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.