,therefore mast cells play an important role in allergy,inflammation and immune response.Activation of mast cells is regulated by a variety of molecules of the cells,FcεRI and KIT receptors play key roles in mast cell activation signal starting stage.Mast cells constitutively express FcεRI on their surface,which is the main membrane receptor for mast cell activation and regulation.When the body exposure to an allergen,Specific antibody IgE,producing by B cells,can bind to and induce FcεRI receptor aggregation,which could cause the body in a state of sensitization.Allergen what once again enter the body can be captured by IgE bound to the α form of FcεRI on the surface of mast cell,then mast cell was activated,leading to mast cell degranulation of histamine and production of inflamatory cytokines.These inflamatory molecules can further result in smooth muscle contraction and expansion of capillary,permeability increase,and cause allergic reactions.Although the mechanism of mast cell activation has be well demonstrated,The key regulation molecules of mast cell activation are still the focus of research.Given the role of mast cells in allergic reaction,it will be of great significance to find the new regulator ofmast cells activation and to explore mast cells-mediated allergic reaction.The 80 kDa membrane skeleton protein 4.1R was first found in mature erythrocytes.There are two forms of protein 4.1R in erythrocytes:135kDa subtypes（4.1R135）and 80 kDa subtypes（4.1R80）.They are expressed in different differentiation stage of erythrocytes.4.1R is the linker between the spectrin-actin network and erythrocytes transmembrane protein,which is important in maintaining the shape,adhesion brittleness and normal physiology function of erythrocytes.Having FERM structure domain proteins known as protein 4.1 superfamily.4.1superfamily is divided into five: protein 4.1 family,ERM protein family,talin related molecules,PTPH and NBL4 proteins.In recent years,the study found that there were4.1N,4.1G,4.1B widely expressed in nucleated cells.4.1N are mainly distributed in the nervous system.4.1B are mainly distributed in the brain tissue.4.1G are widely distributed.4.1R not only expressed in erythrocytes but also widely expressed in the bone marrow hematopoietic stem cells,T cells,B cells and mast cells and other immune cells.Our preliminary study demonstrated that protein 4.1R can inhibit T cell receptor signal transduction and play a role of negative regulation of T lymphocyte activation and proliferation.We also found that 4.1R gene knockout mice was more sensitive to allergens in mice encephalomyelitis（EAE）experimental model.More functions of 4.1R were exhibited in the different cells,however the function of 4.1 R in innate immune cells,such as mast cells and macrophages still remain unclear.ObjectivesThe established 4.1R gene knockout mice model was used to investigate the influence of 4.1R gene on IgE mediated mast cell activation in vitro and allergy disease in vivo.Our results confirmed that membrane skeleton protein 4.1R is one of the important mast cell regulatory factors.This study lay a good foundation for elucidating the molecular mechanism of the regulation effects of protein 4.1R on mast cell activation.To understand and enrich the mast cell activation and regulation theory is of great significance.Methods1.Bone marrow was taken from femur and tibia of 4.1R^+/+ and 4.1R-/-C57BL/6J mice.The bone marrow was cultured using RPMI1640 medium.The suspension cells was collected after 4 weeks in vitro.The suspension cells are BMMC.Light microscope was used to comparison 4.1R^+/+ and 4.1R-/-BMMC growth morphology.Flow cytometry was used to test purity of mast cells dyed with FITC labeled FcεRI antibody and PE labeled CD117 antibody.2.Total RNA was extracted from mature 4.1R^+/+ and 4.1-/-BMMC.cDNA was transcribed.RT-PCR reaction and genotype identification of agarose electrophoresis was performed to identify 4.1R gene of mast cells.The exon composition of 4.1R was identified by sequencing.3.The Absorption Photometry method was used to study the lack of 4.1R gene expression influence on the IgE mediated BMMC degranulation.4.qRT-PCR was used to study that the lack of 4.1R gene expression influence on the secretion of IL-6,IL-4,IL-13 and TNF-α in the mast cell at different time point.5.qRT-PCR was used to study the lack of 4.1R gene expression influence on the expression of FcεRI in the BMMC at transcriptional level.The flow cytometry method was used to study the lack of 4.1R gene expression influence on the expression of FcεRIα in the BMMC.6.The passive cutaneous anaphylaxis model of 4.1R^+/+ and 4.1R-/-C57BL/6J mice was established.The Absorption Photometry method was usd to compare the vascular permeability of ear between two kinds of mice.Embedding and sectioning the ear.Pathological observation and compare the swelling degree of ear between two genotypes mice caused by histamine.7.The late-phase passive cutaneous anaphylaxis model of 4.1R^+/+and4.1R-/-C57BL/6J mice was established.The study compare the thick and weight of ear between two kinds of mice.Embedding and sectioning the ear.Pathological observation and compare the degree of inflammatory cell infiltration of two genotypes mice caused by mast cell.Toluidine blue stain the slice,the microscope was used the to statistics the degranulation rate of mast cell in ear.qRT-PCR was usedto compare the expression of IL-6 at transcriptional level.Results1.Mature 4.1R^+/+and 4.1R-/-BMMC reached more than 85% in the purity.The mature 4.1R^+/+ and 4.1R-/-BMMC can be used in subsequent experiments.There was no significant difference in the biology morphology between two genotypes of BMMC.2.The agarose gel results showed that there were two bands in 4.1R^+/+BMMC and no band was fund in 4.1R-/-BMMC.Mapping of 4.1R gene exon in BMMC display that compared with 4.1R complete exons sequence of mice source.4.1R135 kD of 4.1R^+/+BMMC was deficient 4.1R exons 3,14,15 and 16.4.1R 80 kD of4.1R^+/+BMMC was deficient 4.1R exons 14,15 and 16.3.Absorption Photometry method results showed that 4.1R-/-BMMC degranulation degree is significantly higher than 4.1R^+/+BMMC after activation at RNA level.4.qRT-PCR results showed that the expression of IL-6,IL-4,IL-13 in4.1R-/-BMMC was significantly higher than 4.1R^+/+BMMC at 10 min after stimulation.5.When the stimulus for 10 min,1 hr,qRT-PCR results showed that the expression of FcεRIα,β,γ in 4.1R-/-BMMC was significantly higher than4.1R^+/+BMMC at RNA level.There was no difference in the surface expression of FcεRIα between two genotypes of BMMC.6.The results of passive cutaneous anaphylaxis display that the saturation of evans blue dye of 4.1R-/-C57BL/6J mice in ear was significantly higher than 4.1R^+/+.The results showed that after 4.1R gene deletion,the vascular permeability of ear caused by histamine is increase.Pathological results showed that ear swelling degree caused by histamine in 4.1R-/-C57BL/6J is significantly higher than 4.1R^+/+.7.The results of late-phase passive cutaneous anaphylaxis display that ear thick and weight in 4.1R-/-C57BL/6J is significantly higher than 4.1R^+/+ mice.The infiltrate degree of inflammatory cell caused by mast cell and the expression of IL-6 attranscription level in 4.1R-/-C57BL/6J is significantly higher than 4.1R^+/+mice at transcription level,but there was no significant difference in the mast cell degranulation rate of ear between 4.1R-/-mice and 4.1R^+/+mice.Conclusion4.1R inhibit IgE mediated mast cell degranulation and the secretion of cytokines,and play a role of negative regulation of allergic reactivity disease.