Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2001;70:873-880.)
© 2001 by Society for Leukocyte Biology

Induction of syngeneic graft-versus-host disease in LPS hyporesponsive C3H/HeJ mice

Diana Lowery Flanagan*, Rachel Gross{dagger}, C. Darrell Jennings{ddagger}, Betty E. Caywood{dagger}, Sarah Goes*, Alan M. Kaplan* and J. Scott Bryson*,{dagger}

Departments of
* Microbiology and Immunology and
{ddagger} Pathology, and
{dagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Syngeneic GVHD (SGVHD) develops following syngeneic bone marrow transplantation and treatment with cyclosporine A. Previous studies have demonstrated a role for IL-12, IFN-{gamma}, and TNF-{alpha} in the development of murine SGVHD. Macrophages can be activated to secrete IL-12 and TNF-{alpha} 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The immunosuppressive agent cyclosporine A (CsA) has been used clinically to prevent solid organ rejection and acute graft-versus-host disease (GVHD) following allogeneic bone marrow transplantation (BMT). However, Glazier et al. [1 ] demonstrated that CsA treatment following syngeneic BMT could induce a GVHD-like syndrome in rats, which has been termed syngeneic GVHD (SGVHD). Subsequent studies demonstrated that SGVHD could be induced in mice following syngeneic BMT [1 2 3 4 ] and in humans following autologous BMT (AUGVHD) [5 6 7 8 9 10 ].

Previous work in the murine model of SGVHD has demonstrated that mRNA expression for interleukin (IL)-12, tumor necrosis factor {alpha} (TNF-{alpha}), and interferon-{gamma} (IFN-{gamma}) 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-{gamma} mRNA expression in the colons of SGVHD mice, suggesting that NK1.1+ cells were responsible for the production of IFN-{gamma} [11 ]. In contrast, in vivo depletion of NK1.1+ cells had no effect on IL-12 or TNF-{alpha} 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-{alpha}. 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-{gamma}) 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-{alpha}) 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
C3H/HeN (Harlan Sprague-Dawley, Indianapolis, IN) and C3H/HeJ (Jackson Laboratories, Bar Harbor, ME) mice were purchased at 20–22 days of age and were used within 1 week of arrival. The mice were given autoclaved acidified water and lab food ad libitum and were housed in sterile microisolater boxes (Lab Products, Maywood, NJ).

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/HeJ->C3H/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-{alpha}, IFN-{gamma} (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-{alpha}, and IFN-{gamma} 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-{alpha}, IFN-{gamma}, 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-{alpha}, IFN-{gamma}, 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-{alpha} 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-{alpha} 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
C3H/HeJ mice develop SGVHD
Because LPS has been shown to participate in the development of allogeneic GVHD, experiments were designed to assess the role of LPS in the induction of SGVHD. Preliminary data measuring circulating levels of LPS in the plasma of control C3H/HeN mice and C3H/HeN mice treated with CsA to induce SGVHD were inconclusive. Although little-to-no LPS was detected in the plasma of control mice, on occasion, elevated levels (up to twofold) could be detected during CsA treatment and during active disease (unpublished results). Furthermore, data analyzing cytokine mRNA levels at the completion of CsA therapy demonstrated that macrophage-associated cytokine mRNA (TNF-{alpha}) 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-{alpha} 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-{alpha} 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).

 
With the demonstration that translocation of gram-negative bacteria occurred from the gut to the liver during CsA therapy, and increased TNF-{alpha} 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.

 
To determine if the pathology that developed in CsA-treated C3H/HeN and C3H/HeJ mice was similar, colon tissue samples were removed from transplant-control SGVHD animals, and normal animals and analyzed for SGVHD pathology. Colon sections from C3H/HeJ transplant-control mice demonstrated essentially normal colon architecture (Fig. 4A ), whereas colon sections from C3H/HeJ SGVHD mice exhibited dense inflammatory cell infiltration of the colonic mucosa. Multiple crypt abscesses were present with focal destruction of entire segments of individual glands (Fig. 4B) . Similar pathological findings were found in C3H/HeN animals; sections from transplant-control mice demonstrated normal colon architecture (Fig. 4C) , and sections from SGVHD mice demonstrated dense inflammatory cell infiltration of the colonic mucosa with entire segments of individual glands being destroyed (Fig. 4D) . Consequently, C3H/HeJ mice developed significant SGVHD pathology in the colon and had essentially the same pathology grade as C3H/HeN mice in the colon (Fig. 5 ) and liver (unpublished results).



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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).

 
C3H/HeJ mice with SGVHD develop a TH1-associated cytokine pattern
C3H/HeN mice with SGVHD have been described previously to exhibit increased mRNA for TH1-associated cytokines (IL-12p40, IFN-{gamma}, and TNF-{alpha}) in the colon as well as increases in circulating IL-12 and IFN-{gamma} 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-{gamma}, and TNF-{alpha}. Similar to previously published data [11 ], increases in IL-12, IFN-{gamma}, and TNF-{alpha} 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).

 
Induction of SGVHD primes macrophages
IL-12 is produced by activated macrophages in vivo [13 ]. Previous studies demonstrated increased IL-12 production during SGVHD, and neutralization of IL-12 in vivo inhibited the induction of SGVHD [11 ]. These findings suggested that macrophages were activated during the development of SGVHD. Therefore, studies were performed to determine if macrophages were primed in vivo during SGVHD to become hyper-responsive to sublethal doses of LPS. C3H/HeN and C3H/HeJ mice were induced for SGVHD. Two weeks after the last CsA injection, groups of mice (transplant-control, SGVHD, and normal age- and strain-matched) were injected with a sublethal dose of LPS via the tail vein. No mortality was observed in normal C3H/HeN mice or transplant-control mice (Fig. 7 ). However, significant mortality occurred in C3H/HeN SGVHD animals that received the sublethal dose of LPS (P<0.0001), suggesting that the macrophage cell population was activated during SGVHD. Mortality in C3H/HeN SGVHD mice induced by sublethal doses of LPS appeared to be mediated via induction of TNF-{alpha}, because pretreatment of SGVHD with anti-TNF-{alpha} 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).

 
Although it was clear that macrophages in C3H/HeN mice were primed to respond to low-dose LPS, and IL-12 neutralization inhibited SGVHD induction [11 ], the role of macrophages in C3H/HeJ mice following CsA therapy was unclear. IL-12 neutralization studies were performed to determine the role of IL-12 and, therefore, macrophages in these animals. Treatment of C3H/HeJ mice with neutralizing anti-IL-12 mAb following BMT and CsA therapy inhibited the development of disease (Fig. 8 ; P<0.04). Finally, activated macrophages have been shown to produce significant amounts of nitric oxide (NO) via the inducible form of NO synthase (iNOS) [28 ]. Significant increases in colonic mRNA for iNOS and circulating levels of NO were found in the plasma of C3H/HeN and C3H/HeJ mice (unpublished results). (Manuscript is in preparation for the role of NO in the development of murine syngeneic GVHD.) These results demonstrated the involvement of activated macrophages in the development of SGVHD in C3H/HeJ, as has been described in the LPS-responsive C3H/HeN mice.



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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).

 
Neutralization of TNF-{alpha} alters development of SGVHD
Previous studies had demonstrated increases in TH1-associated cytokines in the target organs of C3H/HeN SGVHD mice [11 ]. Furthermore, neutralization of IL-12 resulted in complete abrogation of clinical disease and tissue pathology. Based on the above study, demonstrating that neutralization of TNF-{alpha} could significantly reduce LPS-induced mortality in SGVHD mice, studies were initiated to determine the effect of in vivo TNF-{alpha} 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-{alpha} 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-{alpha}, 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-{alpha} in the development of SGVHD.



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  		Figure 9. In vivo neutralization of TNF-{alpha} 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-{alpha} mAb i.p. for 2 weeks. The mice were followed for the development of clinical symptoms. C3H/HeN: CsA versus anti-TNF-{alpha}-treated CsA mice, P < 0.002 (Chi-square). C3H/HeJ: CsA versus anti-TNF-{alpha}-treated CsA mice, P < 0.03 (Chi-square).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The studies presented in this manuscript demonstrated that LPS responsiveness was not a critical component in the induction of murine SGVHD. When induced for SGVHD, the LPS hyporesponsive C3H/HeJ mice developed similar clinical symptoms, tissue pathology, a TH1-associated cytokine profile, and macrophage activation as the LPS responsive strain, C3H/HeN. Finally, the ability of neutralization of TNF-{alpha} 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-{alpha} 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-{alpha} 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-{alpha} 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 ({approx}60–80%). Because of the role of LPS in the development of allogeneic GVHD and the findings of increased TNF-{alpha} 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-{alpha}, and IFN-{gamma} 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-{kappa}B (NF-{kappa}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-{alpha} 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-{alpha} 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-{gamma} can respond to LPS [42 ]. Because we have previously shown increased IFN-{gamma} 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-{alpha}. However, the stimulus responsible for macrophage activation was not LPS and although likely of bacterial origin, remains to be identified.


   ACKNOWLEDGEMENTS
 
This study was supported by NIH grant RO1 CA74228. D. L. F. was supported by NCI training grant CA09509. We would like to thank Dr. Georgio Trinchieri (The Wistar Institute, Philadelphia, PA) for kindly providing the 15.1 and 15.6 hybridomas.

Received July 5, 2000; revised July 26, 2001; accepted August 1, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Glazier, A., Tutschka, P. J., Farmer, E. R., Santos, G. W. (1983) Graft-versus-host disease in cyclosporin A-treated rats after syngeneic and autologous bone marrow reconstitution J. Exp. Med. 158,1-8[Abstract/Free Full Text]
  2. Fowell, D., Powrie, F., Saoudi, A., Seddon, B., Heath, V., Mason, D. (1995) The role of subsets of CD4+ T cells in autoimmunity Ciba Found. Symp. 195,173-182[Medline]
  3. Bryson, J. S., Jennings, C. D., Caywood, B. E., Kaplan, A. M. (1989) Induction of a syngeneic graft-versus-host disease-like syndrome in DBA/2 mice Transplantation 48,1042-1047[Medline]
  4. Bucy, R. P., Xu, X. Y., Li, J., Huang, G. (1993) Cyclosporin A-induced autoimmune disease in mice J. Immunol. 151,1039-1050[Abstract]
  5. Jones, R., Hess, A., Mann, R., Piantadosi, S., Vogelsang, G., Farmer, E., Geller, R., Santos, G. (1989) Induction of graft-versus-host disease after autologous bone marrow transplantation Lancet 1,754-757[Medline]
  6. Talbot, D., Powles, R., Sloane, J., Rose, J., Treleaven, J., Aboud, H., Helenglass, G., Parikh, P., Smith, C., Rowley, M., Cavanagh, J., Milliken, S., Hewetson, M., Norton, J. (1990) Cyclosporine-induced graft-versus-host disease following autologous bone marrow transplantation in acute myeloid leukemia Bone Marrow Transplant 6,17-20[Medline]
  7. Carella, A., Gaozza, E., Congiu, A., Carlier, P., Nati, S., Truini, M., Castellaneta, A., Viale, M. (1991) Cyclosporine-induced graft-versus-host disease after autologous bone marrow transplantation in hematological malignancies Ann. Hematol. 62,156-159[Medline]
  8. Yeager, A., Vogelsang, G., Jones, R., Farmer, E., Altomonte, V., Hess, A., Santos, G. (1992) Induction of cutaneous graft-versus-host disease by administration of cyclosporine to patients undergoing autologous bone marrow transplantation for acute myeloid leukemia Blood 79,3031-3035[Abstract/Free Full Text]
  9. Kennedy, M., Vogelsang, G., Beveridge, R., Farmer, E., Altomonte, V., Huelskamp, A., Davidson, N. (1993) Phase I trial of intravenous cyclosporine to induce graft-versus-host disease in women undergoing autologous bone marrow transplantation for breast cancer J. Clin. Oncol. 11,478-484[Abstract/Free Full Text]
  10. Kennedy, M. J., Vogelsang, G. B., Jones, R. J., Farmer, E. R., Hess, A. D., Altomonte, V., Huelskamp, A. M., Davidson, N. E. (1994) Phase I trial of interferon gamma to potentiate cyclosporine-induced graft-versus-host disease in women undergoing autologous bone marrow transplantation for breast cancer J. Clin. Oncol. 12,249-257[Abstract]
  11. Flanagan, D. L., Jennings, C. D., Bryson, J. S. (1999) Th1 cytokines and NK cells participate in the development of murine syngeneic graft-versus-host disease J. Immunol. 163,1170-1177[Abstract/Free Full Text]
  12. Trinchieri, G. (1994) Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes Blood 84,4008-4027[Free Full Text]
  13. Brunda, M. J. (1994) Interleukin-12 J. Leukoc. Biol. 55,280-288[Abstract]
  14. Hendrzak, J. A., Brunda, M. J. (1995) Interleukin-12 Biologic activity, therapeutic utility, and role in disease. Lab. Investig. 72,619-637
  15. Takenaka, H., Maruo, S., Yamamoto, N., Wysocka, M., Ono, S., Kobayashi, M., Yagita, H., Okumura, K., Hamaoka, T., Trinchieri, G., Fujiwara, H. (1997) Regulation of T cell-dependent and -independent IL-12 production by the three Th2-type cytokines IL-10, IL-6, and IL-4 J. Leukoc. Biol. 61,80;dn87
  16. Sousa, C. R., Hieny, S., Scharton-Kersten, T., Jankovic, D., Charest, H., Germain, R. N., Sher, A. (1997) In vivo microbial stimulation induces rapid CD40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to T cell areas J. Exp. Med. 186,1819-1829[Abstract/Free Full Text]
  17. Nestel, F. P., Price, K. S., Seemayer, T. A., Lapp, W. S. (1992) Macrophage priming and lipopolysaccharide-triggered release of tumor necrosis factor alpha during graft-versus-host disease J. Exp. Med. 175,405-413[Abstract/Free Full Text]
  18. Price, K. S., Nestel, F. P., Lapp, W. S. (1997) Progressive accumulation of bacterial lipopolysaccharide in vivo during murine acute graft-versus-host disease Scand. J. Immunol. 45,294-300[Medline]
  19. Hill, G. R., Crawford, J. M., Cooke, K. R., Brinson, Y. S., Pan, L., Ferrara, J. L. (1997) Total body irradiation and acute graft-versus-host disease: the role of gastrointestinal damage and inflammatory cytokines Blood 90,3204-3213[Abstract/Free Full Text]
  20. Poltorak, A., He, X., Smirnova, I., Liu, M. Y., Huffel, C. V., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B., Beutler, B. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene Science 282,2085-2088[Abstract/Free Full Text]
  21. Beutler, B. (2000) Tlr4: central component of the sole mammalian LPS sensor Curr. Opin. Immunol. 12,20-26[Medline]
  22. Bryson, J. S., Jennings, C. D., Caywood, B. E., Kaplan, A. M. (1993) Thy1+ bone marrow cells regulate the induction of murine syngeneic graft-versus-host disease Transplantation 56,941-945[Medline]
  23. Bryson, J. S., Jennings, C. D., Lowery, D. M., Carlson, S. L., Pflugh, D. L., Caywood, B. E., Kaplan, A. M. (1999) Rejection of an MHC class II negative tumor following induction of murine syngeneic graft-versus-host disease Bone Marrow Transplant 23,363-372[Medline]
  24. Murphy, T. L., Cleveland, M. G., Kulesza, P., Magram, J., Murphy, K. M. (1995) Regulation of interleukin 12 p40 expression through an NF-kappa B half-site Mol. Cell. Biol. 15,5258-5267[Abstract]
  25. Berg, R. D., Wommack, E., Deitch, E. A. (1988) Immunosuppression and intestinal bacterial overgrowth synergistically promote bacterial translocation Arch. Surg. 123,1359-1364[Abstract]
  26. Spitz, J., Hecht, G., Taveras, M., Aoys, E., Alverdy, J. (1994) The effect of dexamethasone administration on rat intestinal permeability: the role of bacterial adherence Gastroenterology 106,35-41[Medline]
  27. Cicalese, L., Aitouche, A., Ploskina, T. M., Ford, H. R., Rao, A. S. (1999) The role of laparotomy, gut manipulation and immunosuppression on bacterial translocation from the intestinal tract Transplant. Proc. 31,1922-1923[Medline]
  28. Nathan, C., Xie, Q. W. (1994) Nitric oxide synthases: roles, tolls, and controls Cell 78,915-918[Medline]
  29. Cooke, K. R., Hill, G. R., Crawford, J. M., Bungard, D., Brinson, Y. S., Delmonte, J., Jr, Ferrara, J. L. (1998) Tumor necrosis factor-alpha production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease J. Clin. Investig. 102,1882-1891[Medline]
  30. Ding, A., Hwang, S., Lander, H. M., Xie, Q. W. (1995) Macrophages derived from C3H/HeJ (Lpsd) mice respond to bacterial lipopolysaccharide by activating NF-kappa B J. Leukoc. Biol. 57,174-179[Abstract]
  31. Gibbs, L. S., Del Vecchio, P. J., Shaffer, J. B. (1992) Mn and Cu/Zn SOD expression in cells from LPS-sensitive and LPS-resistant mice Free Radic. Biol. Med. 12,107-111[Medline]
  32. Jin, F. Y., Nathan, C., Radzioch, D., Ding, A. (1997) Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide Cell 88,417-426[Medline]
  33. Matrisian, L. M. (1992) The matrix-degrading metalloproteinases Bioessays 14,455-463[Medline]
  34. Hurwitz, A., Dushnik, M., Solomon, H., Ben-Chetrit, A., Finci-Yeheskel, Z., Milwidsky, A., Mayer, M., Adashi, E. Y., Yagel, S. (1993) Cytokine-mediated regulation of rat ovarian function: interleukin-1 stimulates the accumulation of a 92-kilodalton gelatinase Endocrinology 132,2709-2714[Abstract]
  35. Bernhard, E. J., Gruber, S. B., Muschel, R. J. (1994) Direct evidence linking expression of matrix metalloproteinase 9 (92-kDa gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells Proc. Natl. Acad. Sci. USA 91,4293-4297[Abstract/Free Full Text]
  36. Hewson, A. K., Smith, T., Leonard, J. P., Cuzner, M. L. (1995) Suppression of experimental allergic encephalomyelitis in the Lewis rat by the matrix metalloproteinase inhibitor Ro31-9790 Inflamm. Res. 44,345-349[Medline]
  37. Hamada, T., Arima, N., Shindo, M., Sugama, K., Sasaguri, Y. (2000) Suppression of adjuvant arthritis of rats by a novel matrix metalloproteinase-inhibitor Br. J. Pharmacol. 131,1513-1520[Medline]
  38. Sykes, A. P., Bhogal, R., Brampton, C., Chander, C., Whelan, C., Parsons, M. E., Bird, J. (1999) The effect of an inhibitor of matrix metalloproteinases on colonic inflammation in a trinitrobenzenesulphonic acid rat model of inflammatory bowel disease Aliment Pharmacol. Ther. 13,1535-1542[Medline]
  39. Storb, R., Prentice, R. L., Buckner, C. D., Clift, R. A., Appelbaum, F., Deeg, J., Doney, K., Hansen, J. A., Mason, M., Sanders, J. E., Singer, J., Sullivan, K. M., Witherspoon, R. P., Thomas, E. D. (1983) Graft-versus-host disease and survival in patients with aplastic anemia treated by marrow grafts from HLA-identical siblings. Beneficial effect of a protective environment N. Engl. J. Med. 308,302-307[Abstract]
  40. van Bekkum, D. W., Roodenburg, J., Heidt, P. J., van der Waaij, D. (1974) Mitigation of secondary disease of allogeneic mouse radiation chimeras by modification of the intestinal microflora J. Natl. Cancer Inst. 52,401-404
  41. van Bekkum, D. W., Knaan, S. (1977) Role of bacterial microflora in development of intestinal lesions from graft-versus-host reaction J. Natl. Cancer Inst. 58,787-790
  42. Adi, S., Pollock, A. S., Shigenaga, J. K., Moser, A. H., Feingold, K. R., Grunfeld, C. (1992) Role for monokines in the metabolic effects of endotoxin. Interferon- gamma restores responsiveness of C. 3H/HeJ mice in vivo J. Clin. Investig. 89,1603-1609



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