Departments of
* Microbiology and Immunology and
Pathology, and
Blood and Marrow Transplant Program, Division of Hematology/Oncology, Department of Internal Medicine, Markey Cancer Center, University of Kentucky, Lexington
Correspondence: J. Scott Bryson, Ph.D., Blood and Marrow Transplant Program, Division of Hematology Oncology, Markey Cancer Center, University of Kentucky, Lexington, KY 40536-0093. E-mail: jsbrys{at}pop.uky.edu
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, and TNF-
in the
development of murine SGVHD. Macrophages can be activated to secrete
IL-12 and TNF- via a T-cell-dependent or T-cell-independent pathway
(LPS or bacterial products). Studies were designed to determine if LPS
participated in the development of SGVHD in C3H/HeN (LPS-responsive)
and C3H/HeJ (LPS-hyporesponsive) mice. C3H/HeJ and C3H/HeN mice had
similar levels of disease induction and pathology. Following induction
of SGVHD, treatment of C3H/HeN, but not C3H/HeJ, mice with a sublethal
dose of LPS resulted in mortality. However, neutralization of IL-12
abrogated the development of disease in C3H/HeJ mice, demonstrating
that activated macrophages and their products participated in the
development of SGVHD in these animals. These data suggested that LPS
responsiveness was not a predisposing factor for SGVHD
induction.
Key Words: bone marrow transplantation • cyclosporine A • lipopolysaccharide
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Previous work in the murine model of SGVHD has demonstrated that mRNA
expression for interleukin (IL)-12, tumor necrosis factor
(TNF-), and interferon- (IFN-) was elevated in the target
organs of mice with clinical symptoms of SGVHD [11
].
Moreover, selective in vivo depletion of natural killer
(NK)1.1+ cells with monoclonal antibody (mAb) prevented the
induction of SGVHD and reduced IFN- mRNA expression in the colons of
SGVHD mice, suggesting that NK1.1+ cells were responsible
for the production of IFN- [11
]. In contrast, in vivo
depletion of NK1.1+ cells had no effect on IL-12 or TNF-
mRNA expression in the colons of SGVHD mice, suggesting that a non-NK
cell, perhaps macrophages, may have been responsible for the production
of IL-12 and TNF-. Neutralization of IL-12 inhibited the induction
of SGVHD, demonstrating that this cytokine played a critical role in
driving the effector phase of murine SGVHD [11
].
IL-12 has been shown to be secreted in vivo by macrophages, dendritic
cells, and B cells [12
]. Once secreted, it can activate
T cells and NK cells to become cytolytic and to release cytokines
(IFN-) that drive a TH1 immune response [12
13
14
]. Two
pathways that result in the secretion of IL-12 from activated
macrophages have been identified (T-dependent and T-independent)
[15
]. The T-dependent pathway requires the interaction
between CD40 and CD40L on macrophages and T cells, respectively,
whereas the T-independent pathway requires lipopolysacchardide (LPS) or
other bacterial products to activate the macrophage to secrete IL-12
and other cytokines [15
, 16
].
LPS has been shown to play a significant role in the development and
severity of GVHD following allogeneic BMT [17
18
19
].
Using radiation as pretransplant conditioning has been shown to cause
leakage of LPS from the gut [19
]. To address the role of
LPS in the murine model of SGVHD, studies were performed using the
LPS-resistant strain of mouse, C3H/HeJ. C3H/HeJ mice have been shown to
have a genetic defect in the Toll-like receptor (Tlr4) that inhibits
signaling through CD14 and renders this strain resistant to the effects
of LPS but not other bacterial products [20
]. Tlr4 has
been proposed to associate directly or indirectly with the LPS-CD14
complex and transduce the necessary signals within the cell, because
CD14 does not contain a cytoplasmic signaling domain
[21
]. Studies presented in this study were performed to
determine if LPS plays a major role in the development of murine SGVHD.
C3H/HeJ (LPS-resistant) and C3H/HeN (LPS-sensitive) mice were lethally
irradiated and reconstituted with syngeneic bone marrow and treated
with CsA to determine if LPS responsiveness is required for the
secretion of cytokines (IL-12 and TNF-) that ultimately lead to the
development of murine SGVHD. It is interesting that C3H/HeJ mice
developed SGVHD to similar levels as C3H/HeN mice, indicating that LPS
responsiveness was not a requirement for the development of SGVHD.
However, macrophage activation was found to be critical for induction
of this disease in both strains of animals, regardless of the ability
to respond to LPS.
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Induction of SGVHD
Bone marrow was isolated from the femurs and tibias of
age-matched syngeneic mice. The donor bone marrow suspensions were
prepared in RPMI 1640 (Life Technologies, Grand Island, NY),
supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. The
resulting cell suspensions were depleted of Thy1+ cells
using mAb to Thy1.2 (HO-13-4) and Low Tox M rabbit complement
(Cederlane Laboratories, Westbury, NY) as previously described
[22
]. Recipient mice were lethally irradiated with 9 Gy
in a Mark I 137Cs irradiator (J. L. Shepard and
Associates, Glendale, CA) 4–6 h before transplantation. The irradiated
mice were reconstituted with 5 x 106 Thy1-depleted
syngeneic bone marrow cells (ATBM; C3H/HeN
C3H/HeN;
C3H/HeJC3H/HeJ) intravenously (i.v.) via the tail vein. Starting on
the day of transplantation, groups of animals were treated
intraperitoneally (i.p.) with CsA (15 mg/kg) or diluent olive oil (OO;
control) for 21 days.
Evaluation of SGVHD
After the cessation of CsA treatment, animals were weighed three
times weekly and observed for clinical symptoms of SGVHD (runting,
diarrhea, and weight loss). Animals with weight loss for three
consecutive weighings and/or diarrhea were considered as positive for
the induction of SGVHD. In general, clinical symptoms were evident by 2
weeks after cessation of CsA therapy. Tissue samples were taken from
the animals 2 weeks post-CsA therapy and immediately placed in 10%
buffered formaldehyde. Fixed tissues were embedded in paraffin, cut
into 4–6 µm tissue sections, mounted on glass slides, and then
stained with a standard hematoxylin and eosin (H&E) procedure. All
tissue sections were analyzed blind without the knowledge of the
treatment category of the animal and graded for inflammation caused by
SGVHD using a previously published grading scale [23
]
ranging from 0–4.
Bacterial translocation following BMT
Seven days after the initiation of the SGVHD induction protocol,
groups of four normal-age-matched or control and CsA-treated
reconstituted C3H/HeN or C3H/HeJ mice were euthanatized, and the liver
was removed. Individual organs were placed into 5 ml sterile, distilled
H2O and homogenized using a Seward Stomacher tissue blender
(Brinkmann Instruments, Westbury, NY). The homogenate was diluted 1:10
in sterile water, and 0.1 ml was plated on brain heart infusion agar.
The plates were incubated for 24–48 h at 37°C and scored for
bacterial growth. Selected colonies were Gram-stained to determine if
the bacterial growth was derived from gram-negative or gram-positive
bacteria.
Reverse transcriptase-polymerase chain reaction (RT-PCR) for
cytokine message
Total RNA was isolated from the colons using Trizol reagent
(Life Technologies). RNA from each group (1 µg) was
reverse-transcribed into cDNA using a reverse transcription system
(Promega, Madison, WI). PCR reactions were then performed using primers
for ß-actin, TNF-, IFN- (Stratagene, La Jolla, CA), and
IL-12p40 [24
]. Before use, conditions for each set of
primers were optimized, and the number of PCR cycles was titrated. PCR
conditions for ß-actin, TNF-, and IFN- were as follows: 5-min
denaturing step at 94°C, 30 s at 94°C, 30 s at 55°C,
30 s at 72°C, and a 7-min final extension at 72°C. The PCR
conditions for IL-12p40 were similar, except the denaturing step was
set at 95°C. PCR reactions for TNF-, IFN-, and IL-12p40 were
allowed to cycle 30 times, whereas ß-actin was allowed to cycle 25
times. Expected sizes of the PCR products for ß-actin, TNF-,
IFN-, and IL-12p40 were 245 bp, 276 bp, 405 bp, and 404 bp,
respectively. Aliquots of each PCR reaction were mixed with sample
buffer and SYBR Green (Sigma Chemical Co., St. Louis, MO) and separated
on a 2% agarose gel or on a 2% agarose gel containing ethidium
bromide (Sigma Chemical Co.). The gels containing SYBR Green (Sigma
Chemical Co.) were visualized upon exposure to ultraviolet light and
were scanned (Storm Phosphorimager, Molecular Dynamics, Sunnyvale, CA).
The images were analyzed using the ImageQuant (Molecular Dynamics)
software program. Pictures of the agarose gels containing ethidium
bromide (Sigma Chemical Co.) were taken and scanned using a Bio Imager
(Bio Image, Kodak, Rochester, NY). The images were analyzed using the
VISAGE electrophoresis gel analysis system (Millipore, Bedford, MA).
Optical density (OD) of each individual fragment was normalized to
ß-actin expression from the corresponding tissue fragment.
LPS challenge of SGVHD mice
C3H/HeN and C3H/HeJ mice were induced for SGVHD as described
previously. Two weeks post-CsA treatment, transplant-control mice and
SGVHD mice were challenged with a sublethal dose (10 µg; i.v.) of LPS
(Sigma Chemical Co.) and were monitored for 36 h following
injection. Mice that were moribund were euthanatized and counted
positive for mortality. In addition, in one experiment, anti-TNF-
mAb [MP6-XT22; 1 mg protein in phosphate-buffered saline (PBS)] was
injected i.p. 18 h and 1 h or on days -2 and -1 and 1 h prior to challenge with 10 µg LPS.
Cytokine neutralization following induction of SGVHD
Mice were induced for SGVHD as described. Beginning on the day
of cessation of CsA therapy, control and CsA-treated mice were given
daily i.p. injections of anti-TNF- mAb (MP6-XT22; 1 mg protein in
PBS) or anti-IL-12 mAb (C15.1 and C15.6; 500 µg protein each) for 14
days. The animals were weighed 3x/week and monitored for the
development of SGVHD as described for 80 days post-BMT.
Statistical analysis
Statistical analysis was performed using the Student’s
t-test, log rank test, Chi-square, or the Fischer’s exact
test. P values of P 
0.05 were considered
to be statistically different.
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) was
elevated in the colon of SGVHD (Fig. 1
) mice relative to control BMT animals (P<0.05).
Because LPS can play an important role in the activation of
macrophages, we considered alternative approaches other than detection
of circulating LPS to measure translocation of bacteria during the
SGVHD induction period. It has been shown that irradiation and
cytotoxic drug therapy can induce the translocation of bacteria from
the gut into the liver and spleen [25
26
27
]. Therefore,
liver homogenates isolated from normal age-matched, control BMT and
CsA-treated BMT C3H/HeN and C3H/HeJ mice were tested for bacterial
growth 7 days after irradiation and BMT. The data presented in
Figure 2 A
demonstrate that bacterial growth was detected in the livers of
75% of CsA-treated mice relative to 12.5% of control diluent-treated
BMT or 0% of normal animals (unpublished results; P<0.02).
The results for C3H/HeJ mice (Fig. 2)
demonstrated an increase in the
numbers of SGVHD, as well as control animals that had bacteria in the
liver relative to normal mice. There was not a significant difference
between these two treatment groups. Gram-staining of selected bacterial
colonies demonstrated that the presence of gram-negative and
gram-positive organisms. Thus, bacterial translocation from the gut
occurred to a greater extent in CsA-treated mice following BMT.
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Figure 1. Enhanced TNF- mRNA production at the completion of CsA therapy.
C3H/HeN mice were lethally irradiated, reconstituted with syngeneic
ATBM, and treated with CsA or the diluent OO for 21 days. Groups of
control OO- or CsA-treated animals were euthanatized on day 21
post-transplantation, and the colons were analyzed for the production
of TNF- mRNA by RT-PCR. The data shown were normalized to ß-actin
expression and are expressed as mean ± SE. Data are
pooled from three separate experiments. The numbers in parentheses
represent the number of mice per treatment group. P <
0.05 (Student’s t-test).
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Figure 2. Increased gut leakage of bacteria in CsA-treated mice. C3H/HeN and
C3H/HeJ mice were lethally irradiated, reconstituted with syngeneic
ATBM, and treated with CsA for 7 days. At this time, the animals were
euthanatized, and the livers were removed. Liver homogenates were
diluted in sterile water and spread on brain-heart infusion growth
plates to detect bacterial growth. The data shown are pooled data from
two experiments, HeN (A) or a single experiment, HeJ (B). The numbers
above bars represent the number of mice per treatment group.
P < 0.02 (Chi-square).
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mRNA was present in the principle target organ (colon) of SGVHD
mice, the role that LPS played in murine SGVHD development was
examined. C3H/HeJ mice (LPS-hyporesponsive) and C3H/HeN mice
(LPS-responsive) were used for these experiments and were induced for
SGVHD. Mice were lethally irradiated, reconstituted with
T-cell-depleted bone marrow cells, and treated with CsA or diluent for
21 days. Upon cessation of CsA, mice were monitored for the development
of clinical symptoms of SGVHD (weight loss and diarrhea). As
demonstrated in Figure 3
, C3H/HeJ mice had almost identical disease induction rates as
compared with C3H/HeN mice. Sixty to eighty percent of the animals
developed SGVHD with 20–30% of those animals succumbing to the
disease. In addition to percent induction, the time to induction curves
for the two strains of mice was virtually identical.
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Figure 3. C3H/HeJ (LPS-nonresponsive) mice developed SGVHD. C3H/HeJ and
C3H/HeN (LPS-responsive) mice were lethally irradiated, reconstituted
with T-cell-depleted syngeneic bone marrow cells, and treated with CsA
or OO daily for 21 days. Starting on the last day of CsA injections,
groups of mice were weighed 3x weekly and monitored for clinical
symptoms of SGVHD—weight loss and diarrhea. Mice with weight loss for
three consecutive weighings were counted as positive for SGVHD. Percent
induction, HeN versus control, P < 0.0001; HeJ versus
control, P < 0.0001 (Fisher’s exact test); induction
curve for C3H/HeN and C3H/HeJ SGVHD mice. No significant difference
between C3H/HeN/C3H/HeJ, P > 0.05 (log rank test). The
data shown are pooled from three separate transplants.
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Figure 4. C3H/HeJ SGVHD mice develop a similar pathology as C3H/HeN SGVHD mice.
C3H/HeJ and C3H/HeN mice were induced for SGVHD as described. Two weeks
post-CsA treatment, pieces of the colons were removed from
transplant-control and SGVHD mice and normal animals, stained with a
standard H&E procedure, and graded for SGVHD pathology. All tissue
sections were analyzed blind without knowledge of the treatment
category. (A–D) The colon sections shown were representative samples
of each group. (A) Colon of C3H/HeJ transplant-control demonstrating
normal architecture (H&E; original, x50). (B) Colon of C3H/HeJ SGVHD
mouse exhibiting massive infiltration of inflammatory cells, crypt
abscess formation (small arrowheads), and focal gland destruction
(large arrowhead; H&E; original, x50). (C) Colon of C3H/HeN
transplant-control demonstrating normal architecture (H&E; original,
x50). (D) Colon from C3H/HeN SGVHD mouse demonstrating similar
pathology as in B with glandular destruction (arrowheads). Data
presented are from a representative animal from within each treatment
group.
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Figure 5. C3H/HeJ SGVHD mice developed an identical grade of pathology as C3H/HeN
SGVHD mice. The data shown are the average SGVHD pathology score from
three mice from a single transplant. *, P < 0.003, HeN
versus transplant-control; **, P < 0.01, HeJ versus
transplant-control (Student’s t-test).
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, and
TNF-) in the colon as well as increases in circulating IL-12 and
IFN- protein [11
]. Because C3H/HeJ mice developed
similar SGVHD pathology as C3H/HeN mice, the cytokine response in the
colon was characterized. Pieces of the colon were removed from SGVHD
mice, transplant-controls, and normal mice at 2 weeks post-CsA
treatment and analyzed by RT-PCR for the presence of IL-12, IFN-,
and TNF-. Similar to previously published data [11
],
increases in IL-12, IFN-, and TNF- were observed in the colon of
symptomatic SGVHD C3H/HeJ mice (Fig. 6
).
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Figure 6. C3H/HeJ SGVHD mice exhibited increased TH1 cytokine mRNA in the colon.
C3H/HeJ mice were induced for SGVHD as described. Pieces of the colon
were removed from OO and CsA-treated mice 2 weeks post-CsA treatment
when clinical symptoms (diarrhea and weight loss) were present, and
message levels were analyzed by RT-PCR. The data shown are normalized
to ß-actin expression and are presented as mean ±
SE. Data are representative of two separate transplants. *,
P 0.03 SGVHD versus transplant-control (Student’s
t-test).
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, because
pretreatment of SGVHD with anti-TNF- mAb inhibited 36 h
LPS-induced mortality (Fig. 7
; P<0.006). It is interesting
that although it appeared as though macrophages were activated, as
determined by cytokine mRNA production (Fig. 6)
, sublethal doses of LPS
did not cause mortality in any group of C3H/HeJ mice, because
macrophages from this strain are resistant to the effects of LPS (Fig. 7)
. C3H/HeJ SGVHD mice treated with LPS not only survived the 36 h
assay period, but these animals survived at a rate similar to untreated
SGVHD mice (>60–70%) for >21 days after LPS challenge (unpublished
results).
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Figure 7. Macrophages from C3H/HeN SGVHD mice are primed for enhanced LPS
responsiveness during SGVHD. C3H/HeN and C3H/HeJ mice were induced for
SGVHD as described previously. Two weeks post-CsA treatment, transplant
control mice and SGVHD mice were challenged with a sublethal dose (10
µg) of LPS and were monitored for mortality for 36 h
following injection. In addition, groups of control and CsA-treated
mice were given neutralizing anti-TNF mAb as described. Mice that were
moribund were euthanatized and counted positive for mortality. Data are
pooled from three experiments. *, P < 0.0001, HeN
versus HeJ LPS-induced mortality; **, P < 0.006, HeN
SGVHD versus HeN SGVHD anti-TNF LPS-induced mortality (Fisher’s exact
test).
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Figure 8. IL-12 neutralization inhibits the development of SGVHD in C3H/HeJ mice.
C3H/HeJ mice were induced for the development of SGVHD as described.
Beginning on the last day of CsA therapy, control and CsA-treated mice
were given daily injections of anti-IL-12 mAb i.p. for 2 weeks. The
animals were followed for the development of clinical symptoms of
SGVHD. CsA versus anti-IL-12-treated CsA mice, P <
0.04 (Chi-square).
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alters development of SGVHD could
significantly reduce LPS-induced mortality in SGVHD mice, studies were
initiated to determine the effect of in vivo TNF- neutralization on
the development of SGVHD. C3H/HeN and C3H/HeJ mice were induced for the
development of SGVHD. Beginning on the last day of CsA therapy, mice
were injected daily with anti-TNF- mAb for 2 weeks, and the animals
followed for the development of clinical symptoms (weight loss and
diarrhea). As shown in Figure 9
, treatment significantly reduced the development of clinical
symptoms in the anti-TNF-, SGVHD mice (C3H/HeN, P<0.002;
C3H/HeJ, P<0.03). Treatment of mice with an isotype-matched
rat mAb had no effect on the development of disease (unpublished
results) [11
]. These data suggest a role for TNF- in
the development of SGVHD.
 View larger version (15K): [in a new window] |
Figure 9. In vivo neutralization of TNF- following CsA therapy inhibits the
development of SGVHD-associated clinical symptoms. C3H/HeN and C3H/HeJ
mice were lethally irradiated, reconstituted with syngeneic bone
marrow, and treated with diluent/CsA as described in Materials and
Methods. Beginning on the last day of CsA therapy, control and
CsA-treated animals were given daily anti-TNF- mAb i.p. for 2 weeks.
The mice were followed for the development of clinical symptoms.
C3H/HeN: CsA versus anti-TNF--treated CsA mice, P <
0.002 (Chi-square). C3H/HeJ: CsA versus anti-TNF--treated CsA mice,
P < 0.03 (Chi-square).
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to
inhibit disease in both strains of mice demonstrated a role for this
proinflammatory cytokine in the development of murine SGVHD.
The role of LPS in acute GVHD following allogeneic BMT has been
well-documented. Nestel et al. [17
] first demonstrated
that LPS could be detected in the serum of mice with acute GVHD at the
onset of mortality. Furthermore, acute GVHD mice were more sensitive to
sublethal concentrations of LPS with increased release of TNF- into
the serum as compared with transplant-control mice and normal mice,
indicating that macrophages were primed during acute GVHD
[17
]. In a follow-up study, Price et al.
[18
] described the kinetics of LPS appearance in GVHD
mice, and LPS first appeared in the liver and then the spleen early
post-transplantation. Peak levels were reached in each organ at the
time LPS could be detected in the serum coinciding with mortality in
GVHD mice. Recently, Cooke et al. [29
] demonstrated that
donor-cell responsiveness to LPS is a risk factor for the development
of acute GVHD following allogeneic BMT. When C3H/HeJ mice
(LPS-hyporesponsive) were used as donors, the recipient mice developed
a less-severe form of GVHD with decreased intestinal pathology and
serum LPS and TNF- levels as compared with recipient mice that
received C3Heb/Fej (LPS-responsive) donor cells [29
].
To determine if LPS played a role in SGVHD, experiments were performed
in the SGVHD model to determine if increases in circulating LPS could
be detected in the plasma at a time (2 weeks post-CsA) during active
disease when pathologic changes have been observed [11
].
However, consistent increases in plasma LPS isolated from SGVHD mice
over transplant-control or normal animals were not observed
(unpublished results). This could be attributed to the fact that the
intensity of the pathological processes of SGVHD observed in C3H/HeN
mice is not as severe (low mortality) as that observed during acute
allogeneic GVHD as described by others [17
18
19
,
29
]. Additionally, it was demonstrated that macrophage
TNF- was elevated at the end of the CsA treatment period (day 21
post-BMT). These data suggested that macrophages were being activated
via a CsA-insensitive mechanism; consequently, additional approaches
were used to determine if bacterial translocation occurred during CsA
therapy and could be responsible for macrophage activation.
Gram-negative and gram-positive bacteria were detected in a
significantly higher percentage of the livers of CsA-treated versus
control diluent-treated animals. In fact, the percentage of the animals
in which bacterial translocation was detected was virtually identical
to the percentage of C3H/HeN animals in which SGVHD develops following
lethal irradiation, syngeneic BMT, and CsA therapy (
60–80%).
Because of the role of LPS in the development of allogeneic GVHD and
the findings of increased TNF- mRNA and bacterial translocation
during CsA therapy, C3H/HeJ mice that are hyporesponsive to LPS were
used to determine if LPS participated in the development of SGVHD.
C3H/HeJ mice developed comparable clinical symptoms at the same rate as
C3H/HeN animals (Fig. 3)
. Also, bacterial translocation, pathology, and
inflammation were similar in C3H/HeN and C3H/HeJ mice with SGVHD (Figs. 4
and 5)
. It has been shown that a TH1-associated cytokine response
plays a key role in the development of SGVHD [11
].
Similar to the induction and pathology data, C3H/HeJ animals
demonstrated increased production of the cytokines IL-12, TNF-, and
IFN- as observed in the LPS-responsive C3H/HeN mice (Fig. 6)
. Thus,
LPS responsiveness appeared not to be a critical determinant for the
induction and development of murine SGVHD.
The LPS hyporesponsiveness of C3H/HeJ mice has been attributed to
genetic defects in the Tlr4 receptor [21
]. This mutation
inhibits signaling through CD14, leaving this strain of mouse
nonresponsive to stimulation by LPS but not other bacterial products.
However, it should be noted that stimulation of C3H/HeJ macrophages by
LPS has been shown to result in minimal but detectable activation of
nuclear factor-
B (NF-B) [30
], expression
of manganese superoxide dismutase (MnSOD) [31
], and
secretory leukocyte protease inhibitor (SLPI) [32
]. SLPI
has been shown to function as an LPS-induced inhibitor of LPS
responsiveness. Furthermore, Jin et al. [32
] have shown
that macrophages isolated from C3H/HeJ animals respond with an
increased induction of matrix metalloproteinase-9 (MMP-9) in response
to LPS relative to cells isolated from C3H/HeN mice. These results
suggest that the response to LPS in the hyporesponsive animals may be
disregulated and is not as absolute as absence of a functional
Tlr4 receptor might suggest. Thus, although LPS challenge of
C3H/HeJ mice did not induce mortality via a TNF-mediated
mechanism, LPS does appear to induce the production of molecules such
as MMP-9 that could be involved in inflammatory responses in these
animals. Metalloproteinases have been associated with the ability to
promote cell migration during inflammation [33
34
35
]. In
addition, the use of MMP inhibitors alters the course of inducible
autoimmune disorders [36
, 37
] and inhibits
a rat model of trinitrobenzenesulphonic acid-induced inflammatory bowel
disease [38
]. With this in mind, it is possible in the
SGVHD model that LPS may function to stimulate other processes distinct
from that observed in allogeneic GVHD or C3H/HeN SGVHD (macrophage
activation/proinflammatory-cytokine production). The induction of MMPs
may participate in the colonic/liver inflammatory response that
develops during SGVHD by mediating the movement of macrophages into the
target tissue for activation by molecules distinct from LPS.
Clinical studies [39
] and animal studies
[40
, 41
] have identified a correlation
between maintaining transplant recipients in a germ-free environment
following gut decontamination after transplantation and the incidence
and severity of GVHD. Aplastic anemia patients transplanted with bone
marrow from an HLA-identical sibling that was placed in a protective
environment (decontamination and laminar-airflow isolation) developed a
less-severe grade of GVHD and survived longer as compared with those
patients not in a protective environment [39
]. Although
preliminary studies indicated that SGVHD mice had increased bacterial
translocation during CsA treatment as compared with transplant-control
animals, SGVHD could be induced in C3H/HeJ mice (Fig. 2)
, indicating
that LPS was not the critical determinant in the induction of murine
SGVHD. However, through irradiation and CsA treatment, damage to the
intestinal tract allowed for the release of bacteria and possibly other
microbial products such as bacterial DNA. Bacteria and bacterial DNA
have been shown to activate macrophages to secrete TNF- and IL-12,
which were shown to be critical to the development of SGVHD
[11
] (Figs. 8
and 9)
in C3H/HeN and C3H/HeJ mice. These
data demonstrate, for the first time, that TNF- is involved in the
induction of SGVHD. Furthermore, the IL-12 neutralization studies
confirm earlier studies of Flanagan et al. [11
], which
demonstrated a critical role for IL-12 in this model. More importantly,
they clearly define activated macrophages and their products as
important effector cells in the development of SGVHD in the
LPS-hyporesponsive C3H/HeJ mouse. Therefore, bacteria or other
bacterial products in addition to LPS might play a significant role in
activating macrophages, thereby increasing the release of
proinflammatory cytokines.
It has been suggested that C3H/HeJ macrophages exposed to IFN- can
respond to LPS [42
]. Because we have previously shown
increased IFN- production in SGVHD mice [11
], C3H/HeJ
SGVHD mice were challenged with a sublethal dose of LPS via the tail
vein. The development of SGVHD did not restore the responsiveness of
C3H/HeJ macrophages to LPS, because no mortality was observed after
challenge (Fig. 7)
. However, LPS challenge of C3H/HeN SGVHD mice
resulted in increased mortality as compared with transplant-controls,
demonstrating that macrophages were activated in SGVHD animals.
Collectively, these data presented in this study demonstrated that
macrophages were being primed and activated during the development of
murine SGVHD, resulting in enhanced production of IL-12 and TNF-.
However, the stimulus responsible for macrophage activation was not LPS
and although likely of bacterial origin, remains to be identified.
Received July 5, 2000; revised July 26, 2001; accepted August 1, 2001.
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