Department of Surgery, Sinai Hospital of Baltimore, and the Department of Surgery, the Johns Hopkins Medical Institutions, Baltimore, Maryland

ABSTRACT. Supplemental L-arginine has been shown to enhance thymic and T-cell responses in rodents. We examined the ability of supplemental dietary L-arginine to induce T-cell function in athymic nude mice that lack a normally developed T-cell system. Groups of male nude (nu/nu) mice (Balb/c background) 7 to 8 weeks old were given for 2 weeks 1.2% arginine hydrochloride solution for drinking, whereas controls received acidified tap water. All mice ingested a standard laboratory chow. In the first experiment, the arginine-supplemented animals had significantly greater number of T cells in the spleen (assessed by the number of Thy 1.2-positive lymphocytes) and these cells had enhanced mitogenic responses to

mitogenic stimulation (phytohemagglutinin and concanavalin A). In vivo delayed-type hypersensitivity responses to 2,4dinitro-1-difluorobenzene were also significantly increased after the 2 weeks of arginine supplementation. In a second experiment, mice maintained under the same conditions were

In vivo dietary supplementation with arginine inthe thymic weight and the total thymic lymphocyte contents in rodents.1,2 In addition, lymphocytes from these animals show enhanced blastogenic responses secondary to mitogenic stimulation.’ The immune-modulating actions of arginine occur within 3 days, require only a moderate increase in the arginine intake, are dose dependent, and are maintained for up to 6 weeks after cessation of therapy.’,’ Similar immune-modulating effects have been demonstrated on the responses of peripheral blood mononuclear cells of normal and immunocompromised humans given 30 g of arginine-HCI per

suggesting that limited extrathymic maturation of T cells does occur.&dquo; However, because of the absence of thymic modulation, the functional response of T cells is poor, and these animals remain essentially immune-incompetent in terms of T-cell antigen recognition or response.&dquo; The nude mice, therefore, provide an excellent model to examine the in vivo action of arginine on T lymphocytes and to test the hypothesis that alteration of the thymic environment is not the sole explanation for the beneficial actions of arginine on T cells.


day. 6,7 Little is known about the mechanisms of action of arginine on the T-lymphocyte system either in vivo or in vitro. It is particularly important to ascertain whether the in vivo T-cell enhancement is secondary to the thymotrophic effect of arginine or whether it is due to a direct action of arginine on circulating or extrathymic T lymphocytes and therefore independent of any thymic effects. To answer these questions we have examined the effect of supplemental dietary arginine on T-cell maturation and functions in the athymic nude (nu/nu) mouse. The role of the thymus in the development of a normal repertoire of T cells is well established.’ nu/nu mice have no defect in their T-lymphocyte stem cell population or in the development of pre-T cells.’ Indeed, they possess (in much reduced numbers) functional T cells that are phenotypically indistinguishable from normal T cells,


grafted with rat tail skin. Animals were observed for 100 for rejection but no significant difference was noted in skin graft survival. We conclude that dietary arginine can


increase extrathymic T-cell maturation and function, but cannot induce in vivo allogeneic graft recognition in athymic nude mice. ( 429Journal of Parenteral and Enteral Nutrition 16:

432, 1992)


Immune-deficient male nu/nu mice 6 to 8 weeks old (Balb/c background) (Life Sciences, Inc, St Petersburg, FL) were used in all experiments. Animals were housed in sterilized plastic shoebox cages (five per cage) and maintained in conditions of constant temperature and humidity. Water was sterilized by acidification with hydrochloric acid (pH 2.5) and the chow (Teklad, Harlan Sprague-Dawley) was sterilized by autoclaving. Daily food and water intake was monitored for each group. Animals were acclimatized to our laboratory conditions for 1 week before experiments. In both experiments control animals received acidified water, whereas the experimental group drank a solution containing 1.2% arginine-HCI. Food and water were offered ad libitum.

Experiment 1 In this experiment 15 mice received the arginine supplementation ; there were also 15 animals in the control

Reprint requests: A. Barbul, MD, Department of Surgery, Sinai Hospital of Baltimore, 2401 W Belvedere Avenue, Baltimore, MD



to the

Delayed-type hypersensitivity (DTH) testing. Response hapten 2,4-dinitro-l-difluorobenzene (DNFB)

429 Downloaded from at Kungl Tekniska Hogskolan / Royal Institute of Technology on March 9, 2015


(Eastman Kodak, Rochester, NY)




an in vivo

test of T-cell immune function. Briefly, on days 8 and 9 from the start of the arginine supplementation, mice were sensitized with 25 ~L of 0.5% DNFB in acetone and

olive oil (4:1) evenly applied over 1 CM2 of the abdomen. On day 13 basal ear thickness (outer third of the ear) was measured using a digital micrometer (Mitutoyo Digimatic Minipressor, Japan). The first reading was discarded and the mean of three subsequent readings was used as the basal value. The ear was then painted with 10,uL of 0.2% DNFB. Ear thickness was remeasured 24 hours later. The change in thickness was recorded as a percent increase relative to the basal value minus a nonspecific swelling value obtained by measuring ear thickness changes in a group of mice (n=6) challenged but not sensitized. In vitro T-cell function assessment. After 2 weeks of arginine supplementation, the mice were killed by cervical dislocation and their spleens were aseptically removed and weighed. Spleens were minced and the pulp was passed through 80- and 100-mesh stainless steel filters. The cells were washed twice and then suspended in RPMI 1640 medium supplemented with 100 U/mL penicillin, 50 ~g/mL gentamycin, 2 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, and 10% heat inactivated fetal bovine serum (Grand Island Biologicals, Grand Island, NY). Red blood cells were removed by lysis with ammonium chloride. Aliquots of cells were removed for staining with fluorescein isothiocyanate-labeled antiThy 1.2 monoclonal antibody (all T cells) (Becton Dickinson, San Jose, CA). Briefly,1 X 106 cells were incubated at 4°C for 45 minutes with 4 ~g of antibody. After incubation, cells were washed three times with phosphate-buffered saline and fixed, and percent staining was analyzed using a fluorescence-activated cell sorter. The remaining cell suspension was depleted of macrophages by plastic dish adherence for 2 hours. After these procedures, viability was assessed by Trypan blue exclusion (it always exceeded 90%) and the cells were suspended at a concentration of 1 X 106 cells/mL in RPMI 1640 medium as above. Mononuclear cells were plated in triplicate into microwells (2 X 105/well), stimulated with varying concentrations of phytohemagglutinin (PHA) (40 to 80 ~g/well) and concanavalin A (conA) (1.0 to 2.5 ~g/well) and incubated at 37°C 95% air, 5% C02 for 72 hours. During the last 6 hours of culture, cells were pulsed with 2 ~Ci of 3H-thymidine/well. At 72 hours cells were harvested onto glass filter paper, suspended in scintillation fluid, and counted in a 0-counter (Packard Instrument Co, Meriden, CT). The results are expressed as counts per minute.

precut sterile cardboard template (1 x 2 cm), dorsal host skin overlying the lower rib cage was excised down to the superficial fascia overlying the panniculus carnosus. Donor tail skin from female Sprague-Dawley rats with all subcutaneous fat removed was cut using the same template and placed raw-side-down on the host excised area. Six 6/0 nylon sutures were used to loosely hold the skin in place. The grafted area was covered with a transparent occlusive dressing. Grafts were inspected daily, but dressings were left undisturbed for 10 days. Sutures were removed at day 14. Mice were followed for 100 days. There were 18 animals in the arginine-supplemented group (given for the full 100 days) and 9 animals in the control group. a

Data Analysis

All data are reported as mean ± SE of the mean. Student’s paired or t test was used to compare the data. Statistical significance was achieved at p < .05. RESULTS

Experiment 1 Average daily food and fluid intake and mean body weight were similar in both groups (data not shown). Although the mean number of mononuclear cells was not altered by supplemental arginine intake (473 x 103/ mg spleen vs 430 X 103 for controls, NS), the percentage of splenic mononuclear cells staining positive for the Tcell marker Thy 1.2 was significantly enhanced in the animals that had been given arginine (Fig.1). Furthermore, proliferative responses to conA and PHA were markedly enhanced by the arginine supplementation (Fig. 2). Thus, maximal proliferation to PHA was 1993 ± 370 cpm in controls vs 4236 ± 810 cpm in the arginine group, p

50% of their grafts intact uith preservation of the features of rat tail skin. In the arginine group no grafts were traumatized. One graft ~ontracted and eventually disappeared at 73 days after a perfect take. The other grafts from the arginine-supplemented group showed minor degrees of contraction and/ 3r perigraft inflammation, but otherwise were rated as fully healed and viable. Histologically at 100 days these grafts demonstrated marked perivascular and subdermal nflammatory infiltrate when compared with controls.

tative and functional defects in their T cells. The decreased functionality of T cells from nude mice is thought secondary to the absence of interleukin-2-producing cells, which become detectable only at about 3 months of age. 13 In the present experiments we have shown that arginine can directly and beneficially influence several parameters of T-lymphocyte function in athymic nude mice. After arginine supplementation there was an increase in the number of lymphocytes bearing the T-cell surface receptor. This was coupled with enhanced blastogenesis in response to conA and PHA by the lymphocytes from arginine-supplemented animals when compared with controls. These effects translated into vigorous in vivo cell-mediated response (DTH) to DNFB. However, arginine supplementation could not induce xenogeneic skin graft rejection. The most probable explanation for this failure is that graft rejection is a multifactorial process, more complex than those involved in DTH responses. Previous authors have shown that in addition to stimulation of cytotoxic T cells, the generation of cytotoxic antibodies and complement cascade is required before rejection can be demonstrated in these animals. 14 Although the grafts from the arginine-supplemented animals appeared histologically to have more marked inflammatory infiltration, this was not sufficient to elicit full rejection responses. Our data do not explain the mechanism of action of arginine on T-cell function. The results do demonstrate that an intact thymus is not required for arginine to stimulate T lymphocytes. Direct in vivo enhancement of T lymphocyte activity for those cells relying on extra thymic sites of maturation, as observed in nude mice, can be induced by arginine. Arginine may have a direct cellular effect on lymphocytes or more likely an argininestimulated mediator molecule is responsible for the enhanced T-cell responses. Arginine has been shown to increase interleukin-2 production 15 and alter T-helper/ T-suppressor ratios in normal rodents. 16 In a previous study, we found no evidence that arginine alters Tlymphocyte subsets in nude mice.&dquo; Clearly, arginine appears to act by principally altering functional parameters of T cells; however, the exact mechanisms for these effects remain to be elucidated. REFERENCES

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432 C: Age associated increase in expression of the T cell surface markers Thy-1, Lyt-1 and Lyt-2 in congenitally athymic (nu/nu) mice: Analysis by flow microfluorometry. J Immunol 136:4337-

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4341, 1981

Hunig T: T cell function and specificity in athymic mice. Immunol Today 4:84-96, 1983 12. Steinmueller D: Skin graft procedure. Methods Enzymol 108:2027, 1984 13. MacDonald HR: Phenotypic and functional characteristics of Tlytic cells in nude mice. Exp Cell Biol 52:2-15, 1984 14. Gershwin ME, Castles JJ, Erickson K, Ahmed A: Studies of con-


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Arginine enhances T-cell responses in athymic nude mice.

Supplemental L-arginine has been shown to enhance thymic and T-cell responses in rodents. We examined the ability of supplemental dietary L-arginine t...
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