4 | DISCUSSION
In this study, we examined the IgE-related surface markers on peripheral blood basophils and the responsiveness of basophils stimulated with FcεRI by comparing AD patients and HCs. We particularly focused on the relationships between clinical/laboratory factors such as disease severity and serum IgE in AD patients and these parameters. Regarding surface markers, there was no significant difference in surface-bound IgE between AD patients and HCs (Figure 4 B). Obviously, total serum IgE was very high in AD patients (Table S1). However, a negative correlation between total serum IgE and surface-bound IgE in AD patients was observed (Figure 5 D). Severe AD patients tended to have a high level of total serum IgE and a low level of surface-bound IgE (Figures 1 C, 5 A). Our findings also demonstrated that AD patients had higher baseline CD203c and CD63 than HCs (Figure 2 A, C). These findings may indicate that AD basophils were spontaneously activated to release histamine and inflammatory mediators, possibly including IL-4, without FcεRI stimulation. Because basophils stimulate B cells to synthesize IgE in an IL-4-dependent manner,37this basophil-derived IL-4 can contribute to the increase in serum IgE in AD patients. However, the increased serum IgE did not bind well to basophils for some reason. In contrast, Yanase et al .28 reported that a high concentration of IgE caused histamine release, polarization, and CD203 upregulation in human basophils without stimulation in vitro . Although they did not examine whether the high concentration of IgE actually bound to FcεRI, they concluded that a high concentration of IgE modified the function of basophils. Although a high concentration of total serum IgE may have not bound to FcεRI on basophils in the present study, increased serum IgE may have adjusted basophil activation indirectly, leading to the formation of a vicious circle between high serum IgE and basophils.
On the contrary, expression of FcεRI was higher in AD patients than in HCs (Figure 4 A). FcεRI on basophils was reported to be controlled by total serum IgE.20,21 However, our study revealed that elevated serum IgE had no correlation with baseline CD203c, baseline CD63, and FcεRI on basophils, respectively (Figure S2 D, S3 D, S5 D). Another interesting observation was that IgE poorly bound to AD basophils despite elevated FcεRI expression. Previous reports documented that human basophils expressed IL-4 receptors38 and survival of mice basophils were enhanced by IL-4.39Also, FcεRI expression on mast cells was upregulated by IL-4.18 Because secretion of IL-4 is increased in typical AD patients, FcεRI on basophils may have the possibility of being controlled by IL-4.40 Moreover, because AD patients had higher baseline CD203c and CD63 than HCs in our study, activated basophil-derived IL-4 may contribute to the high expression of FcεRI on AD basophils in an autocrine fashion. It is possible that the increased FcεRI expression and activated steady status on AD basophils observed in this study was increased by AD-related cytokines such as IL-4.
This study revealed that basophils in AD patients exhibited low responsiveness, including CD203c and CD63, to anti-IgE stimulation (Figure 2 B, D). There were moderate negative correlations between CD63 responsiveness with anti-IgE stimulation and EASI score or TARC, suggesting that the responsiveness of basophils to anti-IgE antibody stimulation decreased as AD became more severe (Figure 3 A, B). It is possible that binding of IgE on basophils affected the responsiveness of basophils to anti-IgE antibody stimulation. Therefore, we examined the binding status of IgE on basophils in the comparison between AD patients and HCs, and the correlation between surface-bound IgE and AD severity. However, surface-bound IgE was equivalent between AD patients and HCs, indicating that the low responsiveness to anti-IgE stimulation when comparing AD patients and HCs cannot be explained by surface IgE binding status alone. AD basophils were activated spontaneously with no stimulation (Figure 2 A, C). Because upregulation of CD63 reflects histamine release,41 it is possible that AD basophils may already be mildly exhausted in the steady state. Since the basophils in AD patients had already been activated and exhausted without stimulation, we assumed that it was more difficult to activate these cells by anti-IgE stimulation compared with those in HCs even if the binding sites for anti-IgE antibodies were equivalent. In contrast, there were negative correlations between EASI score and CD63 response ratio (Figure 3 A) and between EASI score and surface-bound IgE (Figure 5 A). These findings suggested that the reduced surface-bound IgE expression on basophils observed in severe AD patients can explain the decreased responsiveness for anti-IgE stimulation in AD patients in general.
Finally, we used an anti-FcεRI antibody as another stimulus to examine the responsiveness of basophils. The CD203c response ratio of anti-FcεRI/baseline was lower than that of anti-IgE/baseline in HCs, consistent with a previous report.33 This means that the anti-IgE antibody under our experimental conditions could increase the HC basophil responsiveness more efficiently than the anti-FcεRI antibody. The anti-FcεRI antibody binds to the stalk region of FcεRI and does not inhibit IgE binding.32 We consider that this result was caused by the different binding sites of the two antibodies. The expression of FcεRI on basophils was higher in AD patients than in HCs (Figure 4 A). Based solely on this expression level, the responsiveness of basophils to anti-FcεRI in AD patients is presumed to be higher than that in HCs. However, AD basophils exhibited equivalent responsiveness to anti-FcεRI to HC basophils (Figure 6 A, B). This relatively weak responsiveness to anti-FcεRI in AD basophils may also be associated with the exhaustion of basophils in the steady state, similar to the phenomenon of the low responsiveness to anti-IgE stimulation in AD patients.
There are some limitations to our small-scale study according to the number of participants and sex adjustment. We could not examine the association between basophils and cytokines including IL-4, and histamine released from basophils. Furthermore, the study focused on circulating basophils and did not examine basophils in AD skin lesions.
In conclusion, we addressed the following hypothesis: type 2 inflammation in AD stimulates B cells and B cells secrete high concentration of IgE.40,42 However, the elevated IgE in AD did not bind efficiently to circulating basophils. AD basophils were spontaneously activated and exhibited low responsiveness against anti-IgE stimulation. Some AD basophils, especially severe AD basophils, behaved like low responders for anti-IgE stimulation, but not for anti-FcεRI stimulation. Further studies are required to determine the physiological meaning for this distinctive basophil status in AD.
Funding: This work was supported in part by Grants-in-Aid for Scientific Research (C) and Young Scientists (B) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (JSPS KAKENHI Grant Numbers 20K08651 and 19K17772 to A.F. and K.W.).
Conflicts of interest: The authors declare that they have no relevant conflicts of interest in relation to this work.