Expreesion Of Protease Activated Receptors And Its Effects On The Histamine Release From Human Basophils

Background and objective:Protease activated receptors (PARs) are members of G protein coupled receptor family. Till now, four PARs have been discovered in human and mouse, and they named PAR-1, PAR-2, PAR-3 and PAR-4. PARs are activated by many protease and they may play important roles in inflammation and allergy.Basophils are important immune cells, basophils possess high-affinity immunoglobulin (Ig) E receptors (FceRI) that are cross-linked upon engagement of receptor-bound IgE with corresponding antigens ("allergens"), result in cellular degranulation and inflammatory mediator secretion, including histamine, IL-4, IL-13, leukotrienes, prostaglandinE2, which will in turn activate other cells involved in inflammation and allergy.Although more and more research focus on roles of PARs in inflammation and allergy, there are no report about PARs on basophils. Do the 4 PARs express on basophil? How do PARs modulate the activation of basophil? We try to get the answers to the above questions through our research. Methods:The expression of PARs in cultured human basophil cell line KU812 was determined by RT-PCR and flowcjtometry techniques. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation. The PBMCs were then resuspended in complete Hank's balanced salt solution(HBSS) and challenged with thrombin, trypsin, PARs agonists and other stimulating agents. Histamine level in samples was determined by ELISA. Results:1.While PAR-1, PAR-2, PAR-3 and PAR-4 mRNA were detected in human basophil, only PAR-1,PAR-3 and PAR-4 proteins were found in the cell membrne and cell plasma.2. 0.1 U/L tlirombin could induce 4.6% net histamine release from basophils. 1.0U/L and 10 U/L thrombin induce 5% and 8.9% net histamine release respectively.(p<0.05) Thrombin could not activate basophils, while its activity inhibit by hirudin. 10 ug/mL trypsin could induce histamine release from basophils, but inhibiting by trypsin inhibitor, trypsin could not activate basophils.3. 10 umol/L and 100 umol/L PAR-1 agonist SFLLR-NH2 could respectively induce 7.4% and 7.8% net histamine release from basophils.(p<0.05) Reverse PAR-1 agonist, PAR-2, PAR-3 and PAR-4 agonists failed to activate basophils.4. anti-IgE, calcium ionophore (CI). F-Met-Leu-Phe (FMLP), C5a and substance P could induce histamine release. Heparin,C5 and adenosine did not induce any histamine release at concentrations tested, but heparin enhanced histamine release induced by C5a and substance P.Conclusion:1. Human basophils express mRNA of all 4 types of PARs, but only express PAR1, PAR3, and PAR4 protein.2.Thrombin and tyrpsin activates basophil through PAR-1 and may involved in pathological physiological process mediated by PARs.3. Heparin can greatly enhance the ability of C5a and substance P to stimulate histamine release, which may be a novel mechanism of amplification of basophil activation signal.