Journal of Undergraduate Research
Volume 2, Issue 1 - October 2000

Supplemental Effects of Arginine on One-Month-Old Mice

Donna Wilgus

ABSTRACT

The purpose of the study was to determine whether arginine supplementation decreases lymphocyte proliferation on stressed one-month-old mice. One-month-old mice were fed arginine supplemented or isonitrogenous diet for two weeks. Half were challenged by LPS injections while the other half was injected with PBS. The spleenocytes were removed and stimulated with PHA in the presence and absence of a NO inhibitor to determine whether NO, generated from arginine surpressed lymphocyte proliferation. The lymphocyte proliferation was not significantly different between the diets and treatment groups.

INTRODUCTION

The effects of arginine on the immune function of young and old humans and rodents are being investigated. 1, 2, 3 At the extremes of age, (young and old) immune function is reduced. 4 Addition of supplements that enhance immunity may decrease risk of infection in these vulnerable age groups.

Arginine, a nonessential amino acid in healthy adults, becomes an important essential amino acid in critically ill patients. The evidence suggests that arginine improves immune function in severely injured animals and critically ill patients. 5, 6, 7 Arginine may also improve host defense mechanisms by increasing thymic weight and lymphocyte proliferation. 8

Healthy adults given 17 grams of an arginine supplement showed a 50% increase in mitogen stimulated lymphocyte proliferation.1 In a nursing home study there were conflicting results showing that when arginine was administered to elderly subjects with pressure ulcers, there was no enhancement of lymphocyte proliferation.2 The differences may be attributed to the stressed condition of the nursing home residents.

In times of stress, nitric oxide is produced from the arginine pathway from a molecule of oxygen and the nitrogen of arginine.8 Nitric oxide may play a role in lymphocyte replication and function, causing a decrease in lymphocyte proliferation, thereby altering in the immune response of patients. 9

To determine whether arginine supplementation decreases lymphocyte proliferation in stressed animal models, one-month-old mice were administered lipopolyssacride (LPS) and mitogen-induced lymphocyte proliferation was assessed. Lymphocyte proliferation was also measured with and without an inhibitor of nitric oxide, NG-methyl-L-arginine (NMMLA), to determine the role of nitric oxide.

MATERIALS AND METHODS

Animals and Diets

One-month-old male CB6F1 (BALB/c x C57BL/6) mice were housed two per cage in a controlled environment at (22C°) with a 12- hour light and dark cycle. The mice were randomly assigned to either an AIN 93 Growth diet (Harlan Teklad, Madison, WI) that was supplemented with arginine (arginine diet, 20 g/kg) or an AIN 93 Growth diet made isonitrogenous with the addition of alanine (isonitrogenous diet).

LPS and PBS Injections

After 2-weeks on the arginine or isonitrogenous diet the mice were weighed and anesthetized with methoxyflurane. Half of the mice received intraperitoneal injection of LPS (17.84 mg/kg) and the other half-received phosphate buffered saline injections (PBS). Four hours after the injections, the mice were anesthetized and the spleens were removed.

Lymphocyte Isolation and Stimulation

The spleens were minced in 4 ml of RPMI and ground with a plunger. The cell suspension was pushed through a 100mm mesh and the mesh was rinsed with 20 ml of RPMI 1640 (Mediatech, Inc., Herndon, VA). The cell suspension was centrifuge at 350-x g for 10 minutes. The supernatant was discarded. Red blood cells in the cell pellets were lysed with 2.0 ml of 0.034 M of cold NaCl. After one minute, 0.0274 M of NaCl was added to the cells. The cell suspension was centrifuge at 200-x g for 10 minutes. The supernatant was discarded and the cells were washed three times in RPMI 1640. After the last wash, the supernatant was discarded and the cell pellet was resuspended in 1.0 ml of RPMI-complete (RPMI 1640 plus 50,000 mL penicillin, 50 mg/L streptomycin, 2 mmol/L L-glutamine, 50 mmol/L 2-mercaptoethenol, and 25 mmol/L ml HEPES buffer) plus 10% heat inactivated fetal calf serum (FCS). The cells were counted and plated in 96 well plates (MicroTest, U-bottom 353077, Becton Dickinson Labware, Franklin Lakes, NJ) with 2.0 x 105 cells per-well. All the treatments were plated in triplicate, e.g. three control, three phytohemagglutinin (PHA 3 mg/ml), and three PHA (3mg/ml) plus NMMLA (15 mg/ml) wells for each mouse. Lymphocyte proliferation was measured by incorporation of [3H] thymidine (20 Ci/mmol; Dupont Company, Wilmington, DE). At 42 hours 1 mCi [3H] thymidine, in RPMI-complete was added to each well and the plates were incubated for an additional six hours. The wells were harvested onto a filter paper (FilterMat 11731, Skaton, England) by using a cell harvester. The filter disks were placed in vials and suspended in a liquid scintillation fluid (Beckman LS2800). The radioactivity was counted. Lymphocyte proliferation was expressed as mean counts per minute(cpm) of triplicate wells. The cpm from the control (unstimulated wells) was subtracted from PHA stimulated wells.

Statistical Analysis

Change in animals' weight between diet groups was compared using an unpaired T-test. Lymphocyte proliferation between diets and treatments was compared using two-way ANOVA with regular factorial design. Preplanned comparisons were made using Fisher's least significant difference tests. All data are reported as means ± SEM.

RESULTS

There were no significant weight changes between the two diet groups of the young mice. Lymphocyte proliferation was not significantly different between the isonitrogenous and arginine diet groups or between the treatment groups. (Figure 1) When NMMLA was added to lymphocyte cultures to determine the role of nitric oxide in lymphocyte proliferation in LPS challenged mice, there was a significant decrease in lymphocyte proliferation in mice fed both diets. (Figure 2A) NMMLA also showed a decrease in lymphocyte proliferation was also decreased in the presence of NMMLA in arginine fed mice injected with PBS. (Figure 2B)

Figure 1. Effect of diet on proliferation of spleenocytes in PBS and LPS injected animals.
Figure 1. Effect of diet on proliferation of spleenocytes in PBS and LPS injected animals.

Figure 2a. Effect of diet on lymphocyte proliferation in LPS injected animals.

Figure 2a. Effect of diet on lymphocyte proliferation in LPS injected animals.

Figure 2a. Effect of diet on lymphocyte proliferation in PBS injected animals.

Figure 2a. Effect of diet on lymphocyte proliferation in PBS injected animals.

DISCUSSION

The purpose of this study was to determine whether arginine supplementation decreases lymphocyte proliferation in LPS challenged mice. Arginine did not alter lymphocyte proliferation in these young mice. When a nitric oxide inhibitor was added to lymphocyte cultures, there was a significant decrease in lymphocyte proliferation in cells obtained from LPS challenged mice in both diet groups.

Nitric oxide in the cell cultures has been shown to decrease lymphocyte proliferation. When inhibited there is an increase in lymphocyte proliferation.5, 9 Cultured spleenocytes stimulated with LPS produce large amounts of inflammatory cytokines and nitric oxide products. 8Nitric oxide decreases lymphocyte proliferation while NMMLA inhibits the suppression of nitric oxide allowing proliferation to take place.10, 11 Our findings were contrary to these studies. Additionally, we saw a significant decrease in lymphocyte proliferation in the presence of the nitric oxide inhibitor, NMMLA in arginine fed animals that were in injected with PBS.

This suggests that NMMLA may have been toxic to the lymphocyte cultures, however, a study showed that NMMLA could inhibit mitogen-induced lymphocyte proliferation when the lymphocytes were cultured in media void of arginine. 9 This suggests that low levels of nitric oxide are required for proliferation while higher levels are cytostatic. 5, 9

Further studies need to be completed to assess cell viability and nitric oxide production before conclusions can be drawn regarding the inhibitory effect of NMMLA on spleenocyte cultures. However, arginine supplementation does not appear to suppress lymphocyte proliferation in LPS challenged mice.

REFERENCES

  1. Kirk, S.J., Hurson, M., Regan, M.C., et al: (1993) Arginine stimulates wound healing and immune function in elderly human beings. Surgery 114:155-159.
  2. Langkamp-Henken, B., Herrlinger-Garcia, K., Stechmiller, J.K., et al: (2000) Arginine Supplementation is Well Tolerated But Does Not Enhance Mitogen-Induced Lymphocyte Proliferation in Elderly Nursing Home Residents with Pressure Ulcers. JPEN, in press.
  3. Ronnenberg, A.G., Gross, K.L., Hartman, W.J., et al: (1991) Dietary arginine supplementation does not enhance lymphocyte proliferation or interleukin-2 production in young and aged rats. J Nutr 121:1270-1278.
  4. Hori, Y., Perkins, E.H. and Halsall, M.K. (1973) Decline in phytohemagglutinin responsiveness of spleen cells from aging mice. Proc. Soc. Exp. Biol. Med. 144: 48-53.
  5. Albina, J.E., Henry, W.L. (1991) Suppression of lymphocytes proliferation through the nitric oxide synthesizing pathway. J Surg Res 50:403-409.
  6. Barbul, A., Sisto, D.A., Wasserkrug, H.L., et al: (1988) Arginine stimulates lymphocytes immune response in healthy human beings. Surgery 90:244-251.
  7. Barbul, A., Wasserkrug, H.L., Yoshimura, N., et al: (1984) High Arginine Levels in Intravenous hyperalimentation abrogate post-traumatic immune suppression. J Surg Res 36:620-624.
  8. Cui, X., Iwasa, M., Iwasa, Y., et al: (2000) Arginine-supplemented diet decreases expression of inflammatory cytokines and improves survival in burned rats. JPEN 24:89-96.
  9. Efron, D.T., Kirk, S.J., Regan, M.C., et al: (1991) Nitric oxide generation from L-arginine is required for optimal human peripheral blood lymphocytes DNA synthesis. Surgery 110:327-334.
  10. Kondo, S., Ishiguro, N., Iwata, H., et al: (1993) The Effect of Nitric Oxide on Chondrocytes and Lymphocytes. Biochem Biophys Res Com 197:1431-1437.
  11. Mills, C.D. (1991) Molecular Basis of Suppressor Macrophages. J Immunology 146:2719-2723.
  12. Nussler, A. K. and Billiar, T.R. (1993) Inflammation, immunoregulation, and inducible nitric oxide synthase. J Leukocyte Bio 54:171-178.
  13. Roberts-Thomson, I. C., Whittingham, S., Youngchaiyud, U., et al: (1974) Aging, immune response, and mortality. Lancet 2: 368-370.
  14. Daly, J.M., Reynolds, J., Thom, A., et. al: (1988) Immune and metabolic effects of arginine in the surgical patient. Ann Surg 208:512-523.
  15. Wick, G. and Grubeck-Loebenstein, B. (1997) The aging immune system: primary and secondary alterations of immune reactivity in the elderly. Exp. Gerontol. 32: 401-413.

--top--

Back to the Journal of Undergraduate Research