Ability of some amino acids incorporated into protein to stimulate the thymus-dependent immune response

Abstract

The i~unomodulating action of certain natural peptide preparations and their synthetic analogs has recently been extensively studied. The immunologic activity of the latter is linked with the definite primary structure of the peptide. The role of amino acids composing immunoactive peptides remained unkno~m until recently. In the case of the known preparation tuftsin -- an immunoglobulin fragment (HThrLysProArgOH) possessing stimulating activity toward phagocytosis of bacteria by neutrophils, the ability of two of the four amino acids composing it, namely proline and arginine, to stimulate phagocytosis in the same way as the original preparation, was demonstrated [9]. The question arises whether individual amino acids are able to affect the specific parameters of immunity. The aim of this investigation was to study the direct immunomodulating effect of amino acids composing a protein. For this purpose two criteria were used: the effect of the amino acids in vitro on differentiation of T precursor cells of bone marrow and T lymphocytes and their ability to stimulate the thymus-dependent immune response in an animal. EXPERIMENTAL METHOD Experiments were carried out on 674 male CBA mice weighing 14-16 g. ~nino acids were obtained from Sigma (USA), Fluka (West Germany), Reanal (Czechoslovakia), and the Olaine Factory (USSR). The amino acids for testing were injected subcutaneously into the animals daily for i0 days, in a dose of 1 ~g per injection, in p#rogen-free physiological saline. The animals were then immunized intravenously with sheep's red blood cells (SRBC) in a dose of 2-106 . On the 4th day after immunization the number of IgM-antibody-forming cells (AFC) was determined in the spleen of each mouse by the method in [7], and the hemagglutinin titer was determined in the serum. The number of AFC was calculated per 106 splenic karocytes. Differentiation of T precursor cells of bone marrow and T lymphocytes under the influence of amino acids was estimated in v~tro by a modified method [5]. For this purpose the bone marrow cell pool from the femur, tibia, and sternum was freed from red cells by treatment with 0.65% ammonium chloride solution. After the cells had been washed five times at 1200 rpm for 7 min with cold Hanks ~ solution they were mixed with the test amino acids so that i ml of medium contained 3~i07 nucleated cells and i ~g of amino acid. The mixture of amino acids with cells was continuously shaken and kept at 37~ for 1.5 h, after which the cells were again washed five times with Hanks' solution under the same conditions and their sensitivity to antibrain serum was determined in the cytotoxicity test [i]. Antiserum was obtained by repeated immunization of rabbits with cerebral cortical tissue of CBA mice without Freund's adjuvant [I], it was absorbed with a homogenate of mouse liver and mouse and sheep red blood cells [i], and used in a dilution of 1:50. In this dilution, in the presence of complement (fresh guinea pig serum -- 1:3), the antiserum led to death of 91.8  1.6% of thymocytes and 1.2  0.3% of bone marrow cells of CBA mice. In each sample no fewer than 200 cells were counted whose viability was estimated by the use of a 0.2% aqueous solution of trypan blue. The experiment was repeated at least 4-5 times. Altogether 115 animals were used for the experiments in vitro.