Kinetics of mast cell migration during transplantation tolerance
Title Kinetics of mast cell migration during transplantation tolerance
Authors Gregor Bond1,2, Anna Nowocin1, Steven H. Sacks1 and Wilson Wong1,3
1MRC
Centre for Transplantation, King’s College London School of Medicine at
Guy’s, King’s and St Thomas’ Hospitals, London, UK
2Current
address Medical University Vienna, Währinger Gürtel 18-20, 1090 Vienna,
Austria
3Corresponding
Guy’s
Hospital,
author MRC Centre for Transplantation, 5th Floor, Tower Wing,
Great
Maze
Pond,
London
SE1
9RT,
U.K.
Phone:
+44(0)2071881522; Fax:+44(0)2071885660; e-mail: wilson.wong@kcl.ac.uk
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Kinetics of mast cell migration during transplantation tolerance
List of abbreviations
Complement component 3
C3
Complement component 3a
C3a
Complement component 3a receptor
C3aR
Mast cell
MC
Wild type
WT
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Kinetics of mast cell migration during transplantation tolerance
Abstract
Background After inflammatory stimulus, mast cells (MC) migrate to secondary
lymphoid organs contributing to adaptive immune response. There is growing
evidence that MC also contribute to transplant tolerance, but little is known about MC
kinetics in the setting of transplant tolerance and rejection. Likewise it has been
demonstrated that complement split products, which are known to act as
chemoattractants for MC, are necessary for transplant tolerance.
Methods Naive skin and lymph nodes, skin grafts and draining lymph nodes from
wild type and complement deficient mice treated with a tolerogenic protocol were
analyzed.
Results Early after tolerance induction MC leave the graft and migrate to the
draining lymph nodes. After this initial efflux, MC reappear in tolerant skin grafts in
numbers exceeding that of naive skin. MC density in draining lymph nodes obtained
from tolerant mice also increased post transplant. There was no difference in MC
density, migration and degranulation status between wild type and complement
deficient mice implicating that chemotaxis is not disturbed in complement deficient
mice.
Conclusion This study gives detailed insight in kinetics of MC migration during
transplant tolerance induction and rejection providing further evidence for a role of
MC in transplant tolerance.
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Kinetics of mast cell migration during transplantation tolerance
1. Introduction
Recent studies have shown that mast cells (MC) play an important role in innate and
adaptive inflammatory responses [1]. Beside their well described role in allograft
rejection [2] MC are also necessary in the establishment of peripheral tolerance after
skin and solid organ transplantation [3-6]. The functional need for MC during the
initiation phase of tolerance was demonstrated in a murine skin graft model [7] and a
heterotopic heart transplant model [8]. Recently it has been proposed that MC act via
direct interaction with T-regulatory cells [7, 9-12], but the mechanisms of MC
mediated transplant tolerance are not understood.
Secondary lymphoid organs are essential for initiating the immune response to
microbial antigens. After inflammatory stimulus MC migrate to the draining lymph
nodes where they interact with B and T lymphocytes [13-16]. In transplantation,
secondary lymphoid organs play an important role in rejection response and
tolerance induction [17]. However migration of MC between donor graft and
secondary lymphoid organs received little attention. This study investigates the
kinetics of MC migration into and out of the donor organ and draining lymph nodes
during tolerance induction and rejection.
It has been shown that the complement system is important for the induction and
maintenance of tolerance [18-20]. This effect is mediated via complement split
product iC3b binding on complement receptor 3 on antigen presenting cells. Recent
publications demonstrated complement component 3 (C3) and complement
component 3a receptor (C3aR) to be crucial for HY specific transplant tolerance
induction [21, 22]. However the underlining mechanisms for this complement
dependant tolerance are not known. The complement system is able to activate MC
via C3 split products [23] and complement component 3a (C3a) is a strong
chemoatractant for MC [24]. We hypothesized that one of the mechanisms that
account for complement dependant transplant tolerance is migration of MC from the
graft to the secondary lymphoid organs via C3 split product induced chemotaxis. To
test this hypothesis we analyzed MC migration in a murine model of complement
dependant tolerance.
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Kinetics of mast cell migration during transplantation tolerance
2. Materials and Methods
2.2. Mice
Animals were kept in specific pathogen free animal facilities and were used between
the age of 8 and 12 weeks in accordance with the Animals (Scientific Procedures)
Act 1986. C57BL/6 mice were purchased from Harlan Limited (UK) and Charles
River (MA, USA). Homozygous C3-/- and C3aR-/- C57BL/6 mice were kind gifts from
Drs M Carroll and Bao Lu, respectively (Harvard Medical School).
2.3. Skin transplantation and definition of rejection
Skin transplantation was performed as previously described [25], except full
thickness trunk skin, was used instead of tail skin. Rejection was defined by >90%
macroscopic necrosis of the skin. Mice were checked daily.
2.4. Donor lymphocyte infusion protocol
For donor lymphocyte preparation male spleens were harvested, mashed through a
40µm cell strainer and washed in phosphate buffered saline (Oxoid, UK). Red blood
cells were removed using an ammonium chloride-based lysing reagent (BD Pharm
Lyse; BD Pharmingen, USA) according to the manufacturer’s instructions. After
washing cells in phosphate buffered saline thirty five million cells were injected into
recipients via the tail vein immediately before skin transplantation.
2.5. Histology
Frozen tissue samples were cut into 8m sections. MC granule stain was performed
with Toluidine-blue. Slides were fixed in acetone/methanol (Sigma, MO, US) 1:1
solution for 5 minutes and stained for 5 minutes with Toluidine-blue staining solution
(0.5% w/v Toluidine-blue in 0.5 N hydrogen chloride acid; Sigma). Slides were
analyzed by a Diplan microscope (Leitz, Germany) and a DXM1200DF digital
camera (Nikon, Japan), using Lucia G software (Nikon). Positive cells were counted
in at least 20 random high power fields (x400) of each sample by an observer
blinded to the experimental conditions.
2.6. Immunohistochemistry
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Kinetics of mast cell migration during transplantation tolerance
For immunohistochemistry standard methods were used [26]. Slides were incubated
with a rat IgG2A anti-mouse stem cell factor R/c-kit monoclonal antibody (clone
180627; R&D Systems, UK) and visualized with a biotinylated goat anti-rat polyclonal
antibody (Pharmingen, CA, US) followed by a and streptavidin-horse radish
peroxidase conjugate (Pharmingen) and Vector NovaRed Substrate kit (Vector
Laboratories) used according to the manufacturer’s protocol. Sections were analyzed
as described above.
2.7. Statistical analysis
Kaplan Meier analysis was applied to calculate graft survival. Numbers of MC are
displayed as median per mm2. Mann Whitney U Test was used to compare density
of MC between groups. All tests were two-sided, with a 5% type I error. Statistical
calculations were performed using SPSS for Windows, version 17.0 (SPSS Inc.,
USA).
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Kinetics of mast cell migration during transplantation tolerance
3. Results
3.1. MC in tolerant skin grafts
First we analyzed MC density in naive trunk skin from male C57BL/6 wild type (WT)
mice. As shown in figures 1and 2a, MC granule stain with Toluidine-blue revealed an
average of 41 MC per mm2. For analysis of MC density in tolerant skin grafts male
WT trunk skin was transplanted to female WT recipients in combination with a donor
lymphocytes infusion. This protocol leads to specific tolerance induction towards the
male HY antigen and thus to indefinite graft survival whereas without donor
lymphocyte infusion skin grafts are chronically rejected [27, 28]. At day 7 after
transplantation 10 MC per mm2 were detectable in the skin grafts harvested from
tolerant mice, which was significantly less compared to naive skin (p=0.001). Similar
results were obtained at day 7 in skin grafts from rejecting controls (18 MC/mm2,
p=0.08), whereas syngeneic skin graft controls had MC numbers comparable to
naive skin (37 MC/mm2, p=0.4). These data demonstrate that MC density within
minor mismatched skin grafts is reduced compared to naive skin, irrespective of the
graft outcome.
In order to characterize the kinetics of MC in donor specific tolerance in more detail,
we analyzed donor skin grafts from mice treated with donor lymphocyte infusion over
time (figures 3, 2b). From day 14 post transplantation MC density in tolerant skin
grafts began to increase, exceeding numbers obtained from naive skin at day 50 (87
MC/mm2, p=0.02) and at day 100 (73 MC/mm2, p=0.04). In contrast there was no
significant difference in MC density between syngeneic skin graft controls at day 100
and naive skin (65 MC/mm2, p=0.1). Taken together these results demonstrate that
after an initial reduction in MC density in tolerant grafts, cell numbers rise over time
exceeding those detected in naive skin.
It has been shown that MC escape granule stain after complete degranulation [29,
30]. Therefore we performed a MC surface stain with c-kit to analyze the total MC
density independently of the degranulation status (figures 1, 2a). Within naive skin
56 MC per mm2 were counted which was not significantly different compared to the
Toluidine-blue granule stain (p=0.2). Similar to Toluidine-blue stains, skin grafts
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Kinetics of mast cell migration during transplantation tolerance
harvested from tolerant mice stained with c-kit showed markedly reduced MC density
at day 7 after transplantation compared to naive skin (11 MC per mm2, p=0.003).
Surface stain in rejecting and syngeneic controls showed also similar results
compared to those obtained from Toluidine-blue stain and there was no significant
difference in MC density between tolerant skin grafts and rejecting controls (p=0.9).
This suggests that the observed reduction in MC density early after transplantation is
not due to MC degranulation and that there is no difference in the amount of MC
degranulation between tolerant and rejecting grafts.
3.2. MC in draining lymph nodes of skin grafts
It has been shown that MC traffic from the local site of inflammation to secondary
lymphoid organs [13-16]. Therefore, we investigated if the observed reduction in MC
density in the skin grafts early after transplantation was the result of MC migration to
the draining lymph nodes. We found an average of 10 MC per mm2 detected by
Toluidine-blue stain in draining lymph nodes from tolerant mice at day 7 after skin
graft transplantation, which is significantly more compared to numbers in lymph
nodes harvested from naive mice (1.4 MC/mm2, p=0.008; figures 4, 2c). There was
no difference compared to lymph nodes harvested from rejecting controls (8
MC/mm2, p=0.9), but there were less MC in the draining lymph nodes harvested from
syngeneic controls (3 MC/mm2, p=0.03; figures 5, 2d). A longitudinal analysis
revealed that from day 14 after transplantation MC numbers within draining lymph
nodes from tolerant mice further rose with a maximum at day 100 post
transplantation (80 MC/mm2; figure 5). The expansion of MC in draining lymph nodes
from tolerant mice was predominantly in the sub capsular region, suggesting an
influx of MC via the afferent lymphatics (figure 5). In contrast MC numbers in draining
lymph nodes from syngeneic controls did not increase in the same way over time (19
MC/mm2; figure 5).
3.3. MC in C3-/- and C3aR-/- skin grafts
Having established that MC migrate from skin grafts into draining lymph nodes early
after transplantation, and re-accumulate in tolerant skin grafts with time, we
investigated whether factors known to influence MC migration would affect this
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Kinetics of mast cell migration during transplantation tolerance
process. Therefore we analyzed complement and complement receptor deficient
mice. As shown in figures 6 and 2e Toluidine-blue granule stain of naive skin from
C3-/- mice revealed an average of 35 MC/mm2 which is not significantly different
compared to naive skin from WT mice (p=0.24). However there was only half the
amount of MC detectable in naive skin from C3aR-/- mice compared to WT animals
(21 MC/mm2, p=0.004). Next we analyzed skin grafts obtained from complement and
complement receptor deficient recipients treated with the same tolerance induction
protocol used in WT animals. Female C3-/- and C3aR-/- mice received a male C3-/and C3aR-/- skin graft in combination with a C3-/- and C3aR-/- donor lymphocyte
infusion respectively. This model of complement dependent tolerance leads to
chronic rejection of the graft [27, 28]. At day 7 after transplantation 9 MC per mm2
could be detected in C3-/- male skin grafts, which is less compared to naive C3-/- skin
(p=0.002). However there was no significant difference compared to MC density
described within grafts of similar treated tolerant WT mice at day 7 after
transplantation (p=0.9). Likewise skin grafts from C3aR-/- mice showed reduced MC
density at day 7 after transplantation in comparison to naive C3aR-/- skin (4 MC/mm2,
p=0.004), which is not significantly different compared to tolerant WT skin grafts at
day 7 after transplantation (p=0.1).
MC surface stain with c-kit revealed no significant difference in MC density between
naive skin from C3-/- mice and WT mice (p=0.5; figure 6). However there were fewer
MC within naive skin from C3aR-/- mice compared to WT mice (p=0.016). Skin grafts
from C3-/- and C3aR-/- mice showed a significantly lower MC density compared to
naive skin from C3-/- and C3aR-/- mice respectively (8 MC/mm2, vs. 51 MC/mm2,
p=0.029 and 6 MC/mm2 vs. 27 MC/mm2, p=0.016 respectively). Compared to skin
grafts from tolerant WT mice, skin grafts from C3-/- mice showed no significant
difference in MC numbers (p=0.2). In contrast there were significantly more MC in
skin grafts from tolerant WT animals compared to skin grafts from C3aR-/- mice
(p=0.029). Of note this difference in absolute MC numbers did not result in a
difference in relative MC numbers, taken into account the MC density within naive
skin from C3aR-/- and WT mice. A relative reduction of 81% MC was calculated for
C3aR-/- and 79% for WT mice respectively. These data suggest that similar to WT
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Kinetics of mast cell migration during transplantation tolerance
animals there is a MC efflux from skin grafts in complement deficient and
complement receptor deficient mice early after transplantation.
3.4. MC in draining lymph nodes of C3-/- and C3aR-/- skin grafts
To determine whether the observed MC efflux is due to migration to local lymph
nodes we harvested draining lymph nodes from C3-/- and C3aR-/- mice at day 7 after
transplantation (figure 4). There were more MC detectable in draining lymph nodes
from skin grafts compared to lymph nodes harvested from naive animals (9 MC/mm2
vs. 1.8 MC/mm2 for C3-/- mice, p=0.004 and 12 MC/mm2 vs. 1.4 MC/mm2 in C3aR-/mice, p=0.005 respectively). However there was no significant difference compared
to draining lymph nodes harvested from tolerant WT mice (p=0.4 for C3-/- and p=0.9
for C3aR-/- mice respectively). Taken together these data suggest that after antigen
stimulus MC migration to draining lymph nodes is not impaired in the absence of
chemotactic complement split products.
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Kinetics of mast cell migration during transplantation tolerance
4. Discussion
MC have been described as mediators of allograft rejection [2], but recent data
provide evidence for a function in the induction and maintenance of transplant
tolerance [6]. It is also known that after antigen stimulus MC migrate to secondary
lymphoid organs contributing to the adaptive immune response [14]. The object of
the present work was to analyze MC kinetics during transplant tolerance induction. In
addition we analyzed the contribution of complement split products to MC migration.
Our data suggest that early after tolerance induction, MC leave the graft and migrate
to the draining lymph nodes. After this initial efflux MC reenter tolerant grafts
exceeding numbers in naive skin. MC density in draining lymph nodes of tolerant
skin increases in a similar way during the whole post transplant period. Analyzing
known chemotactic factors regulating MC traffic our data showed that migration is
not defective in complement deficient mice. Taken together our data provide new
insight in MC migration during transplant tolerance induction and rejection and
further evidence for a role of MC in allograft tolerance.
We observed a reduction in MC density within skin grafts early after transplantation,
which was described earlier by Noelle and colleagues [7]. In contrast to their
observations we did not notice a difference between MC density in tolerant and
rejecting grafts. In our model reduction of MC took place irrespective of the graft
outcome. This reduction was restricted to the presence of HY antigen, as syngeneic
controls showed no reduced MC density. Similar to rejection, HY specific tolerance
induction requires an active interaction of antigen presenting cells with T-cells [31].
During rejection the presentation of donor antigen by recipient antigen presenting
cells leads to T-cell activation, whereas the presentation of the HY antigen to T-cells
by donor antigen presenting cells leads to tolerance induction. It has been shown in
models of inflammation that MC migrate from the primary site, to the draining lymph
nodes [13-16] where they participate in induction of a primary immune response
directly via T lymphocyte recruitment [32], differentiation [33], stimulation [34] and
indirectly via dendritic cell maturation [35], antigen uptake and presentation [36] and
chemotaxis [37]. Furthermore it is described that MC have antigen presenting
function themselves [38]. Therefore we postulate that in our model the reduction in
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Kinetics of mast cell migration during transplantation tolerance
MC density is due to MC migration to draining lymph nodes. Their interaction with T
and B lymphocytes and antigen presenting cells within the secondary lymphoid
organs contributes to tolerance induction and rejection in our transplant model.
There are other possible mechanisms than migration to draining lymph nodes that
might explain the differences in MC numbers between naive skin and skin grafts
observed in our study. First, after complete degranulation MC might escape granule
stain [30]. MC surface stain produced similar results, which makes this explanation
unlikely. Secondly MC might have been eliminated by direct cytotoxic elimination
through the host. We cannot exclude that donor MC have been partly eliminated by
this mechanism in our model.
Analyzing kinetics of MC migration over time we observed that MC density
normalizes in tolerant grafts from day 14, even exceeding numbers obtained from
naive skin from day 50. Interestingly such an increase could not be observed in a
similar way in syngeneic controls. These data suggest a possible role for MC in the
maintenance of transplant tolerance. The described increase in MC density could be
explained by recruitment of progenitors from the circulation, local proliferation of
resident MC or migration of MC from adjacent tissues and secondary organs. Other
groups have also described a normalization of MC numbers over time, but increased
numbers of MC in tolerant tissue compared to naive tissue have not been reported
yet [7, 14]. This discrepancy might be explained due to the longer observation time in
our experiments. It has been shown that MC play an essential role in wound healing
and accumulate at the site of injury [39]. Since we did not detect an increased
density of MC in syngeneic grafts over time in the same way and MC density was
still rising after complete wound healing it is unlikely that repair mechanisms account
for the observed increase of MC density. Longitudinal observations are limited by the
fact that chronic rejecting controls and complement deficient animals could not be
analyzed as grafts are already rejected from day 7.
We detected an increased number of MC in draining lymph nodes from skin grafts
after transplantation in comparison to naive lymph nodes, which has also been
demonstrated in models of inflammation and allergy [13-16]. However such an
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Kinetics of mast cell migration during transplantation tolerance
increase in MC density in draining lymph nodes has not been described in transplant
studies [7]. Interestingly no increase was detected in draining lymph nodes from
syngeneic skin grafts. The parallel increase in MC density in draining lymph nodes
and decrease of MC density in tolerant and rejecting skin grafts, as well as the
predominant increase in the subcapsular region within the draining lymph nodes
strongly argue for a MC migration from the skin grafts to the draining lymph nodes
early after antigen contact. However we cannot exclude a contribution of local MC
proliferation or influx of immature MC progenitiors. Further increase in MC density in
draining lymph nodes was observed in longitudinal analysis of tolerant mice. In
contrast MC numbers in draining lymph nodes from syngeneic controls did not
increase in the same way over time. This observation demonstrates that MC
accumulate in draining lymph nodes during maintenance of transplant tolerance.
Data obtained from C3 and C3aR deficient mice suggest that MC migration to
secondary lymphoid organs is not impaired in the absence of complement split
products. The versatility of the MC lies in its ability to be to receptive to a variety of
chemotactic stimuli. It is known that not only complement split products but also
various chemokines, which are secreted during the inflammatory response, such as
CCL5, CXCL-10, IL-8, IL-3, and TNF [40] are chemotactic for MC. In this regard it is
likely that the lack of anaphylatoxins during the antigen stimulation can be
substituted by other chemoatractants.
In summary this study provides novel insight in MC kinetics during transplant
tolerance induction and rejection. We provide evidence that early after antigen
contact MC leave the graft, migrate to the draining lymph nodes and re enter tolerant
grafts thereafter. This migration is not defective in complement deficient mice.
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Kinetics of mast cell migration during transplantation tolerance
Acknowledgements
GB was the recipient of the Austrian Science Fund (FWF) Erwin Schrödinger
Fellowship, project number J2975. The authors would like to thank Kathryn Brown
for critical review of the manuscript.
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Kinetics of mast cell migration during transplantation tolerance
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Kinetics of mast cell migration during transplantation tolerance
Figure legends
Figure 1. MC granule and surface stain of naive skin and skin grafts from WT
mice. Female mice received male trunk skin after infusion of 35x106 male
splenocytes. Rejecting and syngeneic controls did not receive donor splenocytes
infusions. Skin grafts were harvested at day 7 after transplantation and analyzed by
means of Toluidine-blue granule stain (open circle) and c-kit surface stain (open
triangle). (A) Numbers of MC calculated within skin grafts obtained from donor
lymphocyte induced tolerant mice did not differ from those obtained from rejecting
controls. In contrast MC density within skin grafts obtained from syngeneic controls
was significant higher compared to tolerant grafts and did not differ from numbers
obtained from naive skin. There was no significant difference between MC granule
and surface stain. Dot plots represent groups of three to nine animals.
Figure 2
(a) Representative examples of naive skin (I, III) and tolerant skin graft (II, IV)
samples. I, II: MC granules are stained dark purple (bold black arrow) and cell nuclei
and keratin are stained light blue with Toluidine-blue granule stain. III, IV: MC (open
arrow) and keratin are stained red-brown with c-kit surface stain.
(b) Representative examples of naive skin (V) and long term follow up tolerant (I-IV)
and syngeneic skin graft (VI) samples from WT mice. MC granules are stained dark
purple (bold black arrow) and cell nuclei and keratin are stained light blue with
Toluidine-blue stain.
(c) Representative examples of naive lymph nodes (II, IV, VI) and draining lymph
nodes (I, III, V) from skin grafts obtained from WT (I, II), C3-/- (III, IV) and C3aR-/- (V,
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Kinetics of mast cell migration during transplantation tolerance
VI) mice. MC granules are stained dark purple (bold black arrow) and cell nuclei and
keratin are stained light blue with Toluidine-blue stain.
(d) Representative examples of long term follow up tolerant (I-IV) and syngeneic skin
grafts (V, VI) from WT mice. MC granules are stained dark purple (bold black arrow)
and cell nuclei and keratin are stained light blue with Toluidine-blue stain.
(e) Representative examples of naive skin (I, III, V) and skin grafts (II, IV, VI) from
WT (I, II), C3-/- (III, IV) and C3aR-/- (V, VI) mice. MC granules are stained dark purple
(bold black arrow) and cell nuclei and keratin are stained light blue.
(f) Representative examples of naive skin (I, II) and tolerant skin grafts (III, IV) from
C3-/- (II, IV) and C3aR-/- (I, III) mice. MC (bold arrow) and keratin are stained redbrown with c-kit surface stain.
Figure 3. MC granule stain of naive skin and long term follow up skin grafts
from WT mice. Female mice received male trunk skin after infusion of 35x106 male
splenocytes. Syngeneic controls did not receive donor splenocytes infusions. Skin
grafts were harvested at day 7, 14, 50 and 100 after transplantation and analyzed by
means of Toluidine-blue granule stain. (A) After an initial reduction early after
transplantation, MC density within tolerant skin grafts (open circle) rises from day 14,
exceeding numbers obtained from naive skin (open triangle) from day 50. In contrast
syngeneic controls (open diamond) did not show a similar increase in MC density
and did not have a significant difference in MC numbers compared to naive skin at
day 100 after transplantation. Dot plots represent groups of three to eight animals.
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Kinetics of mast cell migration during transplantation tolerance
Figure 4. MC granule stain of lymph nodes from naive skin and draining lymph
nodes from skin grafts from WT and complement deficient mice. Female mice
received male trunk skin after infusion of 35x106 male splenocytes. Draining lymph
nodes were harvested at day 7 after transplantation and analyzed by means of
Toluidine-blue granule stain. (A) More MC were detected in draining lymph nodes
from skin grafts compared to naive lymph nodes obtained from WT(open circle) and
complement deficient animals (C3-/- open triangle and C3aR-/- open diamond) . No
significant difference between draining lymph nodes from skin grafts obtained from
WT and complement deficient mice was detected. Dot plots represent groups of five
to nine animals.
Figure 5. MC granule stain of draining lymph nodes from long term follow up
WT skin graft recipients. Female mice received male trunk skin after infusion of
35x106 male splenocytes. Syngeneic and rejecting controls did not receive donor
splenocytes infusions. Draining lymph nodes were harvested at day 7, 14, 50 and
100 after transplantation and analyzed by means of Toluidine-blue granule stain. (A)
There was no difference between MC density in draining lymph nodes obtained from
tolerant mice (open circle) compared to lymph nodes obtained from chronic rejecting
controls (open square) at day 7 after transplantation. However there were less MC in
the draining lymph nodes obtained from syngeneic controls (open triangle) compared
to lymph nodes obtained from tolerant mice at day 7 after transplantation. MC
density within draining lymph nodes obtained from tolerant animals rose from day 14,
whereas MC density in lymph nodes obtained from syngeneic controls did not
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Kinetics of mast cell migration during transplantation tolerance
change in a similar way. The expansion of MC in tolerant mice was predominantly in
the sub capsular region of the draining lymph nodes. Dot plots represent groups of
three to five animals.
Figure 6. MC stain of naive skin and skin grafts from WT and complement
deficient mice. Female mice received male trunk skin after infusion of 35x106 male
splenocytes. Skin grafts were harvested at day 7 after transplantation and analyzed
by means of Toluidine-blue granule stain. (A) Naive skin obtained from WT (open
circle) and complement receptor deficient mice had a significantly higher MC density
compared to skin grafts obtained from corresponding strains. Whereas naive skin
obtained from C3-/- mice (open diamond) had a similar MC density, naive skin
obtained from C3aR-/- mice (open triangle) had significantly less MC per mm2
compared to WT naive skin. Dot plots represent groups of five to eight animals. (B)
Naive skin obtained from WT (open circle) and complement receptor deficient mice
had a significantly higher MC density compared to skin grafts obtained from
corresponding strains. Whereas naive skin obtained from C3-/- mice (open square)
had a similar MC density, naive skin obtained from C3aR-/- mice (open triangle) had
significantly less MC per mm2 compared to WT mice. Dot plots represent groups of
four to nine animals.
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