Tacrolimus could induce dermatitis mediated mainly by
lgE-independent MCs degranulation in vivo
WT mice were applied by different concentrations of tacrolimus and
vehicle for 1 day on dorsal skin to mimic tacrolimus clinical
application. Although no visible erythema and scaling difference were
observed between control group and tacrolimus treated groups (Figure
1A), skin inflammatory cells infiltration (Figure 1B) and degranulation
of MCs were more significant in treated groups (Figure 1C). Moreover,
scratching bouts increased dose-dependently in tacrolimus group (Figure
1D). Nevertheless, this rapid MCs activation wasn’t accompanied by
remarkable elevated IgE levels between control group and treated groups
(Figure 1D). However, the levels of tryptase, MCP-1 and TNF-α were
upregulated dose-dependently (Figure 1E). Hence, initial topical
tacrolimus application induced itching and inflammatory reaction without
IgE change.
MCs play an important role in inflammation and pruritus. In order to
further verify effect of tacrolimus on MCs, hind paw swelling and
extravasation experiment was used. Some researchers have found even 0.01
% (125 μM) tacrolimus could enhance neuropeptide (SP) homogenate
concentrations in healthy tissue21. Hence, descending
concentrations of tacrolimus solution were used in hind paw swelling and
extravasation experiment. We found an obvious swelling in left hind paw
injected with tacrolimus or C48/80 in WT mice, compared with right hind
paw treated with saline solution (Figure 1F). Besides, paw thickness
increase rate for tacrolimus injection was dose-dependent, and
tacrolimus increased vascular permeability in vivo , as assayed by
Evans Blue dye (EBD) extravasation, which was also dose-dependent
(Figure 1G). Here, we demonstrated that tacrolimus could activate MCsin vivo .
KitW-sh/W-sh mice, MCs deficient mice, were used to
explore the role of MCs in tacrolimus mediated dermatitis, which were
applied with tacrolimus on dorsal skin for one day. There were no
obvious erythema and scaling in KitW-sh/W-sh mice
group (Figure 2A). Also, skin inflammatory cells infiltration (Figure
2B), scratching behaviors, and the levels of tryptase, MCP-1 and TNF-α
in serum didn’t increase after one-day tacrolimus application in
KitW-sh/W-sh mice group compared with WT mice (Figure
2C). There was no significant different IgE level in serum between any
two groups (Figure 2C). Moreover, in hind paw swelling and extravasation
experiment, KitW-sh/W-sh mice showed no prominent
difference in paw swelling and EBD exudation between left (injected
tacrolimus) and right (injected saline solution) hind paws of
themselves, whereas they both showed decreased paw swelling and
extravasation when compared to WT mice (injected tacrolimus) (Figure
2D).
Collectively, topical tacrolimus application triggered dermatitis and
transient itching via non-IgE dependent MCs activation.
MRGPRB2/X2 participates
in tacrolimus induced MCs activation
Further, we set out to detect whether MrgprB2 participates in MCs
activation induced by tacrolimus as mentioned above. MrgprB2 deficient
mice (MUT mice) were employed here, in hind paw swelling and
extravasation experiment, MUT mice showed no significant difference in
paw swelling and EBD exudation between left (injected tacrolimus) and
right (injected saline solution) hind paws of themselves in hind paw
swelling and extravasation experiment, whereas they both exhibited
decreased paw swelling and extravasation when compared to WT mice
(injected tacrolimus) (Figure 3A). Therefore, we concluded that
tacrolimus could activate MrgprB2 leading to local inflammatory reaction
induced by MCs.
Besides, MUT mice were also applied with tacrolimus on dorsal skin to
mimic tacrolimus clinical use. After one day application of tacrolimus
on dorsal skin of WT mice and MUT mice, no visible erythema and scaling
were observed in all groups (Figure 3B). However, inflammatory cells
infiltration was more obvious in WT group compared with MUT mice (Figure
3C). With avidin staining, no obvious MCs degranulation could be
observed in MUT mice (Figure 3D). Significantly, MUT mice didn’t show
increased scratching behaviors, as well as serum tryptase, MCP-1 and
TNF-α after one-day tacrolimus application (Figure 3E). IgE level in
serum didn’t differ significantly between any two groups (Figure 3E).
Hence, MrgprB2 plays a vital role in initial dermatitis and MCs
stimulation triggered by topical tacrolimus.
Considering the rapid response of MrgprB2 to tacrolimus leading to MCs
degranulation in vivo , we set out to further explore whether
short-term tacrolimus exposure could activate MCs via MRGPRX2 in
vitro . We found calcium influx of LAD2 cells, the early phase of MCs
activation, could be altered by short-term tacrolimus priming (Figure
4A), C48/80 (a MRGPRX2 agonist) as a positive group (Figure 4B). Further
studies revealed that β hexosaminidase release increased (Figure 4C), as
well as tryptase, MCP-1 and IL-8 increased dose-dependently after
treated with tacrolimus in LAD2 cells for 30 minutes (Figure 4D).
Furthermore, we treated MrgprB2-HEK293 cells with tacrolimus, which
resulted in increased intracellular Ca2+ concentration
(Figure 4E), but tacrolimus did not alter the intracellular
Ca2+ concentration in normal control (NC)-HEK293 cells
(Figure 4F). Intriguingly, lower release of inflammatory mediators (β
hexosaminidase, tryptase, MCP-1 and IL-8) was observed obviously in
MRGPRX2-knockdown group compared to NC-LAD2 cells (Figure 4G, H). Thus,
tacrolimus mimics the behavior of C48/80 in LAD2 cells, supporting
action at the same receptor MRGPRX2.