Cellular source of soluble interleukin 2 receptors in serum of mice after recombinant interleukin 2 administration.

Previous studies have shown that peripheral blood mononuclear cells activated in vitro not only express cell-associated interleukin 2 receptors (IL2R) but also release a soluble form of this receptor. In this study, we demonstrate that administration of human recombinant IL 2 (rIL 2) to mice results in increased spleen weights, splenic natural killer (NK) cell cytolytic activity, and serum levels of soluble IL2R. However, compared with rIL 2-treated heterozygote controls, beige mice treated with rIL 2 displayed similar elevations in serum soluble IL2R but significantly less splenic NK activity. Likewise, administration of anti-asialo GM1 antiserum to rIL 2-treated mice resulted in a dramatic reduction in splenic NK cytolytic activity, but no reduction in serum soluble IL2R. Conversely, while rIL 2 treatment of BALB/c mice produced increased splenic NK activity and serum soluble IL2R, similar treatment of BALB/c nude mice resulted in elevation of only splenic NK activity. These studies demonstrate that administration of rIL 2 to normal mice can elevate both serum IL2R levels and splenic NK cytolytic activity. However, the results suggest that T cells are likely to be the source of elevated serum IL2R after rIL 2 administration.     

Characteristics of immunosuppressive macrophages induced in spleen cells by Mycobacterium avium complex infections in mice

The profile of generation and characteristics of splenic macrophages (M phi s) which suppress the concanavalin A (Con A) mitogenic response of splenic T cells (designated as 'immunosuppressive M phi s') in host CBA/JN mice during the course of Mycobacterium avium complex (MAC) infection were investigated. In MAC-infected mice, reductions in some cellular functions of host splenic T cells, such as the Con A mitogenic response and mixed leucocyte reaction, were seen around 2 weeks after challenge of organisms, and this was accompanied by appearance of immunosuppressive M phi s in spleen cells. In this case, increase in immunosuppressive M phi activity was seen in terms of both activity per spleen and activity per individual M phi. In this phase of the infection, MAC-induced splenic M phi s showed a markedly increased ability to produce reactive oxygen radicals in response to phorbol myristate acetate. Thus, the expression of suppressor activity of MAC-induced M phi s seems to be closely linked to their activated state. A large proportion of the immunosuppressive M phi s exhibited suppressor activity dependent on prostaglandins and membrane functions related to microfilaments. It was also found that the generation of IL-2-reactive T cell populations in response to Con A was markedly inhibited by MAC-induced splenic M phi s, whereas they caused no significant reduction in the IL-2-producing ability of normal spleen cells.     

Impaired interleukin 1 and interleukin 2 production following in vitro abortive infection of murine spleen mononuclear cells by Semliki Forest virus

The effect of Semliki Forest virus (SFV) infection of murine spleen mononuclear cells was investigated in vitro. A small percentage of spleen macrophages expressed viral antigens, but no infectious virus particles were released, indicating an abortive-type infection. Wild-type SFV infected a higher percentage of macrophages than the attenuated, demyelinating mutant A7. The proliferation of spleen mononuclear cells under Con A stimulation was inhibited by the viral infection. The supernatant (SN) harvested from infected and Con A-stimulated spleen adherent cells could not stimulate thymocytes in an interleukin 1 (IL-1) assay and indomethacin treatment of infected cultures had no effect. The stimulatory effect of SN from noninfected cultures in the IL-1 assay was reduced when SN from infected cultures was added, suggesting the presence of an IL-1 inhibitor. Interleukin 2 (IL-2) production by splenocytes also decreased after viral infection, but exogenous IL-2 restored the response to Con A stimulation of infected spleen cells. This study demonstrates that abortive SFV infection of spleen macrophages has an immunosuppressive effect which may lead to an aberrant immune regulation.     

Frequency and specificity of precursors of interleukin 2-producing cells in nude mice

The frequency and specificity of precursors of interleukin 2-producing cells (IL 2-P) in congenitally athymic (nude) N:NIH(s)II mice was investigated. IL 2-P were detected and quantitated in a sensitive limiting dilution microassay in which Lyt-2-depleted lymphoid cell populations were first cultured for 12 days with irradiated allogeneic (DBA/2) stimulating cells and a source of IL 2 and then washed and restimulated with irradiated T cell-depleted stimulating cells for an additional 24 hr. Supernatants from restimulated cultures were assayed for IL 2 activity on CTLL indicator cells, and IL 2-P frequencies were calculated. The results indicated that IL 2-P were undetectable in young (6-wk-old) nude mice, but increased in frequency with age to eventually reach levels five to 10-fold lower than their euthymic (nu/+) littermates. In specificity studies, microcultures established originally with limiting numbers of nude or nu/+ responding cells and DBA/2 stimulating cells were split into three aliquots and restimulated with T cell-depleted stimulating cells of DBA/2, BALB/c, or C57BL/6 origin. Analysis of IL 2 production in these restimulated microcultures clearly demonstrated different patterns of cross-reactivity in individual nude mice that were not seen in nu/+ controls. These results are discussed in the context of a model proposing that the T cell repertoire in athymic mice is oligoclonal in nature.     

Immunologic tolerance to HGG in mice. I. Suppression of the HGG response in normal mice with spleen cells or a spleen cell lysate from tolerant mice.

Adoptive transfer of spleen cells or spleen cell lysates from mice tolerant to human-gamma-globulin (HGG) specifically suppressed the response of normal syngeneic recipients to HGG. The suppressive activity could be transferred for over 100 days after tolerance induction. The suppression induced by both spleen cells and spleen cell lysate was found to be specific as evidenced by a normal response to a challenge with turkey-gamma-globulin or goat erythrocytes. The activity of the suppressive lysate could be removed by passing the material through an HGG immunoadsorbent column but not by passing it through an anti-HGG column or a BSA column. These results indicated that the factor had antigen specificity and was probably not antigen-antibody complexes. That this suppression was not due to a shifting of the kinetics of the antibody response has also been demonstrated. The antigen-specific suppressor factor in the tolerant spleen cell lysates was a protein with a m.w. of approximately 45,000 daltons. The kinetics of the appearance of both suppressor cells and suppressor factor were consistent with a mechanism of active suppression functioning in the maintenance of tolerance to HGG.     

Regulation of the interleukin 2 receptor complex tyrosine kinase activity in vitro.

Interleukin 2 (IL-2) has been shown to stimulate tyrosine phosphorylation of a number of proteins requiring only the p75 beta chain of the IL-2 receptor. Unlike the receptors for epidermal growth factor, insulin, and other growth factors, the p55-alpha and p75-beta chains of the IL-2 receptor have no tyrosine protein kinase domain suggesting that the IL-2 receptor complex activates protein kinases by a unique mechanism. The activation of tyrosine kinases by IL-2 in situ was studied and using a novel methodology has shown tyrosine kinase activity associated with the purified IL-2R complex in vitro. IL-2 stimulated the in situ tyrosine phosphorylation of 97 kDa and 58 kDa proteins which bound to poly(Glu,Tyr)4:1, a substrate for tyrosine protein kinases, suggesting these proteins had characteristics found in almost all tyrosine kinases. IL-2 was found to stimulate tyrosine protein kinase activity in receptor extracts partially purified from human T lymphocytes and the YT cell line. Biotinylated IL-2 was used to precipitate the high-affinity-receptor complex and phosphoproteins associated with it. The data indicated that the 97-kDa and 58-kDa phosphotyrosyl proteins were tightly associated with the IL-2 receptor complex. These proteins were phosphorylated on tyrosine residues by IL-2 stimulation of intact cells and ligand treatment of in vitro receptor extracts. Furthermore, the 97-kDa and 58-kDa proteins were found in streptavidin-agarose/biotinylated IL-2 purified receptor preparations and showed high affinity for tyrosine kinase substrate support matrixes. The experiments suggest that these two proteins are potential candidates for tyrosine kinases involved in the IL-2R complex signal transduction process.     

Induction of T-suppressor cells by immunization with allogenic spleen cells in the H-2 system of mice]

The nature of suppressor cells induced by immunization with the allogenic spleen cells and inhibiting the DNA synthesis activation in the mixed lymphocyte culture was studied. Suppressor cells are resistant to mitomycin C and carrageenan. They are not inactivated by the treatment with rabbit anti-B- and anti-Ig- as well as with mouse antibodies (anti-Mls serum) against B lymphocytes in the presence of complement but eliminated by rabbit anti-lymphocyte and anti-T globulins and by mouse anti-theta serum. These T suppressor cells are concentrated in the large lymphocyte fraction in the ficoll gradient. Their blocking of the DNA synthesis activation is evidently non-specific.     

Properties of fractionated spleen cells from NZB/W mice.

The unit gravity sedimentation technique was used to separate spleen cells from sevveral strains of mice. Settling patterns (plot of cell number against settling rate) were similar for BALB/c, DBA/2, C3H/He, and NZB/W mice of different ages. In particular, no subpopulation was found by this technique to be missing from the spleens of old NZB/W mice.A number of functional studies performed with the separated cells proved more informative than the settling patterns themselves. Fractions of cells which sedimented at a rate of between about 6 mm/hr and 10 mm/hr were enriched in responsiveness to PHA, Con A, and allogeneic cells. These fractions obtained from old NZB/W mice lacked such activities. However, the active fractions from young NZB/W spleens, which were enriched in θ-bearing cells, could restore the responsiveness of old NZB/W mice to primary immunization with sheep erythrocytes. These studies indicate that functional separation of spleen cells from NZB/W mice is possible and that activities lacking in whole spleens from old NZB/W mice are also lacking in the separate fractions. The ability to restore helper T cell function in old NZB/W mice with active fractions from young NZB/W mice has implications for further study and treatment of their autoimmune disease.     

Proliferation of natural suppressor cells in long-term cultures of spleen cells from normal adult mice.

Natural suppressor cells were induced by culturing spleen cells from normal adult mice for 2 to 3 wk. The suppressor cells were large in size, nonadherent and nonspecifically suppressed the plaque-forming cells response of fresh spleen cells to SRBC in vitro. The suppressive activity of the cells was not affected by treatment with indomethacin or anti-Thy-1, anti-Ig, anti-Ia, or anti-asialoGM1 plus complement. Phenotype analysis by FACS showed that Thy-1, L3T4, Ly-2, CD3-epsilon, TCR-alpha beta, Ig, B220, Ia, and asialoGM1 Ag were all absent in the suppressor cells, although they were wheat germ agglutinin receptor positive. The suppressor cells did not demonstrate cytotoxicity against either YAC-1 or P-815 cells. Enriched large cell populations from fresh normal spleens expressed the same phenotypes and also exhibited the suppressive activity. These findings suggest that a minor population of natural suppressor cells exist in the normal adult mouse spleen and they proliferate during the in vitro culture of spleen cells.     

Suppression of T cell cytotoxicity by nude mouse spleen cells: reversal by monosaccharides and interleukin 2

The effects of monosaccharides on the suppression of cytotoxic T cell generation by spleen cells from nu/nu mice were examined. Suppression of the B6 anti-BALB/c response and the B6 anti-C3H response was reversed by alpha-methyl-D-galactoside (alpha MG) but not other sugars, including beta MG. Suppression was associated with a decrease in the level of IL 2, which suggests competition; this decrease was also reversed by alpha MG.     

Suppressor activity in the spleen of neonatal mice.

Helper cells of T-cell origin are required for the in vitro proliferation of low numbers of adult mouse thymus cells in response to allogeneic spleen cells. These helper cells are present in the adult mouse spleen. We have demonstrated that neonatal mouse spleen cells lack the helper activity present in adult spleen cells. We have also shown that this lack of helper activity is the result of active suppression. The suppression is due to a suppressor cell which is present in high quantities in the neonatal spleen and can be eliminated by treatment with anti-θ serum and complement.     

Microwaves induce an increase in the frequency of complement receptor-bearing lymphoid spleen cells in mice.

A single 30-min exposure of mice to 2450 MHz microwaves (12 to 15 mW/g body weight) in an environmentally controlled waveguide facility induced a significant increase in the proportion of complement-receptor positive lymphoid cells in the spleen. This effect was further enhanced by repeated (three times) exposures, which in addition produced a significant increase in the proportion of Ig+ cells. The proportion of theta-positive cells and the total number of spleen cells remained unchanged.     

Effect of streptomycin on antibody-producing cells in the spleen of albino mice

The effect of streptomycin on the number of cells involved in immunogenesis during the first ten days after antigenic stimulation was studied in relation to the dose of streptomycin. The results were read by means of Jerne and Nordin’s plaque technique, as modified by Šterzl. In dozes of 0.05, 0.1 and 0.5 mg, streptomycin had practically no effect on antibody production. On raising the dose to 1 mg, and particularly to 5 mg, the number of immunologically competent cells which received antigenic stimulation was markedly reduced. The difference between the number of plaque-forming cells in mice treated with 5 mg streptomycin and in the controls is statistically significant (P<0.05).     

Cytometrically detected specific binding of interleukin 2 to cells.

We have developed a new technique for detecting binding of interleukin 2 (IL-2) to cells. This technique involves incubating the cells with IL-2 and then analysing the cell surface with specific anti-IL-2 antibodies and flow cytometry. This binding was only detected on tumor cells that possessed the p55 subunit of the IL-2 receptor. The role of p55 was ascertained by inhibition of the binding with a monoclonal antibody to p55. Although p55 is necessary for cytometrically detected IL-2 binding, further studies demonstrated that p55 is not sufficient. Thus, cytometrically-detected binding is likely to involved the contribution of other IL-2 surface receptors. Interleukin-2 binding to peripheral blood T lymphocytes and to a non-transformed T-cell clone was also detected cytometrically and it was shown that this binding is regulated by the activation status of the cells. Whereas IL-2 binding to quiescent T cells could not be detected, upon activation abundant binding was seen. The functional consequences of this type of cellular binding were studied. Interleukin-2 binding to cells during a short pulse was shown to have significant long-term consequences both for T-cell proliferation and for the enhancement of major histocompatibility complex (MHC)-non-restricted cytotoxicity.     

Characteristics of immunosuppressive macrophages induced in host spleen cells by Mycobacterium avium complex and Mycobacterium tuberculosis infections in mice

The profile of generation and characteristics of immunosuppressive macrophages (M phi s), which suppress the ConA-mitogenic response of spleen cells (SPCs), in host CBA/JN mice during the course of Mycobacterium avium complex (MAC) and M. tuberculosis (MT) infections were investigated. In both infections, a marked reduction in ConA mitogenic response of splenic T cells was seen around 2 weeks after infection, and this was accompanied by generation of potent immunosuppressive M phi s in the SPCs of infected mice. The suppressive activity was much stronger in MT-infected mice than in MAC-infected ones. In both infections, the large part of the suppressive M phi s exhibited suppressor activity that depended on the arachidonic acid cascade, particularly mediated by prostaglandins (PGs), and the remainder showed the suppressor action independent from PGs. The unique finding of this study is that the generation of IL-2 reactive T cell populations in SPCs in response to ConA signal was markedly inhibited by the MAC- and MT-induced immunosuppressive M phi s, whereas the suppressive M phi s failed to reduce the IL-2-producing ability of splenic T cells. In any case, the present results indicate a close similarity in immunosuppressive M phi s induced by MAC and MT infections.