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The Journal of Immunology, 1999, 163: 1170-1177.
Copyright © 1999 by The American Association of Immunologists

Th1 Cytokines and NK Cells Participate in the Development of Murine Syngeneic Graft-Versus-Host Disease1

Diana Lowery Flanagan*, C. Darrell Jennings{dagger} and J. Scott Bryson2,*,{ddagger}

* Department of Microbiology and Immunology, {dagger} Department of Pathology, and {ddagger} Blood and Marrow Transplant Program, Division of Hematology/Oncology, Markey Cancer Center, University of Kentucky, Lexington, KY 40536


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Syngeneic graft-vs-host disease (SGVHD) is induced by reconstituting lethally irradiated mice with syngeneic bone marrow cells followed by a short course of therapy with the immunosuppressive agent cyclosporine A. Following cessation of cyclosporine A therapy, animals develop clinical symptoms of SGVHD: weight loss, runting, and diarrhea. While it has been suggested that T cells are responsible for the induction and effector phases of SGVHD, the role of nonspecific effector cells and cytokine mediators has yet to be examined in the disease process. Mice with SGVHD had increased levels of message for IL-12p40, IFN-{gamma}, and TNF-{alpha} in the target organs of SGVHD as compared with transplant controls and asymptomatic cyclosporine A-treated mice. Concomitant with the increase in Th1 cytokines was an enhanced cellular infiltrate in the target organs of SGVHD mice as determined by histological analysis. To directly examine the role of IL-12 in the development of SGVHD, in vivo neutralization of IL-12 was performed. Treatment of mice with Abs to IL-12 inhibited SGVHD-mediated tissue pathology and mortality. Because IL-12 has been shown to activate both T cells and NK cells to secrete IFN-{gamma} and to become more cytolytic, studies were initiated to ascertain which lymphocyte populations play a role in the development of murine SGVHD. Depletion of NK cells inhibited clinical symptoms of SGVHD. In contrast, T cell depletion did not alter the disease process. Therefore, these findings collectively demonstrate a role for IL-12 and NK cells in the effector phase of murine SGVHD.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cyclosporine A (CsA)3 is an immunosuppressive drug used clinically to prevent graft rejection following solid organ transplantation and to prevent graft-vs-host disease (GVHD) following allogeneic bone marrow transplantation. However, Glazier et al. described a syndrome in rats where a short course of CsA therapy following syngeneic bone marrow transplantation induced a GVHD-like syndrome termed syngeneic GVHD (SGVHD) (1). CsA has also been shown to induce autologous GVHD in humans (2, 3, 4, 5, 6, 7) and SGVHD in mice (8, 9, 10). Three criteria have been shown to be essential for the development of SGVHD. First, CsA was required for the induction of SGVHD (1). Second, the recipient animals required a thymus for disease development (thymectomized animals did not develop SGVHD following CsA treatment) (11). Finally, irradiation to eliminate a peripheral regulatory mechanism in the recipient was required for optimal disease induction (12, 13).

CsA treatment in rats and mice has been shown to result in the destruction of thymic architecture and complete ablation of the medullary region (14, 15). This observation has led to experiments that demonstrated that clonal deletion of T cells in the thymus was inhibited allowing for the presence of self-reactive T cells in the periphery of mice receiving CsA treatment (8, 16). These results suggested that autoreactive T cells that could potentially function as effector cells in the development and pathology associated with SGVHD were present in CsA-treated mice (8, 16). However, Bryson et al. later reported that the appearance of these forbidden clones did not necessarily correlate with the development of SGVHD (17).

The role of T cells as effector cells has been extensively studied in the rat model of SGVHD. SGVHD has been adoptively transferred with T cells from diseased animals into irradiated recipients (18). Both CD4+ and CD8+ cells have been demonstrated to participate in the disease process (16, 19). Recently, class II-reactive CD8+ T cells specific for the class II-associated invariant chain peptide have been implicated as the autoreactive cells responsible for SGVHD in the rat model (20). However, much less is known about the effector cells contributing to the development of SGVHD in the murine model.

While much research has focused on T cells as effector cells, little research has focused on potential cytokine mediators in SGVHD. The role of cytokines in GVHD following allogeneic bone marrow transplantation has been well documented. In general, Th1 cytokines (IL-2, IFN-{gamma}, IL-12) have been associated with acute GVHD and Th2 cytokines (IL-4, IL-10) have been associated with chronic GVHD (21, 22, 23). Furthermore, acute and chronic allogeneic GVHD can be altered by treatment with cytokines or cytokine agonists. Via et al. and Williamson et al. reported that injections of rIL-12 converted chronic GVHD to acute GVHD (24, 25). Neutralization of IL-12 inhibited acute GVHD (25). Conversely, injection of either high-dose IL-12, IL-2, or IFN-{gamma} has been reported to inhibit acute GVHD following allogeneic bone marrow transplantation (26, 27, 28). These observations further demonstrate the essential role of regulatory cytokines in the development of the two forms of allogeneic GVHD. Similarly, IL-12 has been shown to participate in the development of Th1 immune responses in both infectious and autoimmune diseases (29).

To determine the role of cytokines and nonspecific effector cells in the development of murine SGVHD, tissues from SGVHD target organs were analyzed by RT-PCR for Th1 cytokine mRNA. Mice exhibiting clinical symptoms for SGVHD had higher levels of mRNA for IL-12, IFN-{gamma}, and TNF-{alpha} as compared with control mice. Corresponding with increased Th1 cytokines was an increased cellular infiltrate into the target organs of diseased animals. Moreover, in vivo neutralization of IL-12 inhibited the development of murine SGVHD, demonstrating a role for IL-12 in disease development. Because IL-12 has been demonstrated to activate both T cells and NK cells, studies were performed to assess the role of these cell populations in the development of murine SGVHD. Depletion of NK cells in vivo inhibited the disease process; whereas, depletion of T cells did not inhibit SGVHD. Collectively, these data suggest a role for IL-12 and NK cells in the development of murine SGVHD.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6, C3H/HeN (Harlan Sprague-Dawley, Indianapolis, IN), and DBA/2 (Jackson Laboratories, Bar Harbour, ME) were purchased at 20–22 days of age and were used within 1 wk of arrival. The mice were given autoclaved acidified water and lab food ad libitum and were housed in sterile microisolator boxes (Lab Products, Maywood, NJ).

Induction of SGVHD

Bone marrow was isolated from the femurs and tibias of 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 (30). Recipient mice were lethally irradiated (C57BL/6, 9 Gy; C3H/HeN, 9 Gy; DBA/2, 9.5 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 bone marrow cells i.v. via the tail vein. Starting on the day of transplantation, groups of animals were treated i.p. with either CsA 15 mg/kg or diluent, either olive oil (OO) or PBS, 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 wk after cessation of CsA therapy. Tissue samples were taken from the animals 2 wk 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, 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 (31) (colon: +/-, rare crypt cell necrosis; 1+, definite scattered single-cell necrosis in crypts; 2+, several necrotic cells in gland, crypt abcesses present; 3+, confluent destruction of glands; 4+, loss of mucosa with formation of granulation tissue and pseudomembrane; liver: +/-, minimal lymphocyte infiltrate in portal area; rare bile duct epithelial cell degeneration; 1+, sparse, but definite portal lymphocyte infiltrate; occasional necrotic bile duct epithelial cell; 2+, diffuse infiltrate in portal area and invasion of bile ducts by inflammatory cells; 3+, heavy infiltrate partially obscuring bile ducts and focal bile duct destruction; 4+, all the above plus secondary changes in hepatocytes, bile stasis, hepatocyte necrosis, and disordered architecture).

RT-PCR for cytokine message

Total RNA was isolated from the colon, liver, and kidneys using Trizol reagent (Life Technologies). One microgram of RNA from each group was reverse transcribed into cDNA using a reverse transcription system (Promega, Madison, WI). PCR reactions were then performed using primers for {beta}-actin, IFN-{gamma}, TNF-{alpha} (Stratagene, La Jolla, CA), and IL-12 p40 (32). Before use, conditions for each set of primers were optimized, and the number of PCR cycles was titrated. PCR conditions for {beta}-actin, IFN-{gamma}, and TNF-{alpha} were as follows: 5 min denaturing step at 94°C, 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C, and a 7 min final extension at 72°C. The PCR conditions for IL-12 p40 were similar except the denaturing step was set at 95°C. PCR reactions for TNF-{alpha}, IFN-{gamma}, and IL-12 p40 were allowed to cycle 30 times; whereas, {beta}-actin was allowed to cycle 25 times. Expected sizes of the PCR products for {beta}-actin, TNF-{alpha}, IFN-{gamma}, and IL-12 p40 were 245 bp, 276 bp, 405 bp, and 404 bp, respectively. Aliquots of each PCR reaction were mixed with sample buffer and separated on a 1.5% agarose gel. The gel was then stained with ethidium bromide and visualized upon exposure to ultraviolet light. Pictures of the agarose gels were taken and scanned (Bio Image; Kodak, Rochester, NY). The images were analyzed using the VISAGE electrophoresis gel analysis system (Millipore, Bedford, MA). OD values for each individual fragment were normalized to {beta}-actin expression from the same tissue fragment.

ELISA for cytokine presence in plasma

Blood from SGVHD animals and control animals was collected in heparin-coated syringes from metafane-anesthetized mice. The plasma was collected and analyzed for the presence of IL-12, IFN-{gamma}, and TNF-{alpha} using ELISA kits (Endogen, Woburn, MA).

In vivo neutralization of IL-12

To determine the effects of IL-12 neutralization on the development of SGVHD, C57BL/6, C3H/HeN, and DBA/2 mice were induced for SGVHD as described previously. At day 21 post-transplantation, groups of animals received 1 mg of C15.6 and C15.1 ascites (kindly provided by Dr. Giorgio Trinchieri, The Wistar Institute, Philadelphia, PA) or 1 mg of SFR8 ascites (HB-152; American Type Culture Collection (ATCC), Manassas, VA), a rat isotype control Ab, daily for 3 days then every other day for 3 wk. One milligram/injection of C15.6 and C15.1 i.p. was sufficient to prevent LPS-induced mortality in C57BL/6 mice as described by Wysocka et al. (33) (data not shown). Mice were followed for induction as described.

In vivo depletion of NK cells and T cells

To analyze the potential role of different effector cell populations in murine SGVHD, C57BL/6 mice were induced for SGVHD as previously described. At day 21 post-transplantation, groups of mice received either 1 mg of PK136 (anti-NK1.1) ascites (HB-191; ATCC), 1 mg 116-13.1 (isotype control) ascites (HB-129; ATCC), 1 mg GK1.5 ascites (TIB-207; ATCC), and 1 mg 2.43 ascites (TIB-210; ATCC) or 53-6.72 (anti-CD4, anti-CD8) ascites (TIB-105; ATCC), or 1 mg rat IgG (isotype control; Life Technologies) daily for 1 wk. The mice were then weighed and followed for induction of SGVHD.

Flow cytometry analysis

To determine the number of NK cells in the spleen of control and diseased mice, the spleens were removed and single-cell suspensions were prepared. The spleen cell suspensions were depleted of erythrocytes by lysis with 0.83% Tris-buffered ammonium chloride, then washed twice in RPMI 1640 containing 5% FCS. Spleen cells (1 x 106) were incubated with PE-conjugated anti-NK1.1 mAb, PK136 (PharMingen, San Diego, CA). Surface expression was analyzed on a FACStar flow cytometer (Becton Dickinson, San Diego, CA) using the LYSIS program (Becton Dickinson). Values shown are percent positive cells in the lymphocyte-gated cell population.

To assess the efficacy of NK and T cell depletion in vivo, spleens cells from transplant control mice were isolated the day after the last Ab injection and stained with PE-conjugated anti-NK1.1 Ab or PE-conjugated anti-CD4. On average, only 2.5% positive cells remained in the spleens of NK or T cell-depleted mice. In addition, no NK activity was detected in mice depleted of NK cells as determined by a standard cytotoxicity assay against NK-sensitive target cells, and no proliferative response was detected in mice depleted of T cells in response to T cell mitogens in vitro (data not shown).

Cytoxicity assay

NK cell activity was measured using a standard 4-h 51Cr-release assay. YAC-1 (NK sensitive) and P815 (NK resistant) tumor cell lines were labeled with 200 µCi of 51Cr for 1 h at 37°C. The target cells were washed, and 1 x 104 cells were added to varying numbers of spleen cells from control and CsA-treated animals in 96-well round-bottom plates. The plates were incubated for 4 h at 37°C. The supernatant was harvested from each well using a Skatron harvesting system (Skatron, Sterling, VA) and counted in a gamma counter. Percent cytotoxicity was calculated as (experimental release - spontaneous release)/(total release - spontaneous release) x 100. All assays were performed in triplicate.

Statistical analysis

Statistical analysis was performed using the Student’s t test, Mann-Whitney rank sum test, or Fischer’s exact test. Groups with values of p <= 0.05 were considered to be statistically different.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enhanced levels of Th1 cytokines in SGVHD mice

Because cytokines play a significant role in GVHD following allogeneic bone marrow transplantation (21, 22, 23), studies were performed to analyze the role of cytokines in murine SGVHD. Initial studies did not detect Th2 cytokine mRNA when clinical symptoms of SGVHD were apparent (data not shown). Consequently, the role of Th1 cytokines in the disease process was assessed. C3H/HeN mice were induced for SGVHD, and mice that developed clinical symptoms (weight loss and diarrhea) of SGVHD, asymptomatic CsA-treated mice, and transplant control mice were euthanatized and the colon and liver were examined by RT-PCR for IL-12 p40, IFN-{gamma}, and TNF-{alpha}. mRNA for the p40 subunit of IL-12 was chosen for analysis because the p40 subunit has been demonstrated to be up-regulated when IL-12 is secreted, while the p35 subunit is constitutively expressed (34, 35). As demonstrated in Fig. 1GoA, increased levels of mRNA for IL-12 p40, IFN-{gamma}, and TNF-{alpha} were observed in the colons of mice developing clinical symptoms for SGVHD as compared with transplant control mice or asymptomatic CsA-treated mice (p < 0.05). Similar results were also found in the liver, and no significant differences in cytokine mRNA expression were observed between control, diseased, or asymptomatic animals in a non-target organ, the kidney (data not shown). Finally, the expression of these cytokines was not strain specific, because increased levels of mRNA for IL-12 p40, IFN-{gamma}, and TNF-{alpha} were observed in C3H/HeN, C57BL/6, and DBA/2 mice induced for SGVHD (data not shown).



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  		FIGURE 1. SGVHD mice with elevated levels of Th1 cytokine mRNA have increased SGVHD tissue pathology. A, C3H/HeN mice were induced for SGVHD as described. Pieces of the colon were removed from PBS, CsA-symptomatic, and CsA-asymptomatic mice 2–3 wk post-CsA treatment when clinical symptoms (diarrhea and weight loss) were present, and message levels were analyzed by RT-PCR. The data shown is normalized to {beta}-actin and pooled from three transplants. CsA-symptomatic mice had increased levels of Th1 cytokine mRNA as compared with CsA-asymptomatic mice and diluent-treated mice, p < 0.05. The numbers in parentheses represent the number of mice in each group. B, Circulating levels of IL-12 and IFN-{gamma} are elevated in SGVHD mice. DBA/2 mice were induced for the development of SGVHD. Two weeks post-CsA treatment, three mice per treatment group, OO and CsA, were bled. The plasma was collected and analyzed for the presence or IL-12 p70 or IFN-{gamma} by ELISA. Values shown are pg/ml. C, Sections of the colon and liver were removed from the same groups as described previously in A and stained with H&E. The sections were graded for SGVHD without knowledge of the treatment category. CsA-symptomatic mice had an increased SGVHD grade as compared with CsA-asymptomatic mice or diluent-treated mice, p < 0.01. The data shown is pooled from three separate transplants.

 
Because mRNA expression does not necessarily correlate with protein secretion, the plasma from SGVHD mice was analyzed for the presence of IL-12, IFN-{gamma}, and TNF-{alpha}. As can be seen in Fig. 1GoB, SGVHD mice had elevated plasma levels of IL-12 and IFN-{gamma} 2 wk post-CsA treatment as compared with transplant control animals. No TNF-{alpha} was detected in the plasma of any group (data not shown). TNF-{alpha} has been routinely difficult to detect in the plasma (our unpublished observations), and its difficulty is most likely due to the presence of numerous TNF-{alpha} receptors that bind the circulating TNF-{alpha}.

Tissue samples of the colon, liver, and kidney from transplant control, CsA-symptomatic, and CsA-asymptomatic C3H/HeN mice were also analyzed for pathology associated with murine SGVHD. Mice that developed SGVHD had a more severe histologic grade as compared with either transplant controls or CsA-treated asymptomatic mice (Fig. 1GoC; p <= 0.01). Concomitant with the ability to detect increased levels of message for IL-12 p40, IFN-{gamma}, and TNF-{alpha} was the ability to detect enhanced infiltration of inflammatory cells into the target organs of animals with SGVHD. Pathological changes paralleled the cytokine data and supported the role of Th1 cytokines in the development of murine SGVHD.

In vivo neutralization of IL-12

Because of the importance of IL-12 in the generation of Th1 immune responses, studies were performed to examine the effects of IL-12 neutralization on the development of SGVHD. Following syngeneic bone marrow transplantation and CsA therapy, groups of transplant control and CsA-treated DBA/2 mice received diluent, isotype control, or anti-IL-12 mAbs daily for 3 days, then, every other day for 3 wk. DBA/2 mice were used in these studies because they develop a more severe course of SGVHD resulting in higher percent induction, SGVHD grades, and mortality than C3H/HeN mice (9, 36). As seen in Fig. 2GoA, treatment with anti-IL-12 mAbs inhibited the development of SGVHD as compared with treatment with CsA (p < 0.001). Treatment with an isotype control Ab did not affect the induction of SGVHD (data not shown). In addition, anti-IL-12 treatment significantly reduced the inflammation in CsA-treated mice (Fig. 2Go, B–E; p = 0.02). Treatment with Ab against IL-12 did not simply delay the onset of SGVHD as anti-IL-12-treated mice followed 80 days post-transplantation did not develop clinical symptoms of SGVHD, and the effect was not strain specific, as neutralization of IL-12 in C3H/HeN mice inhibited the development of SGVHD as well (data not shown).



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  		FIGURE 2. In vivo neutralization of IL-12 inhibits the development and pathology of murine SGVHD. DBA/2 mice were induced for SGVHD as described. Day-21 post-transplant mice received diluent, SFR8, a rat isotype control Ab (not shown), or C15.6 and C15.1 ascites for 3 days then every other day for 3 wk. Animals were then observed for clinical symptoms of SGVHD. A, Data shown is percent induction pooled from three transplants. Numbers in parentheses represents the number of animals in each group. CsA-treated mice that received anti-IL-12 injections had a reduced induction of SGVHD as compared with CsA-treated mice that did not receive anti-IL-12 injections or transplant control mice, p < 0.01. B–D, Sections of the colon were removed from OO, OO-anti-IL-12, CsA, and CsA-anti-IL-12 mice 3 wk post-CsA from one of the three transplants described above and stained with H&E. The colon sections shown are representative samples of each group. B, Section of colon from OO animal demonstrating normal architecture (H&E; x50). C, Colon of CsA mouse exhibiting massive infiltration of inflammatory cells (H&E; x50). D, Colon of CsA-anti-IL-12 mouse demonstrating normal histology (H&E; x50). Sections of colons from OO-anti-IL-12-treated mice demonstrated normal colon architecture (data not shown). E, CsA-treated mice that received IL-12 Ab had a reduced SGVHD grade as compared with CsA-treated mice that received no IL-12 Ab, p = 0.02.

 
Enhanced levels of NK activity in CsA-treated mice

The critical role that IL-12 plays in the development of murine SGVHD suggested that IL-12 could potentially activate an effector cell population, because IL-12 has been demonstrated to activate both T cells and NK cells to secrete IFN-{gamma} and to become more cytolytic (34, 37, 38). In attempts to study primary autoreactive T cell responses in mice induced for SGVHD, enhanced NK activity was detected in the spleens of mice induced for the development of SGVHD 1 wk after CsA therapy as monitored by 51Cr-release assay (Fig. 3GoA). An increase in NK cytotoxicity of ~2-fold was consistently observed during the first 2 wk post-CsA treatment (data not shown). In agreement with previously published findings by Kosugi et al. (39), increased NK cell function paralleled increased percentages of NK cells in the spleens of CsA-treated mice as compared with transplant controls (Fig. 3GoB; p = 0.03). When total NK cell number was compared between CsA-treated mice (1.6 x 107/spleen) and transplant control mice (4.1 x 106/spleen), CsA-treated mice had increased numbers of NK cells (p = 0.006).



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  		FIGURE 3. NK cell activity and numbers are elevated in CsA-treated mice following syngeneic bone marrow transplantation. A, C3H/HeN mice were induced for SGVHD. Spleen cells were isolated from normal C3H/HeN and OO (transplant control)- and CsA-treated mice 1 wk post-CsA treatment and assayed for NK cell activity in a 4-h 51Cr-release assay. Values are shown as average percentage cytotoxicity ± SEM of triplicate samples. The numbers in parentheses represent the number of mice analyzed per treatment group, p <= 0.01. On average, an ~2-fold increase was observed during the first 2 wk post-CsA treatment from six independent transplants (data not shown). B, C57BL/6 mice were induced for SGVHD. Spleen cells were isolated from normal C57BL/6 and PBS- and CsA-treated mice on day 21 post-transplantation and analyzed by flow cytometry for the expression of the NK cell marker, NK1.1. The data shown is the percent NK1.1+ cells ± SEM. CsA-treated mice had increased percentages of NK1.1+ cells as compared with diluent-treated control mice, p = 0.03.

 
Depletion of NK cells inhibits murine SGVHD

To determine whether increased NK cell activity was functionally significant in the disease model, in vivo cell depletion studies were used. C57BL/6 mice were induced for SGVHD as described, and groups of mice received either i.p. injection of PK136 mAb (anti-NK1.1) or 116–13.1 mAb (isotype control) and were monitored for the induction of SGVHD. As demonstrated in Fig. 4Go, PK136 therapy inhibited the development of murine SGVHD, implicating NK cells in the disease process. Finally, anti-asialo-GM1 treatment in C3H/HeN mice inhibited the induction of SGVHD (50% reduction), demonstrating that the role for NK cells in the development of murine SGVHD was not strain specific (data not shown).



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  		FIGURE 4. In vivo depletion of NK1.1+ cells, but not T cells, inhibits the induction of SGVHD. C57BL/6 mice were induced for SGVHD. Groups of CsA-treated and diluent-treated mice were injected with 1 mg of anti-NK1.1 (PK136) ascites or 1 mg of anti-CD4 and anti-CD8 ascites daily for the first week post-CsA treatment. The mice were then followed for clinical symptoms of SGVHD. The induction data shown for the anti-NK1.1 treatment is pooled from three transplants, whereas the induction data for the anti-T cell treatment is pooled from two transplants. The numbers in parentheses represent the number of mice in the group. Treatment of CsA mice with anti-NK1.1 inhibited the induction of SGVHD as compared with untreated CsA mice, p < 0.05.

 
To examine whether or not T cells were the effector cell population activated by IL-12 to cause SGVHD, C57BL/6 mice were induced for SGVHD and treated with Abs against CD4 and CD8. Depletion of CD4+ and CD8+ T cells in CsA-treated mice did not alter the induction of the disease (Fig. 4Go), suggesting that conventional T cells do not play a role in the development of murine SGVHD. Treatment with rat IgG (isotype control) did not alter the disease process, further supporting the specificity of the in vivo depletion therapy (data not shown).

Th1 cytokine message in the colon of anti-NK1.1-treated mice

SGVHD mice exhibiting clinical symptoms 2 wk post-CsA had increased levels of message for Th1 cytokines (IL-12, IFN-{gamma}, TNF-{alpha}; Fig. 1GoA). Because NK depletion inhibited the development of SGVHD (Fig. 4Go), the effects of in vivo NK cell depletion on the presence of Th1 cytokine mRNA were evaluated. C57BL/6 mice were induced for SGVHD and treated with diluent or anti-NK1.1 Ab. Pieces of the colon were removed from PBS-, PBS-PK136-, CsA-, and CsA-PK136-treated mice and analyzed by RT-PCR for Th1 cytokine mRNA expression. As shown in Fig. 5Go, depletion of NK1.1+ cells reduced the levels of mRNA for IFN-{gamma} in CsA-treated mice as compared with SGVHD controls (p = 0.001). However, depletion of NK1.1+ cells (NK cells) did not affect either IL-12 or TNF-{alpha} mRNA expression, which was likely produced by the NK1.1- cell population, i.e., macrophages. These results demonstrated that IFN-{gamma} mRNA is reduced by anti-NK1.1 treatment, suggesting that NK1.1+ cells are the effector cells responsible for IFN-{gamma} production in SGVHD.



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  		FIGURE 5. In vivo depletion of NK1.1+ cells affects IFN-{gamma} mRNA expression. C57BL/6 mice were induced for SGVHD and treated with anti-NK1.1 (PK136) ascites as described. Pieces of the colon were removed from normal C57BL/6 and PBS-, PBS-PK136-, CsA-, and CsA-PK136-treated mice, and message levels for IFN-{gamma}, IL-12, and TNF-{alpha} were analyzed by RT-PCR. The data shown are normalized to {beta}-actin expression ± SEM. IFN-{gamma} mRNA expression was reduced in CsA-treated mice receiving anti-NK1.1 injections as compared with mice receiving only CsA, p = 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented in this manuscript is the first to report a role for Th1 cytokines and NK cells in the development of murine SGVHD. We have demonstrated increased mRNA expression for IL-12 p40, IFN-{gamma}, and TNF-{alpha} in the target organs of mice with clinical symptoms of SGVHD. In addition, increased numbers of inflammatory cells within the target organs of SGVHD mice paralleled the increased levels of Th1 cytokine mRNA. Direct evidence for the role of IL-12 in the disease process was demonstrated when neutralizing Abs to IL-12 inhibited the development of SGVHD. Finally, depletion of NK cells in vivo during the first week post-CsA prevented the induction of SGVHD; whereas, T cell depletion had no effect on the disease process.

IL-12 has been shown to be a critical factor in the differentiation of Th1 and Th2 immune responses and to play a pivotal role in allogeneic GVHD (21, 22, 23, 24, 25, 26, 27, 28, 29). When secreted by activated macrophages (34), IL-12 activates both NK cells and T cells to produce cytokines (IFN-{gamma}, TNF-{alpha}, etc.) and to become more cytotoxic (34, 37, 38). In nonirradiated P -> F1 semiallogeneic transplant models, injections of rIL-12 converted chronic GVHD (Th2 response) to acute GVHD (Th1 response) with a concomitant increase in the levels of IFN-{gamma} (24, 25). Moreover, Williamson et al. further demonstrated that weight loss and mortality associated with acute GVHD could be inhibited by anti-IL-12 treatment, and that injection of rIL-12 exacerbated the disease (25). Finally, Kichian et al. were able to detect IL-12 p40 and IFN-{gamma} expression in the target organs of mice with acute GVHD, further demonstrating the importance of IL-12 in the development of acute GVHD following allogeneic bone marrow transplantation (35). Paradoxically, high-dose administration of rIL-12 has been reported to inhibit the development of acute GVHD in lethally irradiated allogeneic models (26). The data presented in this manuscript from the murine model of SGVHD paralleled the allogeneic models of acute GVHD involving no conditioning in that increases in IL-12 could be detected in the target organs of SGVHD mice (Fig. 1GoA) and neutralization of IL-12 inhibited the development of SGVHD (Fig. 2GoA).

The ability to maintain a balance between Th1 and Th2 cytokines has also been demonstrated to be critical in the prevention of autoimmune diseases. IL-10 knockout mice develop clinical symptoms and pathological lesions similar to what is observed in the colons of SGVHD mice (40). Neutralization of IL-12 or treatment with rIL-10 in IL-10 knockout mice inhibited the development of colitis (41). Similar observations were reported in IL-2 knockout mice (42). Increases in IL-12 were found in the colon of IL-2 knockout mice and neutralization of IL-12 also inhibited the inflammation associated with colitis (43). In both models, the inflammation in the colon is believed to occur because of an uncontrolled Th1 response (41, 43). In the current report, increased levels of IL-12, IFN-{gamma}, and TNF-{alpha} mRNA were observed in the target organs of SGVHD mice and correlated with increased inflammation in the target organs (Fig. 1Go). However, Th2 cytokines (IL-4 and IL-10) were not detected by RT-PCR at the time when clinical symptoms were present in SGVHD mice (data not shown). Finally, neutralization of IL-12 inhibited both the development of SGVHD and the inflammation in the colons of CsA-treated mice, demonstrating a role for IL-12 in the disease process (Fig. 2Go).

In addition to the importance of IL-12 in the development of murine SGVHD, this report also demonstrated a role for NK cells in the disease process. In vivo depletion of NK cells inhibited the development of murine SGVHD (Fig. 4Go). NK1.1-depleted CsA-treated mice had reduced levels of IFN-{gamma} mRNA in the colon as compared with transplant controls and SGVHD mice, suggesting that the IFN-{gamma}-producing effector cell population had been removed (Fig. 5Go). No differences were detected in IL-12 p40 or TNF-{alpha} mRNA in NK1.1-depleted CsA-treated mice as compared with transplant controls and SGVHD mice. This observation is not surprising as the production of these cytokines is most likely due to macrophages and not NK cells (34). Kosugi et al. have reported enhanced NK cell activity in the spleens of CsA-treated syngeneic bone marrow transplanted mice (39). The increased cytotoxic activity was mediated by the NK1.1+ Thy1- cell population. In addition, increased percentages of NK1.1+ cells were detected in the spleens of CsA-treated mice as compared with transplant control mice. This data was confirmed and extended by demonstrating a functional role for NK cells in the development of murine SGVHD (Fig. 4Go). Depletion of NK cells inhibited the development of murine SGVHD, implicating NK cells as having an effector function in the disease process.

Studies in the rat model have implicated both CD4+ and CD8+ T cells in the development of SGVHD (18, 19). Specifically, it was demonstrated that CD8+ class II-restricted T cells reactive against the class II-associated invariant chain peptide were the effector cells responsible for the development of rat SGVHD (20). In contrast, T cell depletion in the murine model did not inhibit the disease process, suggesting that conventional T cells are not the effector cells of murine SGVHD. In a previously published report, T cell clones isolated from mice with SGVHD were able to mediate a local graft-vs-host-like reaction when injected into the footpad of sublethally irradiated mice (44). Upon further examination of these clones, it was determined that all of the clones were of the Th2 phenotype and secreted IL-4 and IL-10 when activated (our unpublished observations). Consequently, in the murine disease, conventional T cells may play more of a regulatory role in the disease, and studies are ongoing to determine whether there is a shift to a Th2 profile in mice that recover from SGVHD.

The data presented in this report demonstrates a role for Th1 cytokines and NK cells in the development of murine SGVHD. Collectively, this data suggests a model for the development of the effector phase of murine SGVHD that can be tested in future studies. The secretion of IL-12 (macrophages) can activate the effector cell population (NK cells). The effector cells can then become cytolytic and directly cause tissue damage as well as secrete IFN-{gamma} that would feedback and enhance the secretion of IL-12 and other proinflammatory mediators, thereby amplifying the response.


   Acknowledgments
 
We thank Dr. Alan Kaplan for helpful discussion and careful review of this manuscript.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant AI31998 (to J.S.B.) and a Translational Research Grant from the Leukemia Society of America (to J.S.B.). Back

2 Address correspondence and reprint requests to Dr. J. Scott Bryson, Blood and Marrow Transplant Program, Division of Hematology/Oncology, Markey Cancer Center, University of Kentucky, Lexington, KY 40536-0093. E-mail address: Back

3 Abbreviations used in this paper: CsA, cyclosporin A; GVHD, graft-vs-host disease; SGVHD, syngeneic GVHD; OO, olive oil; H&E, hematoxylin and eosin. Back

Received for publication February 22, 1999. Accepted for publication May 12, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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