Study Of CD69 Expression On Murine Eosinophils And The Inhibitory Effect Of AntiCD69mAb On Airway Inflammation Of Asthma Model Of Mouse

BackgroundEosinophils are the most important effector cells in the mechnism of asthma. The infiltration and activation of eosinophils in airway are correlated with the severity of asthma exacerbation and progress of airway hyper-responsiveness (AHR). IL-5 deficient mice also confirm the innate relation between infiltration of eosinophils in airway, mucus production and AHR. The essence of asthma is the disorder of activation and apoptosis of eosinophils in airway. It is the key step of asthma treatment to induce apoptosis of activated eosinophils in airway. Right now, systemic application of glucocorticoid is still the first choice to treat acute exacerbation of asthma. Glucocorticoid could affect all the blood cells nonspecifically, which is the major problem to induce systemic side effect and limit the application of it. Especially in the asthma therapy of children and the younger, we have to think about the side effects that might influence growth and development of body. Besides, there are still small portion of patients who are steroid resistant. So it is our big challenge to find out a special drug, which can induce apoptosis of activated eosinophils specifically.The discovery of CD69, a surface antigen, brings hope to this need. CD69 was discovered in year 1981. It was named as AIM, EA-1, Leu-23 etc. at the beginning. CD69 once was considered as activation marker of T lymphocytes. It could be expressed on T lymphocytes at 1 hour after activation and lasts 72 hours. It also has roles on proliferation and differentiation of T lymphocytes. Later studies showed CD69 could express on almost all kinds of activated myeloid cells. CD69 usuallyexists in form of homodimer consisting of two polypeptides of 22.5kD. It always expresses on cell surface in the form of phosphorylation. This is important to its connect with protein on surface of other cells. CD69 makes important signal transducing function in cell biofunctions. It could mediate activation and synthesizing of cytokines after activation of cells. Moreover, CD69 also plays important role in the apoptosis of cells. Activated T lymphocytes with high expression of CD69 in vivo are susceptible to spontaneous apoptosis quickly in vitro. The viability of lymphocytes in peritoneal cavity and spleen in CD69^-/- mice increased and the apoptosis of these cells reduced obviously with the decrease of apoptosis signal Caspase-3.Most studies of CD69 focus on its expression on T lymphocytes and natural killer cells. The studies of its expression on eosinophils are rare and all of these are study of human eosinophils in vitro. These studies showed that human eosinophils from peripheral blood could express CD69 after activation under stimulation of different cytokines. And eosinophils from airways of asthmatic patients also could express CD69. But till now there is no research about the expression of CD69 on murine eosinophils after activation.The present study is divided into two parts. The first part is the measurement of CD69 expression on activated murine eosinophils. Using IL-5 transgenic mice with large amount of eosinophils in blood, we purified eosinophils and stimulated them with PMA plus MA and different cytokines respectively, then observed the expression of CD69 on cells. We also measured the expression of CD69 on eosinophils from peripheral blood and airway lumen of asthmatic mice. The second part is to observe the role of antiCD69mAb on airway AHR and inflammation of asthmatic mice after OVA sensitization and challenge. Then we compared the effect of antiCD69mAb with DXM applied before and after OVA challenge. This shred new light on treatment of asthma exerbation.ObjectiveUsing IL-5 transgenic mice with the character of large amount of eosinophils in peripheral blood, we purified peripheral blood eosinophils and observed the dynamic changes of CD69 expression on eosinophils under the stimulation of PMA plus MA, and the influence of different cytokines on CD69 expression. Thus we can test if CD69 could be an activation marker of murine eosinophils. At the same time, using mature asthmatic model of mouse, we observed the effect of antiCD69mAb on AHR and airway inflammation of asthmatic mice and compared with the effect of DXM. We discussed the mechnisem of their inhibitory role on airway inflammation of asthma through the assay of cytokines level of BALF.Methods1 .Peripheral blood of IL-5 transgenic mice was collected and eosinophils in it were purified by magnetic separation systems (MACs) via negative selection. Then expression of CD69 on eosinophils and cells viability was assayed after stimulation of PMA plus MA at 1h, 12h, 18h and 24h. Expression of CD69 on eosinophils and cells viability was also assayed after culture with IL-4, IL-5, IFN-γ, GM-CSF, IL-12 and IL-13 for 18 hours. At the same time, CD69 expression on eosinophils from peripheral blood or BALF of asthmatic mice was measured.3. Using mature asthma model of mice, the inhibitory effect of antiCD69mAb injected after OVA challenge on airway AHR and airway inflammation was observed and compared with the effect of DXM which was injected before or after OVA challenge. The mechanism of their inhibitory roles on airway inflammation of asthma was diccussed through the assay of IL-5, GM-CSF and CRP level of BALF.Results1. Expression of CD69 on eosinophils1.1 Dynamic changes of CD69 expression and cell viability of eosinophils stimulated with PMA plus MAEosinophils freshly purified didn't have expression of CD69. After activation byPMA+MA stimulation, eosinophils expressed CD69 immediately at 1h. The expression of CD69 went up to reach the peak at 12h and lasted at least 24 hours. Eosinophils in media had the similar change (p<0.05). At each time point, CD69 expression on eosinophils activated by PMA+MA were significantly higher than it on eosinophils in media (p<0.05). Eosinophils had spontaneous apoptosis in vitro, but PMA plus MA could accelarate it obviously.1.2 CD69 expression and cell viability of eosinophils in culture with different cytokinesEosinophils cultured with all of these cytokines could expressCD69. But only eosinophils cultured with IFN-γ, GM-CSF and IL-13 had significant increase of the expression of CD69 compared with control (p<0.05) . IL-5, GM-CSF could increased cell vaiblity significantly (p<0.001) . IL-4, IL-12 and IFN-γ didn't have roles on cell viability and CD69 expression.1.3 Expression of CD69 on eosinophils from peripheral blood or BALF of asthmatic miceEosinophils from BAFL of asthmatic mice could express CD69 strongly, but those from peripheral blood had no expression of CD69.2 The effect of antiCD69mAb on AHR and airway inflammation of asthmatic mice2.2 Air way responsiveness measurementThe responsiveness of asthmatic mice to inhaled Methacholine (Mch) was significantly higher than normal mice. Application of DXM before OVA challenge or antiCD69mAb after OVA challenge could inhibit AHR completely(p<0.05).. But application of DXM after OVA challenge could inhibit AHR partly.2.3 Eosinophils counts in BALF of miceThe amount of eosinophils in BALF of normal mice was almost zero. But OVA sensitization and challenge could induce the accumulation of eosinophils in airwaylumen (p<0.05). Application of DXM before OVA challenge or antiCD69mAb after OVA challenge could inhibit the accumulation of eosinophils completely (p<0.05). Application of DXM after OVA challenge had the same effect.2.4 Measurement eosinophils infiltration and mucus production in lung tissueAirway of normal mice did have eosinophils accumulation and mucus production at all. But OVA sensitization and challenge could induce the large amount of accumulation of eosinophils and mucus production in airway (p<0.05). Application of DXM before OVA challenge or antiCD69mAb after OVA challenge could inhibit the accumulation of eosinophils and mucus completely (p<0.05). Application of DXM after OVA challenge only partly inhibited the accumulation of eosinophils and mucus..2.4 Assay of GM-CSF, IL-5 and CRP in BALF of miceIL-5 in BALF of normal mice was uncer detect. IL-5 in BALF of asthmatic mice increased obviously (p<0.05). Pretreat with DXM before OVA challenge or application of antiCD69mAb after OVAchallenge could decrease the IL-5 level in BALF. Application of DXM after OVA challenge had no influence on IL-5 level. BALF of normal mice had baseline concentration of GM-CSF and CRP. And the level of GM-CSF and CRP didn't change with OVA challenge or drugs intervene.Conclusion1. CD69 could be an activation marker of murine eosinophils since it expresses only on activated eosinophils but still eosinophils.2. Expression of CD69 has close relation to apoptosis of cells.3. AntiCD69mAb could inhibit the airway physiology and pathology of asthma through inducing apoptosis of eosinophils in airway.4. Pretreat with DXM before OVA challenge could inhibit the physiology and pathology of asthma completely, but application with DXM after OVA challenge only inhibited the airway inflammation partly.