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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. Inhibition of Nitric Oxide Production Is Associated With Enhanced Weight Loss, Decreased Survival, and Impaired Alloengraftment in Mice Undergoing Graft-Versus-Host Disease After Bone Marrow Transplantation By William R. Drobyski, Carolyn A. Keever, Gerald A. Hanson, Timothy McAuliffe, and Owen W. Griffith The pathophysiologic role of nitric oxide (NO) in graft-versus-host disease (GVHD) was investigated in a murine bone marrow (BM) transplantation modelwhere donor and recipient were H-2-matched but differed at multiple minor histocompatibility antigens. Host AKR/J (H-2K)mice received lethaltotalbodyirradiation as pretransplant conditioning followed by transplantationof donor B1O.BR (H-2K)BM cells with or without spleen cells as a source of GVH-reactive T cells. NO production, as assessed by serum nitrate and nitrite levels, was increased for up t o 3 weeks posttransplant in animals undergoing both moderate and severe GVHD. Administration of NO-methyl-L-arginine (L-NMA), an inhibitor of nitric oxide synthase, t o animals undergoing GVHD resulted in effective suppression of NO production when compared with saline-treated GVHD control animals. Suppression of NO production by L-NMA in GVHD animals was associated with enhanced weight loss early posttransplant and decreased overall survival. Histologic analysis of tissues from L-NMA-treated and saline-treated GVHD animals showed thatearly weight loss was notbecause of anexacer- bation of GVHD, indicating that NO did not appear t o play an immunosuppressive role in this experimental model. LNMA-treated animals with enhanced weight loss were observed t o have splenic atrophy, decreased extramedullary hematopoiesis, and a reduction in BM cellularity when compared with GVHD control mice that were weight-matched before transplant.Analysis of T-cell chimerism in the spleen showed thatL-NMA treatment impaired donorT-cell repopdation. In vitro colony-forming unit (CFU) assays were performed t o further assess the role of NO on BM progenitor cell growth. L-NMA added directly into culture had no effect on CFU-granulocyWmacrophage (CFU-GM) formation in normal murineBM. In contrast, total CFU-GM from L-NMAtreated animals were significantly reduced when compared with GVHD controls or BM control animals who did notdevelop GVHD. Collectively, these data indicatethat inhibition of NO impairs hematopoietic reconstitution and supportthe premise that NO appears to play a novel role in thefacilitation of alloengraftment posttransplant. 0 1994 by The American Societyof Hematology. RAFT-VERSUS-HOST disease (GVHD) is a major cause of morbidity and mortality in patients undergoing allogeneic bone marrow transplantation (BMT). GVHD is a complex pathophysiologic process that results from the cooperative interaction of multiple effector cell populations resident in the donor graft and persistent in the host after conditioning.’ Although still incompletely understood, several fundamental tenets of GVH reactivity have been established using experimental animal models and clinical studies in humans. A central requirement for donor-derived T cells in the pathogenesis of GVHD has been substantiated by studies in which removal of T cells from the donor BM inoculum has resulted in the amelioration or complete prevention of the GVHD ~yndrome.’.~ Similarly, immunosuppressive agents designed to inhibit T-cell function and proliferation have been shown to mitigate GVHD.4 However, while these studies indicate that T cells appear to be necessary for the induction of GVH reactivity, accumulating data indicate that T cells only partially account for the pathophysiologic manifestations of GVHD. Recent data support the premise that additional elements of the immune network play an important role in GVHD. Secondary immune effector cell populations that are recruited by T cells appear to serve as more proximate mediators of GVHD-induced tissue damage in certain experimental settings. This hypothesis is supported by studies identifying natural killer cells histopathologically in areas of tissue damage5 and correlating natural killer cell activity with GVHD severity.6 Additionally, the pathologic discordance between the degree of lymphocytic infiltration and observed tissue damage has suggested that inflammatory mediators may also play a role in GVHD. Emerging data indicate that dysregulation of cytokine production is critical in the pathogenesis of GVHD andmay serve to amplify the immune response. Specific cytokines such as interleukin-l (IL-l),7tumor necrosis factor (TNF),8 and y-interferon (Y-IFN)~ have all been postulated to be mediators of the efferent phase of GVHD. Each of these cytokines has a diversity of immunomodulatory functions, and the mechanism by which they contribute to GVH reactivity has not been completely elucidated. Proposed common pathways by which cytokines might enhance GVHD have included the upregulation of class I1 antigen expression, the activation of additional effector cell populations, and the direct mediation of tissue damage. An alternative pathway by which cytokines might contribute to GVH reactivity is via the induction of a common proximate mediator such as nitric oxide (NO). NO is an unstable radical that has been shown to play an important role in immune modulation,” neurotransmission,” and regulation of vascular tone.” NO is generated from Larginine through an oxidation reaction that is catalyzed by NO synthase (NOS). NO then decays in vivo into the stable inorganic nitrogen oxides, nitrate and nitrite. NOS exists in both a constitutive form present in vascular endothelial cells, neurons, and platelet^'"'^ and in an inducible form that can be present in macrophages, neutrophils, endothelial cells, and hepatocyte^.'^"^ Expression of inducible NOS can be G Blood, Vol 84, No 7 (October I), 1994:pp 2363-2373 From the Departments of Medicine, Pathology, Biostatistics and Biochemistry, and the Bone Marrow Transplant Program, Medical College of Wisconsin, Milwaukee, WI. Submitted April 11, 1994; accepted June 9, 1994. W.R.D. is supported by Grant No. CA01534 from the National Cancer Institute. Address reprint requests to William R. Drobyski,MD, Medical College of Wisconsin, 8700 WWisconsin Ave, Milwaukee, W153226. The publication costsof this article were defrayedin part by page chargepayment. This article must therefore behereby marked “advertisement” in accordance wirh 18 U.S.C. section 1734 solely to indicate this fact. 0 1994 by The American Society of Hematology. 0006-4971/94/8407-0029$3.00/0 2363 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2364 DROBYSKI ET AL Thy 1.2-/L3T4' and Thy I .2 -/Lyt 2 '. Fluorescence activated cell sorter (FACS) analysis were performed on spleen cell preparations from chimeras at selected time points posttransplant. Erythrocytes were lysed using hypotonic distilled water. Cells were stained with monoclonalantibodies as recommended by themanufacturerfor two-color analysis. Cells were analyzed on a FACS Analyzer (Bec30 ton Dickinson)equippedwithaFACSLitelaserandConsort computer support. Hisrologicsfudies. Tissues were obtained from control and experimentalanimals, fixed in 10% neutral bufferedformalin,and processed into paraffin blocks. Four-micron-thick sections were prepared fromeachblockandwere cut atthree levels to optimize sampling. For the evaluation of GVHD, tissue sections were screened with theexaminer blinded to the treatment received by eachanimal. Preliminary studies had indicated that little histologic evidence o f GVHD was observed in theskin or liverearlyposttransplant in animals receiving S X IOh spleen cells (GVHD control mice). Therefore, to facilitate a semiquantitative assessment of GVHD in control MATERIALS ANDMETHODS and experimental animals, gradingof GVHD was confined to analysis of the colon. Mice. AKWJ(H-2L,MW,Thy 1.1+) andBlO.BR/SgSn (H-2', Samples obtained from the colon of representative animals were Mlsh, Thy 1.2+) mice were purchased from Jackson laboratories (Bar evaluated in both axial and longitudinal sections. Glands were seHarbor. ME). All mice were housed in the American Association lected that had the basal portion visibly resting in proximity to the for Laboratory Animal Care-accredited Animal Resource Center of muscularis mucosa. The integrity of the glandular epithelium and the Medical College of Wisconsin (Milwaukee, WI). Mice received the degree of cellular infiltration in the lamina propria were evaluregular mouse chow and acidified tap water ad libitum. ated. The degree of changes in the epithelium was graded on a scale Reagents. L-NMA was prepared from L-ornithine (Sigma of 1 to 4 with 1 being no abnormality, 2 being mild change consisting N,S-dimethylthiopseuduronium Chemical CO, StLouis,MO)and ofoccasionalnecrobioticcellswithoutsignificantglandinjury, 3 iodide (Aldrich Chemical CO, Milwaukee, WI) by adaptation of the beingmoderatechangewith several glandshavingoneor more procedure of Corbin and Reporter.22 The flavinate salt was convened necrobiotic cells without gland injury, and 4 being severe change to theacetatesalt by stirringwithDowex-l(hydroxide;BioRad, with several glands having injury and/or destruction. Inflammatory Hercules, CA) and by adjusting the resulting basic solution to pH 7 cell infiltrates in the lamina propria were graded on a scale of 0 to with acetic acid. N"-methyl-D-arginine (D-NMA) was synthesized 4 reflecting no, mild, moderate,or marked infiltration. GVHD scores in an identical fashion with the exception that D-ornithine (Sigma) were normalized to that of BM controls to account for the effects was used in place of L-ornithine. Both L-NMA and N"-methyl-Dof the conditioning regimen. The scores for epithelial integrity and arginine (D-NMA) were dissolved in sterile distilled water andused degree of infiltration in the lamina propria were added to yield a at a concentration of 35 mg/mL. total GVHD score for individual mice. Nitrate and nitrite levels were determined by the Griess reaction Cohzy-jimtting unit (CFU)as.su.y.s. Femurs were harvested from as described by Green et al.'? Nitrate was reduced to nitrite using a mice,and BM was flushed fromthe BM cavity with Dulbecco's copper-coated cadmium column. Quantitation of biologic samples modified Eagle'smedium media. Cellswerecountedandresuswas by reference to a linear standard curve. Nitrate and nitrite stanpended at a concentration of 1.5 X 10' cells/mL in Iscove's modified dards gave peaks of equal area showing that nitrate was fully reDulbecco's medium. Viability always exceeded 9S% by trypan blue duced. dye exclusion. BM cells were plated in 35-mm tissue culture dishes BMT. AKR recipient mice were treated with 900 cGy total body containing 2.8% methylcellulose, (Fisher Scientific, Itasca, IL) 30% irradiation(TBI)within4to8hoursbeforetransplantation. TB1 fetal calf serum, erythropoietin ( I S p/mL; Toyobo, New York, NY). was administered in a single dose using a Shepherd Mark I cesium hemin (6.5 &mL: Sigma),mercaptoethanol (3.2 X 1 0 ~ 'molfl,), irradiator(JLShepherdandAssociates,SanFernando,CA).The and pokeweed mitogen-stimulated spleen cell conditioned medium. dose rate was 83.3 cGy/min. BM was flushed from donor femurs with complete Dulbecco'smodified Eagle's medium (GIBCO, Grand Conditioned medium was obtained on the sixth day of culture and used at a concentration of S%. This had been shown to be optimal Island, NY) plus S% fetal bovine serum. The BM plugs were passed for promoting progenitor cell growth in BM from normal, untreated through a sterile mesh filter to obtain single cell suspensions. BM AKR mice. Cultures were incubated at 37°C in S% CO2 for 14 days. cells were then washed, resuspendedin fresh medium, and counted. Colonies (greater than S O cells) were scored at day 7 and 14 under Spleens were passaged through sterile mesh screens to obtain single an inverted microscope. Data were expressed as CFU-granulocyte/ cell suspensions. Spleen cells were then treated with sterile distilled macrophage (CFU-GM) per lo5 plated cells and total CFU-GM per water to eliminate erythrocytes. BM and spleen cells were always two femurs greater than 90% viable by trypan bluedyeexclusion.Irradiated Experimenfddesign. AKR recipientswereadministered 900 recipient mice received a single intravenous injection containing 10 cGy TB1 as pretransplant conditioning. Based on the fact that IS of X 10' BM cells with or without 5 to 20 X 10' spleen cells. 16 (94%) irradiated control mice (without BMT) died at a median Flowcytometricanalysis. Two-colorimmunofluorescencewas of I6 days after irradiation (range,I3 to 22 days), this was considered used in some experiments to determine the extent of donor T-cell a lethal dose. Animals were transplanted with BI0.BR BM (10 X chimerism in the spleens of transplanted animals. Fluoroscein isoIO" cells) with or without graded dosesof spleen cells( S to 20 X 10'). thiocyanate-conjugated anti-Thy 1.2 andphycoerythrin-conjugated anti-L3T4 (CD4) and Lyt 2 (CD8) were obtained from Becton Dick- Irradiated micereceiving BM only wereconsideredexperimental controls, because these animals do not develop GVHD because of inson (Mountain View, CA). Donor-derived T cells were defined as an insufficient number of T cells in the BM inoculum (typically 2% Thy I .2+/L3T4' and Thy I .2'/Lyt 2'. Host T cells were defined as stimulated by cytokines such as IL-I, TNF, 7-IFN, and IL2, resulting in the augmentation of NO production both in vitro and in V ~ V O . " " ~ Enhanced NO productionhasbeen undergoing a GVH reacrecently documented in animals tion," but the pathophysiologic significance of this molecule is unknown. Because cytokine dysregulation is thought to play an important role in GVHD, this raises the possibility that NO might be a more proximate mediator of a cytokinefacilitated GVH response. Conversely,otherstudies have shown that NO has potent immunosuppressive properties in vitro," suggesting that the molecule might play a role in the downregulation of immune reactivity. To evaluate the role of NO in GVHD, we used a specific NOS inhibitor (NGmethyl-L-arginine [L-NMA]) i n a murine model of allogeneic BMT where donor and recipient are H-2-matched but differ at minor histocompatibility antigens. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. NO PRODUCTION ALLOGENEIC IN GVHD AFTER 2365 BMT Table 1. Effect of Varvina lntensitv of GVH Reactivitv on NO Production Nitrate/Nitrite Levels (prnollL) Experiment no. 1 B M only ( N = 3) BMS-20 (N 5) Day 4 6 28.3 f 2.2 127.5 -C 18.2 30.6 t 4.7 84.168.3 f 29.5 13 8 11 18.2 f 3.1 98.6 2 14.0 15.6 19.6 f 3.1 f 59.9 rt 5.1 83.5 f 6.5 Nitrate/Nitrite Levels (pmol/L) ~~ Experiment no. 2 = 4) B M only ( N 104 BMS-5 ( N = 4) Day 4 7 11 21 f 18.4 190.2 f62.6 24.5 42.3 f 3.0 t 7.6 39.6 t 30.0 4.8 71.2 f 8.9 14 18 t 49.4 9.7 81.7 t74.0 21.8 f 38.0 9.2 t69.8 38.4 f 8.9 t 38.2 Cohorts of mice were alternately bled at the defined intervals for determination of nitratehitrite levels. A total of 6 and 10 mice were in the and BMS-20 groups, respectively (Experiment no. l ) , whereas 8 mice each were in the B M and BMS-5 groups (Experiment no. 2). Abbreviations: BMS-20. B M plus 20 X 106spleen cells; BMS-5. B M plus 5 X lo6 spleen cells. BM to 3%). Animals receiving BM plus spleen cells (BMS) received treatment with either phosphate buffered saline (PBS) or L-NMA (250 mgkg twice per day, intraperitoneally) to assess the effect of L-NMA on GVHD and hematopoietic reconstitution. A dose of 250 mgkg was chosen based on a prior report that had shown that this dose prevented histopathologic evidence of intestinal GVHD in a L-NMA or saline was administered parent + F1 murine to animals for a total of 10 consecutive days, with the first dose administered 4 to 6 hours before transplant. All dosing was based on pretransplant weights of individual mice. BMS chimeras treated with saline were designated as GVHD controls. BMS chimeras receiving either saline or L-NMA were stratified before transplant by body weight so that the mean weight of each experimental group was similar. Weights were obtained for every animal at least twice weekly for 60 days. All mice were ear punched before transplant so that individual animals could be serially assessed and to allow for comparisons between L-NMA-treated and weight-matched GVHD control animals. In some experiments, AKR hosts were preconditioned with 900 cGy TB1 and then transplanted with AKR BM (10 X lo6) with or without AKR spleen cells (20 X IO6) to assess the effect of L-NMA in a syngeneic transplant model. Sratisficat analysis. Data are reported as mean values -C 1 SD. Descriptive statistics for weight profilesare reported as group means. The group mean weights were based on decreasing numbers of measurements over time because of expiring animals. Repeated measures analysis of variance was performed using individual animal weight profiles. Analysis examined the questions of whether the observed weight profiles showed a group difference, variation over time, and a group-time interaction (ie, different time variation across study groups). Analyses were performed at the two-sided 0.05 level of significance. The Greenhouse-Geisser significance level adjustmentwasused for testing differences within animals over time. Repeated measures analysis of variance was also performed to compare the effect of L-NMA on Cm-GM in normal murine BM and 28 1 l 90 80 - o i -r? 70- '5 5 m 3 60 50 - Y 40 Y 30 - 3 18i 1 6 0 ! . 10 1 . 1 . ~ . " 1 20 30 40 50 Days Post-Transplant . ' 60 Fig 1. Serial weight curves showing lackof toxicity of L-NMA administration in normal and transplanted AKR mice. Normal unirradiated AKR mice IN = 4; A) received a 10-day course of L-NMA (250 mglkg twice per day). Irradiated (900 cGy TBI) AKR animals were transplanted with 10 x 10'B1O.BR B M and were then treated with saline IN = 3; 0) or L-NMA IN = 4; B) for 10 consecutive days. - ::\ . , . , L o 1 -% tt. . , . , . I 0 0 10 20 30 40 50 ; 60 Days Post-Transplant Fig 2. Actuarial survival of BlO.BR/AKR chimeras. AKR mice were conditionedwith 900 cGy T B 1 and were then administered either 10 x lo* B1O.BR BM cells alone IN = 10; 0) or BM admixed with 5 = 10' B1O.BR spleen cells(BMS-5). BMS-5 chimeras receivedtreatment with either saline (N = 28; 0 )or L-NMA (N= 35; 0) for 10 consecutive days. Data are derived from four experiments. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2366 DROBYSKI ET AL mals ( P < ,001). This was observed as early as 4 days after transplant and was well before any clinical signs of GVHD wereevident.Elevated nitrate and nitritelevelspersisted 24 26!i throughout the period of testing in both experiments. In the group receiving 20 X 10' spleencells, further testingwas h not possible because animalsbegan to dieof GVHD. Median 22 survival in this group was 18 days (n = IO), with no animals surviving to day 60. The group receiving S X 10' spleen S cells had a lessintense GVH reaction as evidenced by a .YJ median survival of 28 days (n = 8), with 25% of animals 2o surviving to day 60. In contrast, all animals transplanted with BM only (n = 14) survived to day 60. NO production 18 was not significantly different between animals transplanted with S X 10' versus 20 X IO6 spleencells.Unirradiated normal AKR mice (n = 3) had serum nitratehitrite levels of 15.7 ? 1.6 pmol/L.Thusnitratehitrite productionwas increased in irradiated BM control animals when compared with that for normal mice. Days Post-Transplant Administration of L-NMA inhibits NO production in recipients undergoing U GVH reaction. A study was performed Fig 3. Serial weight curves for the first 60 days posttransplant. to assess thetoxicity of L-NMA innormal unirradiated AKR AKR mice were conditionedwith 900 cGy TB1 and transplanted with B1O.BR BM IO) or B1O.BR BM plus 5 x 10' spleen cellsIBMS-5). BMSmice andinirradiated AKR recipientsnot developing 5 chimeras were treated with either PBS (e)or L-NMA (0) for 10 GVHD. Irradiated AKR hosts were transplanted with consecutive days. Values represent mean weights of surviving aniB1O.BR BM only and treated with either saline or L-NMA. mals at each timepoint. Date are derived from the four experiments No clinical signs of toxicitywere observed inany of the shown in Fig 2. three groups. All animalssurvived through day 70, at which time they were killed.Asa moreobjectiveparameter of clinical well-being, serial weights were obtained in animals to compare serial nitratehitrite levels in animals with and without GVHD. over the courseof this experiment (Fig I ) . Irradiated animals Data representedby single measurements were analyzed by analy- in boththe L-NMA and saline groups showed a characteristic sis of variance methods in conjunction with Tukey's multiple comdecrease in weight, with nadirs occurring approximately S parisonsmethod,atthe 95% confidencelevel, to identifygroups to 6 days aftertransplant. There was nodifference in the having significantly different population means. Actuarial survival weight curvesbetween the two groups. Unirradiated animals curves were compared using the log ranktest. Pair-wise comparison treated with L-NMA slowly gained weight over the course to identify groups having significantly different survival distributions of the experiment. were performed at the 0.025 level to preserve the overall .OS level The effectiveness of this dose of L-NMA in inhibiting of significance.Spleencellsizes,histologicGVHDscores,total NO production was assessed in a pilot study.Because a more CFU-GM, and T-cell chimerism data were assessed using the paired intense GVH reaction (20 X 10' spleen cells versus S X Student'sf-test tocompareL-NMA-treatedanimalswith salinetreated mice that had similar pretransplant weights. 10' spleen cells) was associated with higher mortality (see above), the efficacy ofL-NMA in animals receiving 20 X IO6 RESULTS spleen cells was initiallytested.Nitratelnitritelevelswere measured on day 9, near the end of L-NMA administration, NO production is increased in GVHD induced across miat a time when levels were expected to be elevated (Table 1). nor histocompatibility antigens. Initial studies wereperformed to determine the effectof GVHD on NO production Blood samples wereobtained by retroorbitalvenipuncture before the morning L-NMA dose so that the nitratehitrite in this murine BMT model. The intensity of the GVH reaclevelswould be reflective of atrough L-NMA level. The tion was varied to assess how this affected NO production. control group consisted of animals transplanted with BM AKR recipients were administered 900 cGy TB1 and then cells only. Nitratehitritelevels were higher in saline-treated transplanted with 10 X lo6 BM cells admixed with S X lo6 mice (mean, 52.9 t 24.5 pmol/L; n = 4) than in L-NMAor 20 X lo6spleencellsfrom B1O.BR donorstoinduce treated animals (mean, 22.8 t 15.7 pmol/L; n = S) or animoderate or severeGVHD, respectively.Control animals mals receivingBMonly (mean, 33.0 ? 19.4 pmol/L; n = received TB1 and BM cells but no spleen cells so that the impact of the conditioning regimen alone on NO production 3), indicating that L-NMA was able to suppress NO production in animals undergoing an intense GVH reaction to the could be determined. Peripheral blood was obtainedby retrolevel of BM controls. orbital venipunctureat defined intervals posttransplant to Administration of L-NMA exacerbates weight loss and determine the time course of NO production during a GVH adversely affects survival in mice with GVHD. The effect reaction. of L-NMA administration on survival of mice undergoing a The results of these experiments are detailed in Table 1. GVH reaction was investigated. AKR recipients were condiNitrateand nitritelevels,reflecting NO production, were tionedwith 900 cGy TB1 and administered 10 X 10' BM significantly elevated compared with those of control ani- 8 S i46 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. NO PRODUCTION IN GVHD AFTER ALLOGENEIC BMT 2367 Table 2. Effect of L-NMA on NO Production and Survival After Syngeneic (AKR -t AKRI BMT ~~ ~~ ~~~________ Group No. No. Inoculum of Mice 1 2 3 4 5 5 4 3 Spleen Survival Cell Bo-Day Nitrate/Nitrite Adminisiration L-NMA day) per(250 twice mglkg (% Surviving Animals1 bmolk) + 20 x 106 20 x 106 - - - 100 +100 12.3 26.4 11.8 12.8 2 1.8 f 12.2 2 7.1 2 0.7 100 l00 AKR mice were conditioned with 900 cGy TB1 and transplanted with AKR BM (10 x 10' cells) with o r without 20 Nitratehitrite levels were determined on day 4 posttransplant. All animals were then followed up for survival. cells plus 5 X lo6 spleen cells from B1O.BR donors. Mice received treatment for 10 days with either PBS or L-NMA (250 mgkg twice per day, intraperitoneally). A control group received 900 cGy T B 1 plus lo7BM cells. Nitratehitrite values on the ninth day posttransplant were determined in all experimental groups. Saline-treated animals (n = 28)had significantly higher nitratehitrite levels (mean, 55.6 2 12.6 PmoVL) compared with those of L-NMA-treated mice (n = 33; mean, 42.2 f 12.4 pmoyL; (P = .OOl). Nitratehitrite levels in control animals (n = 10) averaged 43.6 f 7.5 pmoliL, which did not differ from L-NMA-treated animals. Survival of these animals is shown in Fig 2. Sixty-day surviva1 for saline-treated animals was significantly better than for L-NMA-treated animals (50% v 17%;P < .001). Poorer survival in L-NMA-treated animals was primarily because of substantial early mortality (median survival, 12 days in L-NMA-treated animals versus 58 days in saline-treated animals). L-NMA-treated animals were observed to have significantly greater weight loss (Fig 3) within the first 10 days posttransplant when compared with saline-treated mice ( P < .01). After the initial 2 weeks, saline-treated animals 281 7 26 h E 24 22 o IO 3020 40 50 Days Post-Transplant 60 Fig 4. Serial weight curves for the first 60 days posttransplant. AKR mice were conditionedwith 900 cGy TB1 and transplantedwith AKR B M alone orAKR B M plus 20 = l@ spleen cells (BMS-20). Mice transplanted with BM only were treatedwith either saline (U) or LNMA (U1 for 10 consecutive days. BMS-20 chimeras also received treatment with either saline(0)or L-NMA (01over the same interval. Values represent mean weights of surviving animals at each timepoint. x 10' AKR spleen cells. and surviving L-NMA-treated animals were both able to regain weight and did not differ significantly from each other thereafter. In contrast, L-NMA-treated animals who received BM only (without spleen cells) showed no disproportionate loss in weight when compared with saline-treated BM controls (data not shown). To confirm that the effect of L-NMA administration was enantiomer-specific, identically transplanted mice (n = 10) received a 10-day course of D-NMA (250 mgkg twice per day, intraperitoneally). Nitratehitrite levels in this group averaged 54.1 2 10.6 pmol/L, indicating that D-NMA was unable to inhibit NO production. Weight curves were similar to those of saline-treated animals (data not shown). Sixtyday survival was 80% with no early mortality, showing that the effect observed with L-NMA was enantiomer-specific. Inhibition of NO production is associated with weight loss but not with exacerbation of GVHD early in the posttransplant period. Weight loss is known to be a sensitive indicator of GVHD in mice. Therefore, it was possible that the observed early weight loss in L-NMA-treated animals was because ofan exacerbation of GVHD resulting from the suppression of NO production. To evaluate this possibility, tissues from saline- and L-NMA-treated mice that had received BM plus 5 x lo6 spleen cells were analyzed at 9 to 10 days posttransplant for histologic evidence of GVHD. LNMA-treated animals showing the most profound weight loss were selected along with a group of saline-treated animals (GVHD controls) that had similar pretransplant weights. None of the animals in either group were found to have significant pathologic changes of GVHD in the skin or liver on the ninth day posttransplant (data not shown). Therefore, histologic analysis was confined to the colon. Saline-treated animals were observed to have pathologic changes in the colon (eg, crypt cell necrosis and lymphocytic infiltration in Table 3. Effect of L-NMA on Spleen Size in BMS Chimeras Underaoina GVHD Weight (g) Pretransplant Day Spleen Total 9 to 10 Size (no. of cells x Saline-treated ( n = 9) L-NMA-treated ( n = 9) 24.8 f 0.8 21.8 f 1.3 24.2 16.3 t 1.4 ? 1.8 23.2 f 8.6 6.98.1 Weights and spleen sizes reflect the mean values for each group. Results are derived from three separate experiments. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2368 DROBYSKI ET AL Fig 5. Photomicrographs of spleen sectionsfrom AKR mice preconditionedwith 900 cGy TB1 and then transplanted with 10 x lo6 BM plus 5 x lo6 spleen cells from B1O.BR donors. Spleens were harvested from animals9 to 10 days after transplant. (A) Spleen section from a saline-treated animal showing areas of white pulp (W) and red pulp (R). Prominent extramedullary hematopoiesis is shown in the red pulp; note dark staining cells indicative of erythroid hyperplasia (hematoxylin eosin stain). (B) Spleen from LNMA-treated mouse showing marked reduction in extramedullaryhematopoiesis in the red pulp (hematoxylin eosin stain). the lamina propria) consistent with GVHD. Although LNMA-treated animals overall had less evidence of GVHD, differences in the histologic GVHD scores for saline (mean, 2.7 2 1.9) and L-NMA mice (mean, I .2 0.9; N = 8 mice per group) were statistically nonsignificant (P = .IO). These findings suggested that administration ofL-NMA did not exacerbate GVHD and that the enhanced weight loss in LNMA-treated animals could not be ascribed to GVHD. No other histologic abnormalities were observed in the colon, skin, or liver of L-NMA-treated animals to account for weight loss. The majority of surviving saline-, L-NMA-, andD-NMA-treated animals transplanted withBMShad clinical evidence of GVHD byday 60, indicating that LNMA,when administered over a IO-day period, wasnot able to abrogate the eventual development of GVHD. Weight loss was observed only in irradiated AKR recipients that had received BMS from histoincompatible B I O.BR donors. To determine whether spleen cells alone, in the absence of histocompatibility differences between donor and recipient, could effect weight loss, experiments were performed using a syngeneic model. AKR recipients were irra- diated with 900 cGy and transplanted with AKR BM with or without spleen cells (20 X 10'). Control animals received BM only and were treated with either saline or L-NMA. The results of this experiment are shown in Table 2. L-NMAtreated recipients of AKR BMS had identical survival compared with that of saline-treated animals. Moreover, there was no difference in weight curves posttransplant between any of the groups (P = .90; see Fig 4). NO production at 9 days posttransplant was not increased above that of unirradiated AKR controls. These data indicated that a histocompatibility difference between donor and recipient plus the presence of spleen cells in the donor inoculumwereboth necessary to augment NO production andto effect enhanced weight loss in the posttransplant period. L-NMA treatment impairs hematopoietic reconstitution in animals undergoing GVHD. During histologic studies for analysis of GVHD, it was observed that the spleens of LNMA-treated chimeras showing marked weight loss were noticeably smaller than those of saline-treated animals, suggesting that L-NMA might interfere with posttransplant hematopoietic reconstitution. To quantify this observation, From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2369 NOPRODUCTION IN GVHD AFTER ALLOGENEIC BMT c 1.0 0 Ql a, .-C C 0.9 0.8 .................................................................. ................................................ I 8 m m mm l m m m l mm U S -E 0.34 S I 1 I BM BMS-5 Saline BMS-5 L-NMA Fig 6. Effectr of L-NMA administrationon donor T-cell chimerism. AKR mice were conditionedwith 900 cGy TB1 and transplanted with 10 x lo* B1O.BR BM cells or B1O.BR BM plus 5 x lo6 spleen cells (BMS-5). BMS-5 chimeras received al 0 day courseof treatment with either saline or L-NMA. Animals were killed at day 9 to l 0 to assess the proportion of donor T cells in the spleen. The horizontal line represents the lower threshold for complete donorT-cellchimerism.'O In one experiment, animals were precondkioned with 1,100 cGy TBI. Data are derived from three experiments. spleen sizes of both groups were assessed (Table 3). LNMA-treated mice with accelerated weight loss were killed onday 9 posttransplant and compared with pretransplant weight-matched saline-treated animals. The cellular content of spleens from L-NMA-treated animals was significantly less than that of saline-treated mice (mean, 8.1 X lo6 cells v 23.2 X lo6 cells; P < .003). Histologic analysis of spleens from saline-treated and L-NMA-treated animals showed that L-NMA-treated mice had a substantial reduction in extramedullary hematopoiesis which was most noticeable in the red pulp (Fig 5). To evaluate donor cell repopulation in these chimeras, flow cytometric analysis was performed to determine the extent of donor T-cell chimerism in the spleens of L-NMA- and saline-treated mice (Fig 6). The proportion of donor T cells was determined by the number of CD4' and CD8+ T cells coexpressing Thy-1.2. There was a significantly greater percentage of donor T cells in spleens of saline-treated animals (0.63 ? 0.13) as compared with LNMA-treated mice (0.47 ? 0.13; P = .02) when evaluated 9 to 10 days after transplant. Histologic studies of BM were then performed to determine the effect of L-NMA on BM reconstitution. Representative BM from saline- and L-NMA-treated mice are shown in Fig 7 and show a marked reduction in cellularity in LNMA-treated animals. To further evaluate the role of NO in hematopoietic reconstitution posttransplant, in vitro assays were performed to assess the effect of L-NMA on progenitor cell growth. Addition of L-NMA to C N assays had no affect on CFU-GM ( P = .76), indicating that L-NMA was not directly toxic or suppressive to progenitor cells (Table 4). Because it was possible that L-NMA might indirectly affect hematopoieic reconstitution, progenitor cell growth was investigated using BM from L-NMA- and salinetreated GVHD animals along with BM from control mice without GVHD. AKR animals were conditioned with 900 cGy TB1 and transplanted with BM plus either 5 X lo6 or 10 X lo6 B1O.BR spleen cells to induce moderate GVHD. GVHD animals received 10 days of treatment with either LNMA or saline. Irradiated control animals received BM only and no other treatment. The results of these studies are shown in Table 5. Total CFU-GM from L-NMA-treated animals were noted to be significantly reduced when compared with CFU-GM from saline-treated animals or from animals transplanted withBM only. The differences in total C N - G M between saline- and L-NMA-treated animals were significantly different at day 7 (P = .OS)and at day 14 ( P < .02) of the assay. DISCUSSION In this study, we have examined the pathophysiologic significance of NO in GVHD using a murine BMT model where donor and recipient differ for minor histocompatibility antigens. This model is analogous to the majority of transplants in humans where donor and recipient are HLA-identical but differ at multiple minor histocompatibility loci. NO production was increased under experimental conditions, which resulted in both moderate and severe GVHD. Nitratehitrite levels remained elevated for several weeks, suggesting that an ongoing GVH reaction was responsible for enhanced NO production. Nitratehitrite measurements in irradiated control animals were observed, in some cases (Table l), to be greater than those in normal untreated AKR mice, suggesting that the irradiation regimen may have played a contributory role in addition to the alloimmune effect. This is consistent with previously published dataz5and is further supported by the observation that nitrateinitrite levels were highest on day 4, during which time conditioning regimen toxicity was maximal. This study showed that L-NMA, a potent inhibitor of nitric oxide synthase, could be administered immediately posttransplant without obvious toxicity to irradiated animals not undergoing GVHD. In animals with GVHD, administration of L-NMA resulted in the reduction of nitratehitrite levels to that of BM controls as assessed by measurements taken 9 days posttransplant. Suppression was shown to be enantiomer-specific, because treatment with D-NMA didnot inhibit NO production. Nitratehitrite measurements were deliberately taken immediately beforeL-NMA dosing. This was done so that measurements would be performed when LNMA levels in individual mice would presumably be lowest and would, therefore, correspond to a period of least NOS suppression. Thus, the fact that NO production was suppressed at this point makes it likely that suppression was continuous during the period of dosing. Prior studies in rodents have shown that NO is able to suppress immune reactivity in vitro. This conclusion is based on observations that inhibition of NO production with specific NOS inhibitors enhanced mitogen- and alloantigen-induced lymphocyte proliferation." Suppression of proliferation by NO appeared to be mediated in part by macrophages, because macrophage depletion before culture was able to From www.bloodjournal.org by guest on June 18, 2017. For personal use only. DROBYSKI ET AL 2370 ‘I , ” augment a proliferative response.’6 NO has also been implicated as having a role in mediating some of the immune suppression observed in in vitro studies performed in animals undergoing GVHD.” Although these data support an immunosuppressive role for NO in vitro, other studies have implied that NO may be able to potentiate immune reactivity in vivo. Lander and coworkers” showed that NO donor compounds were able to augment selected activation parameters in peripheral blood mononuclear cells, suggesting that NO might facilitate recruitment of other effector populations thereby enhancing immune reactivity. Similarly, NO has been shown to potentiate islet cell destruction caused by IL1 administration in an autoimmune model of diabetes.” More recently, McCartney-Francis et aI3” reported that inhibition of NOameliorated the manifestations of inflammatory arthritis. These latter studies suggest that NO plays a proinflammatory role and serves as a more proximate mediator of tissue damage in vivo. Although NO production has been documented to be increased in GVHD, the pathophysiologic role of NO in GVHD has not been extensively studied. Garside and co- Fig 7. Photomicrographsof BM obtained from AKR mice preconditionedwith 900 cGy TB1 and then transplanted with B1O.BR BM (10 x lo6) plus 5 x lo6 spleen cells. BM samples wera harvested from femurs of mice 9 to 10 days after transplant. (A) BM from saline-treated animal showing early hematopoietic reconstitution with three cell line engraftment (hematoxylin eosin stain). (B) BM from LNMA-treated mouse showing marked reduction in cellularity with prominent central sinusoidal dilatation reflecting an overall reduction in hematopoietic elements (periodic acid Schiff stain). workersz4showed that treatment with L-NMA ameliorated the pathologic manifestations of intestinal GVHD after haploidentical BMT inmice. However, this studydidnot verify that L-NMA effectively blocked NO production, nor was the effect of L-NMA on survival of animals undergoing GVHD assessed. In the present study, L-NMA administration was associated with enhanced weight loss, initially suggesting that inhibition of NO production might exacerbate GVHD. However, histologic analyses of colonic tissue samples fromL-NMA-and saline-treated GVHD controls clearly indicated thatL-NMAdidnot exacerbate GVHD in this experimental model. This finding is consistent with previous in vivo observations in other disease ~ t a t e s ’ ~and .~ supports the premise thatNO does not appear toplayan immunosuppressive role in GVHD. Amelioration of GVHD by inhibition of NOS early after BMT wassuggested by our data, but the study was not conclusive on thispoint. Assessment of a potential saluatory effect ofL-NMAon GVHD was confounded by the fact that L-NMA adversely affected alloengraftment which is a prerequisite for the induction of GVHD. The observation that the majority of sur- From www.bloodjournal.org by guest on June 18, 2017. For personal use only. NOPRODUCTION IN GVHDAFTERALLOGENEIC 2371 BMT Table 4. Effect of In Vitro L-NMA Administration on CFU-GM CFU-GM Experiment no. 1 NO L-NMA 1 mmol/L L-NMA Experiment no. 2 NO L-NMA 1 mmol/L L-NMA Experiment no. 3 No L-NMA 1 mmol/L L-NMA Day 7 Day 14 151 -C322 16.5 134 2 18.6 300 2 34.8 t 18.2 198 191 t 12.4 122 t 25.4 ? 21.3 199 2 34.5 139 2 15.8 158 2 20.0 161 ? 21.8 176 t 22.4 BM was obtained from normal AKR mice and assayed in vitro for the formation of CFU-GM in the presence or absence of 1 mmol/L LNMA. Assays were scored on days 7 and 14. Data are presented as CFU-GM per 1 x lo5 plated cells 2 1 SD. Each experiment represents a replicate assay. viving L-NMA-treated animals went on to develop clinical signs of GVHD indicated that L-NMA, as administered in this study, was not able to abrogate the eventual development of GVHD. Whether alternative routes of administration or more prolonged therapy might mitigate GVHD without resulting in early mortality requires further study. The observation of enhanced weight loss in L-NMAtreated animals was unexpected and appeared to be a major contributing factor to early mortality. The finding of weight loss was consistent with prior studies by Evans and colleagues3' who reported a wasting syndrome in Leishmaniasis-infected animals treated with L-NMA. These investigators attributed this finding to a reduction in food intake and speculated that elevated levels of cytokines such as TNF may have played a role in the development of cachexia. In this study, weight loss was not observed in syngeneic recipients or allogeneic recipients of BM. Weight loss was observed only in irradiated animals transplanted with histoincompatible BMS, indicating that an allogeneic immune response was a prerequisite for the induction of enhanced weight loss. Although GVHD wasnot the cause of pronounced weight loss, the intensity of the GVHD reaction appeared to influence the degree of weight loss and early mortality in L-NMA-treated animals. Specifically, we have observed that, in very mild GVH reactions, weight loss and early mortality are mitigated (unpublished observations). Therefore, a threshold level of GVHD appears to be necessary for this outcome. It appears that TB1 is also a critical factor, because weight loss occurred early posttransplant, after which time, some L-NMA-treated animals were able to regain weight as they recovered from the conditioning regimen. The fact that this occurred despite an ongoing GVH reaction suggests that GVHD alone was insufficient to effect continued weight loss. Because both TB13*and GVHD'.' are able to cause enhanced cytokine production, this would be consistent with the hypothesis that weight loss was cytokinemediated. Studies are ongoing to define this mechanism that may represent a novel approach for the study of the phenomenon of cachexia in inflammatory conditions. We cannot exclude the possibility that the microbiologic flora of animals was in part responsible for weight loss and early mortality in L-NMA-treated mice. Infection is a major cause of death in mice undergoing GVHD, and the microbiologic status of animals can modulate the intensity of GVH reactivity. This is born out by studies that have shown that GVHD severity can be ameliorated or completely prevented by transplanting animals in germ-free environment^.^^ NO is known to be important in the host defense against selected parasites and intracellular although its full role in the spectrum of host immune responses has not been completely defined. It is possible that early mortality in LNMA-treated animals may have been because of a reduced ability to eradicate infection or that the process predisposing animals to weight loss may have enhanced host susceptibility to infectious complications. It is unlikely that deaths were exclusively caused by impaired hematopoietic reconstitution, because irradiated control animals survived longer than LNMA-treated mice. NO has been postulated to play a role in vitro in the growth and differentiation of leukemia suggesting that NO may be important in hematopoiesis. L-NMAtreated mice with early weight loss were noted to have marked reduction in BM cellularity and extramedullary hematopoiesis when compared with weight-matched salinetreated GVHD controls. Donor T-cell repopulation in the spleens of these animals was also reduced (Fig 6 and Table 3). Collectively, these data suggest that inhibition of NOS was able to compromise hematopoietic reconstitution posttransplant. To further evaluate this observation, we performed in vitro CFU assays to assess the effect of L-NMA on normalmurine BM. Addition of L-NMA to colony assays had no effect on CFU-GM, indicating that L-NMA did not directly inhibit progenitor cell growth. In contrast, there was a significant reduction in CFU-GM inBM obtained from cachectic L-NMA-treated animals when compared with either GVHD or BM control animals, indicating that inhibition of NO production indirectly effected BM suppression. The reason inhibition of NO production was able to impair hematopoiesis is not clear. Graft rejection has not been observed in B 10.BWAKR donorhost combination^,^^ because animals transplanted with BM depleted of mature T cells become mixed chimeras. This is presumably because of the lack of mixed lymphocyte culture reactivity in the host-versus-graft Table 5. Effect of In Vivo L-NMA Administration on CFU-GM Total CFU-GM' EMS L-NMA EMS saline BM onlv No. of Mice Day 7 Day 14 4 4 3 6,319t 1,877 13,297 ? 3,692 18.011 ? 1.270 4,347 t 1,955 11,860? 3,694 11.371 2 1.439 BM was obtained from AKR hosts that had been preconditioned with 900 cGy TB1 and transplanted with B1O.BR BM with or without 5 x IO6 spleen cells. EMS chimeras received treatment with saline or L-NMA for 10 consecutive days. BM cells were harvested 8 to 9 days posttransplant and assayed in vitro for formation ofCFU-GM. Assays were scored on days 7 and 14. Data are derived from two separate experiments. * Data expressed per two femurs 2 1 SD. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. DROBYSKI 2372 d i r e ~ t i o nTherefore, .~~ impaired hematopoietic reconstitution was not likely to be due to augmentation of a host-antidonor immune response. It is tempting to speculate that L-NMA may have augmented release of cytokines such as TNF and y-IFN that are known to have BM suppressive effects in vivo. However, there ispresentlyno data to support the premise that inhibition of NO production is able to enhance cytokine release. It is also possible that L-NMA administration may have facilitated the induction of other soluble factors or effector cell populations which played more proximate BM suppressive roles. It should also be noted that these observations may be due in part to the strain combinations used in this study and that other strains will needto be evaluated to confirm the generalizability of these results. In conclusion, this study showed that NO production is increased in GVHD induced across minor histocompatibility antigens. Administration of L-NMA was able to suppress NO production in animals undergoing GVHD but was associated with pronounced early weight loss and decreased survival. L-NMA treatment did not exacerbate GVHD early in the posttransplant period, indicating that NO does not appear to play a critical immunosuppressive role in this experimental model. Inhibition of NO production was associated with impaired hematopoietic reconstitution, suggesting that NO may play a role in facilitating engraftment in allogeneic BMT recipients. Efforts to understand the mechanisms by which inhibition of NO production impairs hematopoietic reconstitution may provide new insights into understanding the process of alloengraftment and warrants further study. ACKNOWLEDGMENT We thank David Majewski, Michael Chaltry, and Michael Hayward for technical assistance and Jennifer Olson for preparation of the manuscript. REFERENCES 1. Burakoff SJ, Deeg HJ, Ferrara J, Atkinson K: Graft-VersusHost Disease. New York, NY, Marcel Dekker, Inc, 1990 2. Vallera DA, Youle RJ, Neville DM, Kersey JH: Bone marrow transplantation across major histocompatibility barriers V. Protection ofmicefromlethalgraft-versus-hostdiseasebypretreatment of donor cells with monoclonal anti-Thy1.2 coupled to the toxin ricin. J Exp Med 155:949, 1982 3. 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McCarthy PL, Abhyankar S, Neben S, Newman G, Sieff C, ET AL Thompson RC, Burakoff SJ, FerraraJLM: Inhibition of interleukin1 by an interleukin-1 receptor antagonist prevents graft-versur-hort disease. Blood 78:1915, 1991 8. PiguetPF,Grau GE, AlletB,Vassalli P: Tumournecrosis factodcachetin is an effector of skin and gut lesions of the acute phase of graft-versus-host disease. J Exp Med 166:1280. 1987 9.MowatAM:Antibodies to TFN-y preventimmunologically mediated intestinal damage in murinegrdft-VerSUS-hoSt reaction. Immunology 68: 18, 1989 10. Nathan CF, Hibbs JB: Roleof nitric oxide synthesisin macrophage antimicrobial activity. Cum Opin Immunol 3:65, I991 1 I . Garthwaite J, Charles SL, Chess-Williams R: Endotheliumderivedrelaxingfactorreleaseonactivation of NMDAreceptors 336385, suggests role as intercellular messenger in the brain. Nature 1988 12. 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Hoffman RA, Langrehr JM, Billiar TR, CurranRD, Simmons RL: Alloantigen-induced activation of rat splenocytes is regulated by theoxidativemetabolism of L-arginine.JImmunol145:2220, l990 22. Corbin JL, Reporter M: N"-Methylated arginines; A convenient preparationof N"-Methylarginine. Anal Biochem57.3 IO, 1974 23. Green LC, Wagner DA, Glogowski J , Skipper PL, Wishnok IS, Tannenbaum SR: Analysis of nitrate, nitrite, and ["NI nitrate in biological fluids. Anal Biochem 126:131, 1982 24. Garside P, Hutton AK,Severn A, Liew FY, Mowat AM: Nitric oxide mediates intestinal pathology in graft-versus-host disease. Eur J Immunol 22:2141, 1992 25. Voevodskaya NV, Vanin AF: Gamma irradiation potentiates L-arginine dependent nitric oxide formation in mice. Biochem Biophys Res Comm 186:1423, 1992 of "suppressor"macrophages. 26. Mills CD:Molecularbasis From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 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Evans TG, Thai L, Granger DL, Hibbs JL: Effect of in vivo inhibition of nitric oxide production in Leishmaniasis. J Immunol 151:907, 1993 32. Xun CQ. Thompson JS, Jennings CD, Brown SA, Widmer 2373 MB: Effect of TBI, BuCy2 or Cy5 conditioning on inflammatory cytokine release and development of acute and chronic graft-versushost disease in H-2 incompatible transplanted SCID mice. J Cell Biochem 18B:78, 1994 33. Jones JM, Wilson R, Bealmear PM: Mortality and gross pathology of secondary disease in gem-free mouse radiation chimeras. Radiat Res 45:577, 1971 34. James SL, Glaven J: Macrophage cytotoxicity against schistosomula of schistosoma mansoni involves arginine-dependent production of reactive nitrogen intermediates. J Immunol 143:4208, 1989 35. Magrinat G , Mason SN, Shami PJ, Weinberg JB: Nitric oxide modulation of human leukemia cell differentiation and gene expression. Blood 80:1880, 1992 36. Truitt RL, Atasoylu AA: Impact of pretransplant conditioning and donor T cells on chimerism, graft-versus-host disease, graftversus-leukemia reactivity, and tolerance after bone marrow transplantation. Blood 11:2515, 1991 37. Janeway CA, Yagi J, Conrad PJ, Katz ME, Jones B, Vroegop S, Busser S: T-cell responses to MLs and to bacterial proteins that mimic its behavior. Immunol Rev 107:61, 1989 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 1994 84: 2363-2373 Inhibition of nitric oxide production is associated with enhanced weight loss, decreased survival, and impaired alloengraftment in mice undergoing graft-versus-host disease after bone marrow transplantation WR Drobyski, CA Keever, GA Hanson, T McAuliffe and OW Griffith Updated information and services can be found at: http://www.bloodjournal.org/content/84/7/2363.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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