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(Journal of Leukocyte Biology. 2002;72:762-768.)
© 2002 by Society for Leukocyte Biology

Nitric oxide participates in the intestinal pathology associated with murine syngeneic graft-versus-host disease

D. M. Flanagan*,{dagger}, C. D. Jennings{ddagger}, S. W. Goes{dagger}, B. E. Caywood§, R. Gross§, A. M. Kaplan{dagger} and J. S. Bryson{dagger},§

* Department of Biology, Hardin-Simmons University, Abilene, Texas; and Departments of
{dagger} Microbiology, Immunology and Molecular Genetics,
{ddagger} Pathology, and
§ Internal Medicine, Markey Cancer Center, Chandler Medical Center, University of Kentucky, Lexington

Correspondence: J. Scott Bryson, Ph.D., Associate Professor, Blood and Marrow Transplant Program, Division of Hematology Oncology, Department of Internal Medicine, Markey Cancer Center, University of Kentucky, Lexington, KY 40536-0093. E-mail: jsbrys{at}uky.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Syngeneic graft-versus-host disease (SGVHD) develops following lethal irradiation, reconstitution with syngeneic bone marrow, and treatment with a short course of cyclosporin A (CsA) therapy. The disease is characterized by the development of a T helper cell type 1-like cytokine response [interleukin (IL)-12, interferon-{gamma} (IFN-{gamma}), and tumor necrosis factor {alpha}], and macrophage activation is central to development of the syndrome. It has been shown that nitric oxide (NO) participates significantly in the development of allogeneic GVHD. Studies were initiated to determine if NO participates in the pathology associated with SGVHD. Significant increases in inducible NO synthase (iNOS) mRNA and circulating NO were found in the tissues of SGVHD versus control animals. Treatment of SGVHD animals with the iNOS inhibitor aminoguanidine (AG) reversed the pathology associated with this disease. Furthermore, AG treatment reduced the production of IL-12 and IFN-{gamma} mRNA in the colons of CsA-treated mice. These studies demonstrate that NO participates in the pathological processes that are associated with the development of murine SGVHD.

Key Words: IFN-{gamma} • interleukin-12 • nitric oxide • aminoguanidine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pleiotropic molecule nitric oxide (NO) can be produced via constitutive and inducible forms of the NO synthase (iNOS) enzyme [1 2 3 ]. The constitutively expressed enzymes are expressed in vascular endothelial cells (eNOS, NOS-3) or neuronal cells (nNOS, NOS-1). Production of NO via these enzymes is thought to participate in normal physiology [2 , 4 5 6 ]. The iNOS (NOS-2) is expressed by many cell types including endothelial, epithelial, and inflammatory cells [1 ]. This enzyme is associated with the production of large amounts of NO over extended periods of time following stimulation with cytokines and inflammatory agents such as lipopolysaccharides (LPS). NO produced in large amounts has been suggested to be important in mediating tissue injury [7 ]. Alone, NO is a weak, free radical; however, reaction with superoxide (O2-) results in the formation of the highly toxic, reactive nitrogen intermediate, peroxynitrite (OONO-) [8 ]. Peroxynitrite then has the ability to generate potent oxidizing intermediates that can mediate a number of cytotoxic processes including lipid peroxidation [9 ] and DNA damage [10 ].

Not only has NO been associated with normal homeostatic, physiologic functions, but the molecule has also been linked to various pathologic disorders as well [8 , 11 12 13 14 15 16 17 ]. In addition, NO has been shown to be important in normal and pathologic processes following allogeneic bone marrow transplantation (BMT) including the development of graft-versus-host disease (GVHD), a major complication associated with allogeneic BMT. GVHD is induced following the transfer of alloreactive donor T cells in the BM inoculum [18 , 19 ]. During the acute GVHD response, alloactivated donor T cells produce primarily T helper cell type 1 (TH1) cytokines, interleukin (IL-2), and interferon-{gamma} (IFN-{gamma}) [20 , 21 ]. These cytokines and others can in turn activate macrophages to secrete proinflammatory cytokines and other mediators including NO [19 , 22 ]. Finally, efferent effector cells such as alloreactive cytolytic T lymphocytes, natural killer (NK) cells, and macrophages are activated and act in concert with the cytokines described above to cause additional tissue damage [23 ]. Experimental animals that are induced to develop GVHD have significantly increased iNOS activity and increased levels of plasma nitrite (NO2-) and nitrate (NO3-), byproducts of NO [24 25 26 ]. It was shown that inhibition of iNOS in mice undergoing GVHD resulted in less tissue injury [27 ], reduced intestinal pathology [28 ], decreased serum NO2- and NO3-, and reduced lethality [29 ]. It is interesting that aminoguanidine (AG; a relatively specific iNOS inhibitor) therapy also enhanced hematopoiesis, suggesting that increased NO may be in part responsible for inhibiting hematopoiesis during acute GVHD [29 ]. In contrast, Drobyski et al. [30 ] have demonstrated that treatment of mice with the NOS inhibitor NG-L-methyl-arginine (NMA), following allogeneic BMT, inhibited hematopoietic reconstitution, suggesting that NO may play a role in facilitating alloengraftment [30 ]. However, as NMA is not specific for iNOS and is likely to have inhibited the constitutive forms of NOS as well, overall the role of NOS in hematopoiesis remains unclear.

Rodents treated with the immunosuppressive agent cyclosporine A (CsA) following syngeneic or autologous BMT [31 32 33 34 35 ] develop a disease similar to allogeneic GVHD, termed syngeneic GVHD (SGVHD). This disease appears to be mediated by specific and nonspecific immune responses [35 36 37 38 ]. It has been demonstrated in the rat model that T cells specific for the CLIP peptide of the invariant chain mediate the development of rat SGVHD [37 ]. Recently, Flanagan et al. [35 , 39 ] demonstrated that activated NK cells and macrophages played a significant role in the development of murine SGVHD. Treatment of animals with anti-NK monoclonal antibodies (mAb) as well as the in vivo neutralization of IL-12 inhibited SGVHD as determined by clinical symptoms and tissue pathology [35 , 39 ]. Because activated macrophages are the main producer of IL-12 [40 ], and IL-12 plays a critical role in the development of SGVHD, it was clear that these cells are major participants in this inducible disease.

In addition to IL-12 and other proinflammatory cytokines, activated macrophages have the potential to produce large amounts of NO via the iNOS enzyme [1 ]. As macrophages appear to be essential participants in the pathophysiology of murine SGVHD, and NO plays an important role in the intestinal pathology associated with acute GVHD, studies were initiated to determine if NO contributed in the development of this disease. Increases in iNOS mRNA and circulating NO were observed in the target organs and plasma serum of SGVHD mice, respectively. Treatment of these diseased animals with the iNOS inhibitor AG resulted in abrogation of clinical symptoms and tissue pathology and cytokines (IL-12, IFN-{gamma}) associated with this inducible disease. These studies demonstrate a role for NO in the intestinal pathology of murine SGVHD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
C3H/HeN mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN) at 20–21 days of age and were used within 1 week of arrival. Mice were housed in sterile, microisolator cages (Lab Products, Maywood, NJ) and fed autoclaved food and acidified water ad libitum.

Reagents
CsA was purchased through the Division of Laboratory Animal Resources, University of Kentucky (Lexington). Reagents for the detection of the NO metabolite nitrite (NO2-) in culture supernatants (Griess reagent) [41 ], N-[l-naphthyl] ethylenediamine dihydrochloride, and sulfanilamide were purchased from Sigma Chemical Co. (St. Louis, MO). To detect NO metabolites in plasma/serum, the nitrate/nitrite colorimetric assay kit (Cayman) was obtained from Alexis Biochemicals (San Diego, CA). The iNOS inhibitor AG was purchased from Sigma Chemical Co.

Induction of SGVHD
BM was isolated from the femurs and tibias of syngeneic, age-matched mice. The donor BM 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 [34 ]. Recipient mice were lethally irradiated with 900 cGy in a Mark I 137Cs irradiator (J. L. Shepard and Associates, Glendale, CA). The animals were reconstituted intravenously with 5 x 106 T cell-depleted marrow, 4–6 h after conditioning. Beginning on the day of transplantation, the mice were treated daily for 21 days with 15 mg/kg CsA in the diluent olive oil (Sigma Chemical Co.) or diluent alone. Following cessation of CsA, the animals were weighed three times/week and observed for clinical signs of the development of SGVHD, weight loss, and diarrhea. Animals that developed clinical symptoms for three consecutive weighings were considered as positive for the induction of SGVHD. In general, clinical symptoms were evident by 2 weeks after cessation of CsA therapy.

Histologic evaluation of SGVHD
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- to 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 (colon: ±, rare crypt cell necrosis; 1+, definite, scattered, single-cell necrosis in crypts; 2+, several necrotic cells in gland, crypt abscesses present; 3+, confluent destruction of glands) [42 ].

Reverse transcriptase-polymerase chain reaction (RT-PCR) for iNOS and cytokine mRNA
Total RNA was isolated from the colons using Trizol reagent (Life Technologies). RNA (1 µg) from each group was reverse-transcribed into cDNA using the Promega (Madison, WI) reverse-transcription system. PCR reactions were then performed using primers for ß-actin (Stratagene, La Jolla, CA) and iNOS [43 ]. Before use, conditions for each set of primers were optimized, and the number of PCR cycles was titrated. PCR conditions for ß-actin 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. PCR conditions for iNOS were as follows: 5-min denaturing step at 94°C, 30 s at 94°C, 30 s at 60°C, 45 s at 72°C, and a 7-min final extension at 72°C. PCR reactions for iNOS were allowed to cycle 30 times, whereas ß-actin was allowed to cycle 25 times. Conditions for IL-12 and IFN-{gamma} have been previously described [35 ]. Expected sizes of the PCR products for ß-actin, iNOS, IL-12, and IFN-{gamma} were 245 bp, 474 bp, 404 bp, and 405 bp, respectively. Aliquots of each PCR reaction were mixed with sample buffer and SYBR Green (Sigma Chemical Co.) and separated on a 2% agarose gel. The gel was visualized upon exposure to ultraviolet light. The agarose gels were scanned (Storm Phosphorimager), and the images were analyzed using the ImageQuant (Molecular Dynamics, Sunnyvale, CA). Optical density (OD) of each individual fragment was normalized to ß-actin expression from the corresponding tissue fragment.

In vitro NO production
Spleens were isolated from normal, age-matched transplant control or symptomatic SGVHD animals. Single-cell suspensions were prepared, and the red blood cells were lysed with Tris-buffered ammonium chloride and resuspended into complete RPMI-1640 medium (Gibco, Grand Island, NY) containing 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco), and 5 x 10-5 M 2-mercaptoethanol (Sigma Chemical Co.). The cells were plated at 2 x 106 cells in 1 ml into wells of 24-well plates. The cells were stimulated with varying concentrations of LPS. After 72 h of culture, the supernatant was harvested and analyzed for the presence of NO2- using the Griess reagent. The OD of the purple azo dye was measured at 550 nm in a Molecular Dynamics plate reader.

Measurement of plasma/serum NO
To measure the production of NO during the development of SGVHD, normal, control, and SGVHD mice were anesthetized with metofane, and the animal blood was obtained by cardiac puncture using heparinized (plasma) or untreated (serum) syringes using a 25-gauge needle. Circulating NO levels were determined using a nitrate/nitrate colorimetric kit. The reagents in this kit used nitrate reductase to reduce plasma nitrate to nitrite. The levels of NO2- were measured using a modification of the Griess reagent.

In vivo inhibition of iNOS
Control and CsA-treated BMT mice were treated daily with the iNOS inhibitor AG, 8 mg/kg/day intraperitoneally [44 ], beginning on the last day of CsA treatment and continuing for 14 days. The animals were then observed for development of SGVHD. In addition, groups of control and SGVHD mice were killed, and sections of liver and colon were removed and placed in buffered formalin. The tissues were then analyzed histologically for SGVHD-associated pathology. Alternatively, sections of tissues were placed in Trizol Reagent, RNA isolated, and analyzed by RT-PCR as described.

Statistical analysis
Statistical differences between control and SGVHD samples were determined using Student’s t-test and Fisher’s exact test (induction). Differences <=0.05 were considered statistically different.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
As studies made it apparent that macrophage activation was critical to the development of SGVHD [35 , 39 ], we became interested in the possibility that reactive oxygen intermediates produced by activated macrophages participated in the pathology that was observed during this disease. NO was particularly intriguing because of the relationship that had been identified between NO and gut pathology during allogeneic GVHD [28 ].

Increased NO production during SGVHD
Previous studies had demonstrated that the production of the macrophage-associated cytokine IL-12 during the development of SGVHD was critical [35 ]. It has also been shown that activated macrophages can produce large amounts of NO from L-arginine via the iNOS enzyme [1 2 3 , 45 ]. RT-PCR studies were performed to determine if increases in mRNA for iNOS could be detected in the target organs of SGVHD, liver, and colon. Increased mRNA for iNOS was present in the colon and liver (data not shown) of C3H/HeN SGVHD versus BMT-control mice (P=0.0064; Fig. 1 ) at 2–3 weeks after cessation of CsA therapy. To determine if increases in tissue iNOS mRNA translated into increases in iNOS activity and circulating NO, plasma/serum was isolated from control and SGVHD animals and was analyzed for NO2- following reduction of nitrate to nitrite. Plasma/serum isolated from SGVHD mice contained increased levels of NO relative to normal or transplant control animals (Fig. 2A , P=0.0011; Fig. 2B , P=0.0033).



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  		Figure 1. Increased levels of iNOS mRNA in the colon of SGVHD mice. C3H/HeN mice were induced to develop SGVHD as described. Two to three weeks after cessation of CsA therapy, tissue isolated from the distal colon was analyzed by RT-PCR for the presence of mRNA for iNOS. Data presented are average ± SEM of four animals and are representative of four experiments. No difference was observed when tissue was isolated at 2 or 3 weeks after CsA therapy.

 


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  		Figure 2. Circulating NO is increased in SGVHD animals. Normal, control, and CsA-treated BMT (A) or AG-treated (B) recipient mice were bled 2–3 weeks after cessation of CsA therapy. Plasma/serum was collected, and nitrate was reduced to nitrite by nitrate reduction. The samples were then analyzed for the presence of NO2-. Data presented are average ± SEM of four animals and are representative of two experiments each.

 
In addition to increases in in vivo NO production, spleen cells were isolated from normal, control, and SGVHD mice and were stimulated in vitro with LPS. The supernatants from these cultures were analyzed for the presence of NO2-. As shown in Figure 3 , significant increases in NO2- were observed in the culture supernatants of spleen cells isolated from SGVHD mice relative to cells isolated from normal or control animals. This increase was observed over a wide range of LPS concentrations (0.1–10 µg/ml), suggesting that the macrophages from these animals were primed/activated as demonstrated by the ability to respond to low doses of LPS in vitro. These studies demonstrated a correlation between increased NO production and the development of SGVHD.



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  		Figure 3. Spleen cells from SGVHD mice produce increased levels of NO2- after in vitro stimulation with LPS. Spleens were removed from normal, control, and SGVHD mice 2–3 weeks after cessation of CsA therapy. Spleen cells were stimulated with varying concentrations of LPS, ranging from 10 to 0.1 µg/ml. After 72 h of culture, the supernatant was harvested and analyzed for the presence of NO2- as described. Data presented are representative of three experiments.

 
Inhibition of iNOS alters the development of SGVHD
To determine a functional relationship between increased NO (iNOS activity) and development of SGVHD, the iNOS inhibitor AG [44 , 46 ] was given to control BMT and CsA BMT mice. C3H/HeN mice were lethally irradiated and reconstituted with syngeneic BM and treated with CsA for 21 days as described. Beginning on the last day of CsA therapy, the mice were treated with AG, 8 mg/kg/day for 14 days [47 ]. Treatment of control and CsA-treated mice with AG for 2 weeks after the cessation of SGVHD prevented the development of clinical symptoms of SGVHD (P=0.0019; Fig. 4 ). The reduction in disease induction following AG treatment also correlated with a reduction in circulating NO levels (serum) in CsA-AG-treated mice versus CsA-treated mice that developed SGVHD (Fig. 2B) . The reduction of circulating NO demonstrated that AG treatment acted by inhibiting the production of NO via the iNOS enzyme. In addition, when the colon of these animals was observed for the development of the tissue pathology associated with murine SGVHD, a reduction in inflammation was observed. As shown in Figure 5 , a reduction in SGVHD-associated inflammation was observed in the colons of CsA-treated mice given AG to inhibit iNOS (Fig. 5C) relative to diseased animals (Fig. 5B) . Furthermore, a significant reduction in the grade of tissue pathology was observed in AG-treated versus diseased animals as well (Fig. 6 ; P=0.0083). Similar results were observed in the liver as well (data not shown). Finally, in the small percentage of CsA-AG animals that developed clinical symptoms of SGVHD (Fig. 4) , the pathology observed in the colon and liver was similar to that of CsA-SGVHD animals (data not shown).



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  		Figure 4. Inhibition of iNOS alters the development of SGVHD. C3H/HeN mice were induced for SGVHD as described. Beginning on the last day of CsA therapy, control and SGVHD mice were treated with AG for 14 days (8 mg/kg/day). The animals were observed for clinical symptoms of SGVHD, weight loss, and diarrhea. Data represent pooled data from two experiments.

 


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  		Figure 5. Treatment of CsA-treated mice with AG reduces SGVHD-mediated inflammation. C3H/HeN mice were induced for SGVHD and treated with AG as described. On the day after the last AG treatment, colon was removed from (A) control, (B) CsA-treated (SGVHD), and (C) CsA-AG. The tissue sections were fixed in formalin and stained with H&E. The sections presented were magnified to 250x (original magnification). Data are representative of tissue from four individual animals.

 


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  		Figure 6. Decrease in tissue pathology grade following AG therapy of SGVHD mice. Colon tissue from control, CsA-treated (SGVHD), AG-control, and AG-CsA-treated mice were graded for SGVHD pathology as previously described [42 ]. Data presented are average ± SEM of four mice.

 
AG therapy of SGVHD animals alters colonic cytokine production
Recent studies have shown that NO is involved in the production of IL-12 [48 ] and has been shown to participate in the production of IFN-{gamma} by IL-12-stimulated NK cells [49 ]. Thus, AG therapy and subsequent inhibition of NO may have immunoregulatory consequences that alter the cytokine milieu following CsA, resulting in a reduction or inhibition of SGVHD. Preliminary studies were performed to determine if inhibition of iNOS altered the TH1-like cytokine profile associated with murine CsA-induced SGVHD [35 ]. Mice were induced for SGVHD and treated with AG as described above. One day after the last AG treatment, the colons of control, control-AG-treated, CsA-treated, and CsA-AG-treated mice were removed, and RNA was isolated and analyzed by RT-PCR for the presence of iNOS, IL-12, and IFN-{gamma} mRNA. As demonstrated above, CsA-treated SGVHD animals had increased iNOS mRNA (data not shown) relative to control animals. In addition, these animals demonstrated increased levels of mRNA for IL-12 (Fig. 7A ) and IFN-{gamma} (Fig. 7B ; P<0.005), as previously described by Flanagan et al. [35 ]. It is interesting that treatment with the iNOS inhibitor AG significantly reduced the level of mRNA for IL-12 (P=0.0223) and IFN-{gamma} (P=0.0225) relative to that observed in SGVHD animals. These findings suggest that enhanced NO production was required for increases in these cytokines that are characteristic of the development of murine SGVHD, leading to a regulatory role for this molecule in this inducible disease.



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  		Figure 7. AG therapy of SGVHD mice resulted in decreased, colonic IL-12 mRNA production. C3H/HeN mice were induced for SGVHD and treated with AG as described. On the day after the last AG treatment, the colon was removed from control, CsA-treated (SGVHD), AG-control, and AG-CsA-treated. RNA was isolated from the tissue, and RT-PCR was performed for IL-12 and IFN-{gamma} mRNA. Data presented are pooled from two independent experiments demonstrating a reduction in cytokine mRNA following NO therapy of SGVHD mice. The data presented are the average ± SEM, and n represents the number of animals within each treatment group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
NO is a ubiquitous molecule that participates in the development and regulation of many pathologic and immune responses. Studies were therefore undertaken to determine if NO played a role in the development of murine CsA-induced SGVHD. Increases in circulating plasma NO and colonic iNOS mRNA were demonstrated in tissues isolated from SGVHD animals compared with controls. A reduction in the induction of clinical symptoms and tissue pathology associated with this disease was observed following treatment of SGVHD mice with the iNOS inhibitor, AG. These findings demonstrate for the first time a direct role for NO in the pathology of this disease.

NO has been associated with many normal, physiologic functions [2 , 4 5 6 ]. Production of NO in large amounts via iNOS has been associated with pathologic processes [7 , 8 ]. NO can interact with O2- to produce the highly toxic molecule peroxynitrite [8 ]. Peroxynitrite has the ability to mediate a number of cytotoxic processes, including lipid peroxidation that mediates tissue damage during inflammatory processes [9 , 10 ].

Many inflammatory diseases including inflammatory bowel disease [8 ], experimental autoimmune encephalomyelitis [47 ], and GVHD [24 25 26 ] have been shown to have increased NO production. Similarly, these GVHD mice had increased circulating NO and iNOS mRNA in the target organs relative to control animals. The use of NO synthase inhibitors has been successful at altering the development of allogeneic GVHD [28 , 29 ]. Decreased levels of NO3- and NO2- were found in allogeneic BMT animals treated with NOS inhibitors. Furthermore, Haddad et al. [50 ] demonstrated an increase in the levels of peroxynitrite in the lungs of mice with interstitial pneumonitis following allogeneic BMT. Finally, treatment of GVHD mice with NMA eliminated the mucosal pathology associated with GVHD [28 ]. This treatment had no effect on the splenomegaly that developed following induction of GVHD and did not affect TNF-{alpha} production by activated macrophages in vitro. However, it is interesting that NMA treatment had a negative effect on enhanced NK activity of cells isolated from GVHD mice. This finding may be in line with recent findings that NO is involved in the production of IFN-{gamma} by NK cells via an IL-12-mediated mechanism [49 ].

The development of murine SGVHD has been associated with the activation of NK cells and macrophages and the production of increased levels of IL-12, IFN-{gamma}, and TNF-{alpha} [35 , 39 ]. In vivo neutralization of the macrophage-associated cytokines, IL-12 [35 , 39 ] and TNF-{alpha} [39 ], or depletion of NK cells abrogated the development of SGVHD [35 ]. Thus, activated macrophages and NK cells appeared to be central to the development of this disease. In addition, activated macrophages have the potential to produce large amounts of NO via the iNOS enzyme [1 ]. Furthermore, significant pathology is associated with the development of syngeneic and allogeneic GVHD. As NO participates in the intestinal pathology of allogeneic GVHD, studies were performed to determine if NO participated in the development of pathology associated with murine SGVHD.

Significant increases in the levels of NO and iNOS mRNA were found in the plasma/serum and target tissues of SGVHD mice. To determine if the increases in NO were relevant to the development of the pathology associated with SGVHD, the iNOS inhibitor AG was used. If NO was in part responsible for the SGVHD pathology, then inhibition of iNOS should have altered the course of SGVHD following cessation of CsA therapy. The iNOS inhibitor AG was chosen for these studies, as it has been shown to be relatively specific for the iNOS enzyme [29 ]. AG therapy ameliorated the CsA-induced development of SGVHD. There was a reduction in the development of clinical symptoms, circulating NO, and the tissue pathology associated with SGVHD. At this time, it is unclear as to whether the inhibition of disease is related to reduced cytotoxic processes or reduced inflammatory cytokine production associated with the reduced production of NO in the AG-treated SGVHD mice. The former cannot be ruled out, however, and future studies analyzing the production of peroxynitrite in tissue samples will have to be performed to address that issue.

Conversely, preliminary experiments were performed to address the second of these possibilities. Tissues isolated from SGVHD animals treated with AG demonstrated a decrease in mRNA for IL-12 and IFN-{gamma}. These studies suggest that NO is required for the production of cytokines that drive the disease process. This finding is consistent with the studies of Mullins et al. [48 ], demonstrating that paclitaxel (Taxol) treatment of macrophages induces the production of IL-12 through a NO-dependent mechanism [48 ]. Furthermore, NO has recently been shown to participate in the production of IFN-{gamma} by IL-12-stimulated NK cells [49 ]. Failure to produce NO may result in reduced IL-12 and the failure to activate NK cells to produce IFN-{gamma}, both of which are requirements for this disease. Alternatively, it may be that IL-12 and IFN-{gamma} are required for the production of NO. The production of NO may then feed back and further enhance the production of inflammatory cytokines (IL-12, IFN-{gamma}) associated with the development of SGVHD. Thus, based on the data presented, it is unclear at this time whether NO acts upstream in the induction of IL-12 and IFN-{gamma}. However, it should be noted that significant increases in iNOS mRNA are observed by days 7–14 post-BMT (unpublished data) prior to increases in IL-12 and IFN-{gamma} (days 21–28 post-BMT), supporting the first of these possible roles for NO in the regulation of cytokine production in this model.

Thus, whether through cytotoxic properties or regulatory functions, NO plays a significant role in the development of pathology associated with murine SGVHD. Finally, it should be mentioned that in the preliminary studies described above, treatment with AG did not completely abrogate the production of NO, suggesting the involvement of other mechanisms such as CD40-CD40L interactions. The ligation of CD40L on macrophages has been shown to be involved in the production of IL-12 by macrophages [51 ]. The role of NO and CD40-CD40L interactions in the production of IL-12 during the development of SGVHD is currently under investigation.


   ACKNOWLEDGEMENTS
 
This work was supported by NCI grant CA74228. D. M. F. was supported by NCI Training Grant CA09509.

Received March 3, 2002; revised May 20, 2002; accepted June 2, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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J. S. Bryson, L. Zhang, S. W. Goes, C. D. Jennings, B. E. Caywood, S. L. Carlson, and A. M. Kaplan
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