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-Irradiation in B6 and B6/lpr Mice1
Departments of
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Medicine and
Microbiology/Immunology, University of North Carolina, Chapel Hill, NC 27599
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiation
expressed Fas protein. Fas was up-regulated on irradiated B6 cells and
could be detected on B6/lpr cells undergoing apoptosis
following in vitro culture. Detection of Fas on live lpr
cells was demonstrable when apoptosis was blocked by zinc. In a short
term chimera system, Fas was shown to play a role, in vivo, in the
disposition of radiation-injured cells from both normal and
lpr mice. The addition of anti-Fas Ab to in vitro
cultures resulted in an increase in apoptosis in both B6 and
B6/lpr cells. Detection of intact Fas message and low
levels of Fas protein in lpr mice has led to the
consideration of lpr as a leaky mutation. This study
demonstrates that lpr mice can produce functional Fas
protein. This system is also appropriate for identifying the in vivo
role of Fas/FasL in apoptosis following other cell
manipulations. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A recent report from our laboratory has shown that Fas/FasL has a role
in radiation-induced cell death (8). Cells from lpr mice
were relatively resistant to in vitro apoptosis after exposure to in
vivo or in vitro -irradiation. Because irradiated normal cells
showed a marked increase in Fas expression, and because much of the
radiation-induced death of normal cells was blocked in the presence of
a Fas-IgG fusion protein, which competed with cell-bound Fas for FasL
binding, it was concluded that Fas/FasL interactions played an
important role in the death of radiation-injured cells. These studies
raised the question of whether such Fas/FasL interactions were of
significance in vivo and whether FasL was provided mainly by the
injured cells themselves or by normal bystander spleen cells.
Because gld mice lack functional FasL, it was possible to test the role of this molecule in the elimination of injured cells by transferring allotype-disparate irradiated cells into gld mice and comparing their fate with that of similar cells infused into normal mice capable of expressing FasL. Our results indicate that host FasL is an important factor but is not entirely responsible for the elimination of irradiated transferred cells. Unexpectedly, the clearance of lpr cells was also accelerated by the presence of FasL in hosts. The unanticipated finding that lpr cells are capable of expressing significant amounts of Fas after injury may help explain this finding and offer insights into the pathology of these mice and the normal regulation of Fas expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following mice were maintained in our breeding facility: C57BL/6-Thy1a/Thy1a (B6-Thy1.1; Ighb), C57BL/6-lpr/lpr-Thy1a/Thy1a (B6/lpr-Thy1.1; Ighb), C57BL/6-Igha/Igha (B6.C20; Igha, Thy1.2), and C57BL/6-gld/gld-Igha/Igha (B6.C20/gld; Igha, Thy1.2). The B6, B6-Thy1.1, B6/lpr, and B6/gld strains were originally obtained from The Jackson Laboratory (Bar Harbor, ME), and the B6.C20 strain was obtained from Dr. Gayle Bosma (Institute for Cancer Research, Philadelphia, PA). The B6/lpr-Thy1.1 strain was developed in our laboratory as previously described (9). The B6.C20/gld strain was developed in our laboratory by intercrossing B6.C20 with B6/gld and selecting for homozygosity for gld and Igha. B6-Thy1.1 and B6/lpr-Thy1.1 mice used as donors for cell transfer were 6–8 mo old, and B6.C20 and B6.C20/gld recipients were 2–3 mo old. Analysis of Fas expression was done with 8-mo-old B6, B6-Thy1.1 B6/lpr, and B6/lpr-Thy1.1 mice. Cells cultured in the presence and absence of anti-Fas Ab, as well as the two-color Annexin V vs anti-Fas staining, were from 6-mo-old B6-Thy1.1 and B6/lpr-Thy1.1 mice.
In vitro culture of splenocytes for analysis of Fas expression and anti-Fas treatment
Spleens were removed from B6-Thy1.1 and
B6/lpr-Thy1.1 mice; two spleens were pooled for each
strain. Single-cell suspensions were made by rupturing the splenic
capsule between the frosted ends of two glass slides and washing with
PBS (University of North Carolina Cancer Center Tissue Culture
Facility, Chapel Hill, NC). Erythrocytes were lysed with ammonium
chloride for 5 min at 4°C and washed twice. Irradiated cells received
150 rad of -radiation in a Gamma-cell 40 137Cs apparatus
(Atomic Energy of Canada, Ottawa, Ontario, Canada). Both irradiated and
nonirradiated cells were washed once and resuspended in complete medium
(RPMI 1640 with 10% FCS, 15 mM HEPES, 2 mM L-glutamine, 1
mM sodium pyruvate and nonessential amino acids, 2 x
10-5 M 2-ME, 100 U/ml penicillin, and 100 µg/ml
streptomycin). Cells were cultured for 18 h at 37°C in a 5%
CO2/95% air humidified atmosphere, in 24-well tissue
culture plates, with 4 x 106 cells per well (8). Zinc
acetate was added to the cultures indicated, at a final concentration
of 1 mM, to inhibit apoptosis (10). The anti-Fas Ab treatment was
done with 1 µg/ml of Jo2 (PharMingen, San Diego, CA).
In vivo transfer and recovery of donor splenocytes
Spleens were removed from B6-Thy1.1 and B6/lpr-Thy1.1
mice; 5–12 spleens were pooled for each strain. Single-cell
suspensions were made and erythrocytes lysed as described above.
Irradiated cells received 150 rad of -radiation. Cells were washed
once and resuspended in PBS at 3 x 108 cells per ml.
Cells were kept on ice throughout their preparation. Donor cells
(3 x 107) were transferred to recipient B6.C20 and
B6.C20/gld mice by tail vein injection.
Dexamethasone treatment of splenocytes from B6-Thy1.1 mice was done by culturing for 24 h at 37°C in a 5% CO2/95% humidified air atmosphere in 24-well tissue culture plates with 4 x 106 cells per well in complete medium with 10-6 M dexamethasone. Following culture, cells were washed three times in PBS. Untreated cells were kept at 4°C. Dexamethasone-treated and untreated cells were resuspended and transferred as above.
Twenty hours after injection, spleens were harvested from recipient mice. Single-cell suspensions were made as above, but each spleen was prepared and analyzed individually. Following erythrocyte lysis, splenocytes were washed and resuspended in HBSS supplemented with calcium, magnesium, 15 mM HEPES, 0.1% NaN3, and 3% FCS (HBSS complete). Total splenocyte recovery was determined using a model D2N Coulter Counter (Hialeah, FL).
Immunofluorescence staining
All cells were incubated with an anti-Fc receptor Ab
(2.4G2, mouse IgG2b; American Type Culture Collection) to block
nonspecific staining. Fluorescein-conjugated anti-Fas (Jo2,
hamster IgG), anti-2,4,6-trinitrophenyl (TNP) (UC8-4B3,
hamster IgG), anti-Thy1.2 (53-2.1, rat IgG2a)
anti-IgMb (AF6-78, mouse IgG1), Annexin V,
phycoerythrin-conjugated anti-B220 (RA3-6B2, rat IgG2a),
anti-Thy1.1 (Ox-7, mouse IgG1), and anti-Fas (Jo2, hamster IgG)
were obtained from PharMingen). Abs were added to 1 x
106 cells in 96-well microtiter plates and incubated for 20
min at 4°C. Cells were washed once in HBSS complete, and then twice
in PBS with 0.1% NaN3, and were fixed in an equal volume
of PBS and 2% paraformaldehyde in PBS. Fixed cells were analyzed with
a FACScan (Becton Dickinson, Mountain View, CA) using Cytomation data
acquisition and software (Fort Collins, CO) with size gating on the
lymphocyte population. Forward angle vs 90° light scatter was plotted
on a linear scale, while red and green fluorescence data were plotted
on a four-decade logarithmic scale. Twenty thousand events were
collected per sample in all experiments (8). For the Annexin V vs
anti-Fas staining, cells were first stained with the anti-Fas
Ab, as described above, and then resuspended in binding buffer (PBS
with 0.01 M HEPES/NaOH, pH 7.4; 0.14 mM NaCl; and 2.5 mM
CaCl2). Cells were then stained with Annexin V for 15 min
at room temperature and immediately analyzed by FACScan.
Analysis of apoptotic cells
Cells undergoing apoptosis were distinguished from live and necrotic cells by their characteristic light scatter profile. Cell death by necrosis resulted in a large decrease in both forward angle light scatter and side scatter, while cells undergoing apoptosis showed a less marked decrease in forward angle light scatter together with an increase in side scatter (8, 11, 12). Cells stained with Annexin V were analyzed by gating on all but the necrotic cells, based on their forward angle light scatter vs side scatter profiles. Cells within this gate that were Annexin V positive were deemed apoptotic.
Statistical analysis
Analysis of variance techniques were used to analyze the data
statistically as a completely randomized model (13). Data were tested
for homogeneity of variance and normality before analysis (14).
Statistical tests were considered significant if p 
0.05 and marginally significant if p 0.10.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been shown previously that a smaller fraction of spleen
cells from B6/lpr mice undergoes apoptosis following
-irradiation, compared with normal B6 mice (8). To better understand
the relationship between the fate of individual cells and their levels
of Fas expression, two-parameter immunofluorescence staining was
undertaken of spleen cells subjected to irradiation. Figure 1
shows immunofluorescence analysis of
irradiated and control splenic lymphocytes from B6-Thy1.1 mice analyzed
after short term in vitro culture using forward vs side angle light
scatter changes to distinguish between viable and apoptotic cells.
Under these conditions, there was substantial Fas expression both on
live cells and on those undergoing apoptosis, either spontaneous or
radiation induced. Following -irradiation, as noted previously, Fas
expression increased further on live cells from 52 to 86% (8). There
was an increase from 68 to 79% on those cells undergoing apoptosis.
The proportion of lymphocytes with apoptotic light scatter profiles
increased from 30% without -irradiation to 58% with
-irradiation.
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shows Fas expression on
B6/lpr cells analyzed in parallel to those shown in Figure 1
. As anticipated, live lpr splenic lymphocytes did not
express Fas, even after -irradiation. Remarkably, Fas was expressed
on many lpr cells undergoing spontaneous apoptosis, and the
percentage positive for Fas increased from 52 to 65% following
-irradiation. Staining with the isotype-matched control anti-TNP
Ab was negative on all cells. The expression of Fas on
B6/lpr cells undergoing apparent apoptosis has been observed
consistently and has also been seen on apoptotic cells from
MRL/lpr mice (data not shown).
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Fas expression on live B6/lpr cells treated with zinc
The presence of Fas on B6/lpr cells falling within the
apoptotic gate did not establish a causal relationship between Fas
expression and apoptosis, nor was it certain that the observed
fluorescent staining was occurring on the cell surface. If the
apoptotic signal for some of the lpr cells was mediated
through Fas, then Fas expression must occur before apoptosis and should
be detectable on live lpr cells, if only for a short time.
To determine whether Fas could be detected on live B6/lpr
cells, zinc acetate was added to cell cultures following irradiation,
to inhibit apoptosis (15). In this way, Fas-expressing lpr
cells might be identified before the completion of apoptosis. Figure 3 shows immunofluorescence analysis of
Fas expression on B6/lpr cells falling within the live or
apoptotic gates, following irradiation and culture in the presence and
absence of zinc acetate. As shown in Figure 2 and again in Figure 3A, Fas was expressed only on the cells falling within the
apoptotic gate. In Figure 3B, in which cells were cultured
with zinc acetate, a fraction of the live cells expressed Fas,
coincident with marked inhibition of apoptosis, as judged by light
scatter. Results from several experiments done with zinc acetate
are compiled in Table I.
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The next experiments were designed to ask whether the increased
Fas expressed on irradiated cells rendered them more susceptible to in
vivo death and subsequent elimination. Short term chimeras were
generated to compare the recovery of B6-Thy1.1 and
B6/lpr-Thy1.1 donor cells from B6.C20 and
B6.C20/gld recipients. Donor B6-Thy1.1 and
B6/lpr-Thy1.1 (Ighb) splenocytes were injected
into B6.C20 and B6.C20/gld (Igha, Thy 1.2)
recipients. By staining with anti-allotype Abs, donor T and B cells
could be detected in recipient mice by flow cytometry (Fig. 4). Each panel represents an individual
mouse, shown as a representative of a group of three. Donor T and B
cells are shown in the shaded quadrants. As anticipated, a small
fraction of the injected cells "homed" to the spleen. Most of the
injected cells were probably eliminated promptly, before homing, via
nonimmune mechanisms.
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An aliquot of each type of donor cell was set aside for flow
cytometry analysis, before injection, and kept at 4°C to evaluate the
initial level of apoptosis. Figure 5
shows that the proportions of live and apoptotic cells, as determined
by light scatter characteristics, were identical in all four groups.
The data presented here are from a representative of three experiments.
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In chimeras generated with nonirradiated donor cells, cell recovery was independent of donor and recipient strains
To evaluate the role of Fas/FasL in the clearance of cells from B6
and B6/lpr mice, we compared the recovery of donor B6-Thy1.1
and B6/lpr-Thy1.1 cells from B6.C20 and
B6.C20/gld recipient mice. Figure 6A shows the recovery of donor
B and T cells from short term chimeras made with untreated cells. No
significant difference was seen in the comparison of the four groups,
for either B cells or T cells (p = 0.76 and
p = 0.31, respectively).
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Comparison of the recovery of irradiated B6-Thy1.1 donor cells
between recipient B6.C20 and B6.C20/gld mice addresses the
question of the role of FasL in the removal of cells damaged by
-irradiation. Figure 6B shows the recovery of donor B and
T cells from short term chimeras made by injecting -irradiated
cells. There was a significant increase in the recovery of B cells in
B6.C20/gld recipients compared with B6.C20 mice
(p = 0.001). The same comparison for T cells
was marginally significant (p = 0.09).
Comparison of the recovery of irradiated B6/lpr-Thy1.1 donor
cells between recipient B6.C20 and B6.C20/gld mice addresses
the question of whether the Fas expressed on lpr cells
undergoing apoptosis has a role in the removal of cells damaged by
-irradiation. There was a significant increase in the recovery of
infused lpr B cells in B6.C20/gld recipients
compared with B6.C20 mice (p = 0.03). The same
comparison for T cells was marginally significant
(p = 0.08). The observed differences in
recovery of B and T cells in chimeras made with irradiated cells were
dependent upon whether the recipient strain had a normal FasL molecule.
There was no significant difference for B cell recovery when B6.C20
mice were the recipients of B6-Thy1.1 or B6/lpr-Thy1.1 cells
(p = 0.86). Similarly, B cell recovery was the
same when B6.C20/gld received cells from B6-Thy1.1 or
B6/lpr-Thy1.1 (p = 0.26). For T
cells, too, recovery was the same from recipient B6.C20 mice given
B6-Thy1.1 or B6/lpr-Thy1.1 cells (p
= 0.87). Finally, T cell recovery was the same when
B6.C20/gld received cells from B6-Thy1.1 or
B6/lpr-Thy1.1 (p = 0.88).
B6.C20/gld mice did not have a generalized defect in the clearance of apoptotic cells
Given the observed differences seen in the recovery of apoptotic
cells from B6.C20 and B6.C20/gld mice, we wanted to
determine whether this difference was specific to Fas/FasL-mediated
apoptosis. Apoptosis was induced in splenocytes from B6-Thy1.1 mice by
culturing them overnight in the presence of dexamethasone, which
induces apoptosis by a Fas-independent mechanism (11). The untreated
cells were kept at 4°C to prevent the spontaneous apoptosis that we
observed under normal culture conditions (11). Short term chimeras were
generated by injecting untreated and dexamethasone-treated cells into
B6.C20 and B6.C20/gld mice. Figure 6C shows the
absolute numbers of donor B cells and T cells recovered from the
spleens of recipient mice. There was no significant difference between
the recovery of untreated B or T cells from B6.C20 and
B6.C20/gld mice (p = 0.75 and
p = 0.23, respectively). The recovery of
dexamethasone-treated B and T cells was significantly lower than that
of the untreated cells (p = 0.0001 and
p = 0.0002, respectively), but again there was no
difference seen between B6.C20 and B6.C20/gld mice
(p = 0.79 and p = 0.46,
respectively). The data presented in Figure 6C were from a
single experiment with three mice per group, which has been repeated
once.
Induced apoptosis by in vitro treatment of cells with anti-Fas Ab
To directly test whether the Fas expressed on B6/lpr
mice was functional, untreated and irradiated splenocytes were cultured
in the presence or absence of an anti-Fas Ab, and the percentage of
cells undergoing apoptosis was determined by staining with Annexin V.
Figure 7A shows that
anti-Fas induced apoptosis of both irradiated and untreated B6
cells. Figure 7B indicates that B6/lpr cells
showed induction of apoptosis in the presence of anti-Fas Ab, both
in irradiated and unirradiated cells. The induction of
anti-Fas-induced annexin V positivity in untreated cultured
B6/lpr cells apparently reflects acquisition of Fas on cells
undergoing spontaneous apoptosis. These results are from an experiment
in which the splenocytes from three mice were pooled for each strain,
and cells from the same pool were used for the four conditions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-irradiated splenocytes from B6 or
B6/lpr into B6 or B6/gld mice. If Fas-mediated
apoptosis were occurring in the recipients, we reasoned that there
should be a difference in recovery of donor cells between B6 and
B6/gld recipients, as the latter lack functional FasL. Cells
subjected to treatment resulting in Fas-mediated apoptosis would thus
survive in B6/gld mice, whereas they would be killed in B6
mice. When cells are subjected to treatment resulting in
Fas-independent apoptosis (such as dexamethasone-induced apoptosis),
donor cell survival in B6 and B6/gld mice should be
indistinguishable. The increased recovery of irradiated B6 B and T cells from B6/gld compared with B6 recipients is compatible with the notion that Fas-mediated apoptosis participates in the disposition of radiation-injured cells in vivo. While recovery of irradiated B6 cells was greater in B6/gld than in B6, survival was not as great as that of untreated B6 cells in B6 or B6/gld, indicating that the presence of host FasL was only partially responsible for the elimination of injured cells. This may reflect the provision of FasL from the infused cells, resulting in autocrine killing. Fratricidal killing by other irradiated cells is less likely, as it would be improbable that transferred cells would be in close proximity in the host. More likely, other mechanisms account for the demise of many of the transferred cells. However, it is likely that FasL expressed on the transferred cells provided some of the signal required to induce apoptosis and subsequent removal of the injured Fas-expressing cells.
It was surprising that Fas could be detected on apoptotic cells from B6/lpr mice. Because Fas expression was seen only on cells undergoing apoptosis, it is not known whether it functions to induce apoptosis or is only detectable once the cell is committed to the apoptotic pathway. Here we have taken an apoptotic pathway that we know is partially mediated by Fas (8) and used our system to test whether the Fas expressed on B6/lpr splenic lymphocytes undergoing apoptosis is functional. When we compared the recovery of irradiated donor B6/lpr cells from recipient B6 and B6/gld mice, we found that the recovery of B6 B and T cells from B6/gld was greater than that from B6 recipients, supporting our hypothesis that the Fas expressed on B6/lpr cells undergoing apoptosis is actually instrumental in that process.
The conventional method for generating chimeric mice includes irradiation of the recipients, before injection, to "make room" for donor cells (17). We found that by irradiating recipient B6.C20 mice, there was a significant depletion of splenocytes, which increased the percentage of donor cells detected. However, the absolute number of donor cells recovered was not increased (data not shown). In addition, B6.C20 and B6.C20/gld mice responded differently to irradiation in that irradiation did not reduce the total number of splenocytes in B6.C20/gld mice (data not shown). For these reasons, recipient mice were not irradiated. The increased in vivo resistance to radiation-induced depletion of spleen cells in gld mice provides further support for a role for Fas/FasL in radiation injury.
Our finding that the recovery of untreated donor cells was independent of whether the donor strain had an intact Fas gene suggested that there was no difference, under these conditions, in homing or trafficking between B6 and B6/lpr splenocytes. The recovery of untreated donor cells was also independent of whether or not the recipient strain was gld homozygous, suggesting that there was not a general difference between B6 and B6/gld mice in terms of their capacity to accept transferred cells.
The evidence provided here that B6/lpr mice express
functional Fas, especially on apoptotic cells, is supported by the
finding that Fas was expressed on apparent live cells irradiated in the
presence of the apoptosis inhibitor zinc acetate. Based on reports that
find <10% of the normal level of Fas expression on MRL/lpr
thymocytes (18, 19), it is possible that as Fas gene expression is
up-regulated following -irradiation, the low level normally produced
increases, proportionately, to the point where it can be detected. An
alternative possibility is that -irradiation somehow exerts an
effect on the splicing process, resulting in an increased chance of
producing a functional Fas protein. Direct evidence that
B6/lpr mice express functional Fas protein is provided in
the finding that apoptosis is increased when cells from these mice are
cultured in the presence of anti-Fas Ab.
The lpr mutation is frequently described as leaky, based on the expression of small amounts of intact Fas mRNA or protein (20). To our knowledge, this is the first report that the leakiness of the lpr mutation results in the expression of functional Fas protein. This result is supported by the finding that the lymphoproliferation and autoimmune disease seen in lpr mice was enhanced and accelerated in Fas knockout mice (20).
This study contributes to the accumulating evidence that lpr is in fact a leaky mutation by providing data suggesting that there is functional Fas in lpr mice capable of transmitting an apoptotic signal. In addition, we have designed an in vivo system demonstrating the importance of Fas/FasL in the disposition of injured cells. This method can be utilized to test the role of Fas/FasL-induced apoptosis in cells subjected to a variety of treatments.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Philip L. Cohen, Department of Medicine, Division of Rheumatology, CB 7280, 3330 Thurston Building, University of North Carolina, Chapel Hill, NC 27599-7280.
3 Abbreviations used in this paper: FasL, Fas ligand; TNP, 2,4,6-trinitrophenyl.
Received for publication January 6, 1998. Accepted for publication June 24, 1998.
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