Modulation of human natural killer cell cytotoxic activity, lymphokine production, and interleukin 2 receptor expression by thymic hormones

Thymic hormone preparations have been shown to modulate natural killer (NK) activity in vivo in mice. We have investigated the effects of thymosin fraction 5 (TF5) on the in vitro NK cell activity of highly purified human large granular lymphocytes (LGL). The results indicate that TF5 but not kidney fraction 5 (a preparation used as control) is able to enhance the spontaneous NK activity of normal LGL. In addition, TF5 exhibited additive effects with recombinant interferon-alpha in enhancing NK activity in vitro. TF5 also enhanced interleukin 2 production and interleukin 2 receptor expression as well as interferon-gamma production in mitogen-stimulated LGL. Thymosin-alpha 1, a synthetic polypeptide originally isolated in its native form from TF5, also exhibited enhancing effects on LGL activities, suggesting that it is the active species in TF5. These results indicate that thymic hormones might regulate NK activity through the induction of lymphokine production and receptor expression by LGL.     

Increase of cyclic GMP induced in murine thymocytes by thymosin fraction 5

Using a radioimmunoassay (RIA) for the determination of adenosine 3'5' cyclic monophosphate (cAMP) and an acetylation-RIA procedure to measure guanosine 3'5' cyclic monophosphate (cGMP), we observed that cGMP levels, but not cAMP levels, were significantly elevated in murine thymocytes which had been incubated with preparations containing the thymic hormone, thymosin. Stimulation of intracellular cGMP levels was seen as early as 1 minute after incubation with thymosin fraction 5 and was maximal at approximately 10 minutes. Dose response studies indicated an optimum stimulation of cGMP with a thymosin concentration of 100 microg/ml. A control spleen fraction prepared by an identical procedure as fraction 5 did not affect the levels of either cyclic nucleotide.     

Separation of rosette formation-inducing polypeptides from the supernatant of cultures of rat thymic epithelial cell line

Four polypeptide fractions were isolated by high-pressure liquid chromatography (HPLC) from culture supernatant of rat thymic epithelial cell line (IT-45R1). Biological activity of these fractions was examined according to the capacity inducing rosette-formation between rat thymocytes and guinea pig erythrocytes. A relatively rich population of non-rosette-forming cells (non-RFC), one of targets for thymic hormones, was separated from rat thymocytes by combining rosette-formation method with differential centrifugation. Non-RFC consists of outer-cortical and medullary thymocytes. Medullary thymocytes treated with the polypeptide fraction did not differentiate into RFC. Therefore, the cells, which rosette-forming capacity was induced, seem to derive from outer-cortical area. One of the polypeptide fractions (estimated molecular weight: 3 K) possessed the activity endowing non-RFC with rosette-forming capacity. Since its molecular weight was similar to that of thymosin alpha 1, the fraction was pretreated with anti-thymosin alpha 1 antiserum. The pretreatment suppressed the activity of the fraction. Thus, the fraction must contain thymosin alpha 1 or a polypeptide possessing an antigenic determinant similar to that of thymosin alpha 1. Moreover, two of four subfractions, which were divided from the active fractions by reversed-phase HPLC, showed the biological activity.     

Changes in thymic estrogen receptor expression following orchidectomy.

Estrogen receptor levels were measured in cytosols prepared from thymuses of age-matched normal male and female mice. Thymic estrogen receptor concentrations were significantly higher in females than in males. Castration restored the thymic estrogen receptor expression in males to levels nearly equivalent to that of females. The results suggest that androgenic hormones may suppress estrogen receptor expression in thymus and thus account at least in part for the well documented inferior immune response of males. The data further suggest a role for estrogenic hormones in thymic function and thymocyte maturation.     

Developmental changes of serum thymosin alpha 1 and beta 4 in male and male castrated pigs: modulation by testosterone and human chorionic gonadotropin.

Thymic secretory peptides thymosin beta 4 and alpha 1 have possible endocrine roles in both immune and reproductive systems; thus, they should respond to endocrine feedback control mechanisms consistent with gonadal function. In an initial experiment, male pigs (boars; n = 90; 10/time) were bled at 1, 3, 6, 12, 18, 24, 30, 36, and 96 wk of age before and 24 h after hCG stimulation. Thymosin beta 4 concentrations were significantly depressed 24 h after hCG challenge. Testosterone concentrations increased with age up to 36 wk and were further increased with hCG stimulation (p less than 0.01). In a subsequent experiment, boars (n = 12) and barrows (males castrated shortly after birth; n = 12) were blood-sampled, administered hCG, and sampled again 24 h later at 1, 3, 6, 12, 18, and 24 wk of age. Barrows (n = 12) were administered testosterone with the same protocol. Testosterone concentrations increased in boars with maturity and were further increased from the hCG stimulation (p less than 0.01). Thymosin beta 4 concentrations decreased with age in boars and barrows (p less than 0.01), and hCG challenge depressed thymosin alpha 1 and beta 4 concentrations in boars and thymosin beta 4 in barrows (p less than 0.01). Testosterone treatment of barrows also depressed thymosin beta 4 and alpha 1 in barrows (p less than 0.01). The depression of thymosins by hCG treatment points to a role for gonadotropins in altering circulating thymosin concentrations independent of, but in conjunction with, the effect of gonadal steroids.     

Estrogen receptor in the thymus of the castrated mice.

Sucrose density gradient ultracentrifugation and dextran-coated charcoal adsorption permitted us to characterize the estrogen-binding proteins in cytosols obtained from the thymus, spleen and mesenteric lymph node of the castrated male and female mice of C57BL strain. The thymic cytosol from both sexes incubated with 3H-estradiol-17 beta in the presence of excess unlabeled steroids showed a specific estrogenbinding 4 S protein with its binding capacity of 10(-14) moles/mg protein for males and 4 x 10(-15) moles/mg protein for females, respectively. The dissociation constant was of 4 x 10(-10) M for males and 3 x 10(-10) M for females, respectively. No specific binding was, however, found in the cytosols of the spleen and mesenteric lymph node. Steroid analysis by thin-layer chromatography of the thymic cytosols after incubation of them with 3H-estradiol-17 beta showed that a fair amount (around 60%) of radioactivity was from the undegradated radioactive steroid still bound to 4 S binder in both sexes. Enzyme study and heat experiment revealed that the estrogen specific 4 S binding component in the thymic cytosols bears at least protein in nature and is of heat-labile nature. These results strongly suggest that the thymus of the castrated mice contain a specific estrogen receptor, the nature of which is in part protein and heat-labile.     

Immunoregulatory influence of thymic epithelium and thymopoietin on human B-lymphocyte differentiation

Thymic influence upon immunoregulation of B-lymphocyte differentiation in human bone marrow was investigated. Mononuclear cells isolated from marrow of normal adult volunteers were incubated with thymic epithelial monolayers or with the polypeptide thymopoietin. Generation of pokeweed mitogen-stimulated anti-sheep red blood cell antibody-secreting direct plaque-forming cells (PFC) was found to be inhibited following incubation of marrow mononuclear cells with thymic epithelial monolayers. Addition of 50 ng/ml thymopoietin to pokeweed mitogen-stimulated cultures resulted in enhanced marrow PFC responses, whereas higher doses of thymopoietin were inhibitory for the generation of PFC in this assay system. The data suggest that both helper and suppressor T cells are recruited from their precursors in human bone marrow by thymic influences. Generation of helper or suppressor cells may be dependent upon (a) the stage of differentation of precursor T cells and (b) upon the specific action and intensity of the thymic influence.     

The early induction of the actin-sequestering peptide thymosin beta 4 in thymocytes depends on the proliferative stimulus

The expression of the actin-sequestering peptide, thymosin beta 4, was analyzed in proliferating rat thymocytes, activated by diverse stimuli, during the early G1 phase and the S phase. In the presence of concanavalin A a 6.3-fold increase of thymosin beta 4 occurred already after 1 h of stimulation without elevation of the corresponding mRNA level. In contrast, during the S phase the increase of thymosin beta 4 (2.5-fold) was accompanied by a higher mRNA level, but did not exceed the growth related increase of total protein. Stimulation with a crosslinked antibody against rat T cell antigen receptor or stimulation with phorbol 12-myristate 13-acetate (PMA) and Ca(2+)-ionophore A23187, separately or in combination, did not lead to the marked increase of the thymosin beta 4 concentration in the early G1 phase but resulted in elevated thymosin beta 4 peptide and mRNA levels during the S phase. It therefore appears that protein kinase C activation and a rise in cytoplasmic Ca(2+)-concentration are not exclusively responsible for the stimulation of thymosin beta 4 specific translation in thymocytes. This assumption was reinforced by the observation that inhibition of the protein kinase C activity by 1-(5-isoquinolinylsulfony)-2-methylpiperazine (H-7) did not affect the cellular thymosin beta 4 content 1 h and 48 h after concanavalin A (Con A) stimulation.     

In vivo generation of suppressor T cells by thymosin in congenitally athymic nude mice

Treatment of nude mice with thymic factors such as thymosin has been mostly ineffective in generating effector T cells. This study examined the effects of treating nude mice with thymosin fraction 5 on the induction of cells that could participate in and/or regulate cytotoxic T lymphocyte (CTL) generation by normal spleen cells in vitro. Splenic lymphocytes from BALB/c nude mice injected with thymosin fraction 5 every other day for 2 wk were tested for their ability to generate CTL in vitro. Two days after the last subcutaneous injection of thymosin, nude spleens were removed, mixed with normal BALB/c spleen cells, and placed into a mixed lymphocyte tumor culture (MLTC) against allogeneic RBL 5 tumor cells. After a 5-day incubation, cultures were tested for the presence of CTL in a 4-hr 51Cr-release assay. Spleen cells from thymosin-treated nude mice did not generate CTL but suppressed the ability of normal spleen cells to generate CTL in vitro. Characterization of the thymosin-induced nude mouse suppressor cells showed them to be Thy 1 positive, nonadherent, cyclophosphamide-sensitive T cells. These data demonstrate that some T cell maturation occurs in vivo under thymosin influence. However, the activity of these cells is initially limited to a regulatory function. These studies suggest that maturation of functional suppressor T cells occurs before CTL. Further immunologic manipulation appears to be necessary in order to induce CTL effector cells in nude mice.     

Liver cytosolic fatty acid-binding proteins: Effect of diabetes and starvation

The binding of [1-¹⁴C]oleate to rat liver cytosol was studied, using gel filtration on Sephadex G-75 and G-50. In liver cytosols from control rats, most of the high-affinity oleate binding was in the region of 12 000-dalton proteins. In liver cytosols from diabetic and starved rats, a second peak of radioactivity appeared in the void volume. This peak was shown to be associated with a component having the molecular weight of 400 000. Evidence suggesting that a change in the composition of cytosolic binding proteins is involved is presented.     

Phytohemagglutin-stimulated human T cell: prothymosin alpha as an accessory signal

Thymic peptide factors are known to modulate proliferation of normal human lymphocytes. In this work, we studied the effect of Prothymosin alpha (Pro alpha) on PHA-stimulated PBMC and PBLC. The observed effects of Pro alpha and thymosin alpha 1 (alpha 1) on PBMC were found to depend on the degree of cell stimulation, dose, and preincubation-time. Thymosin beta 4 (beta 4) had no effect on either cell type, regardless of the degree of stimulation, which shows that beta 4 may be used as a control peptide to work in this area. Induction of lymphoproliferation also depended on the presence of macrophages. Addition of monocytes or a cell-free monocyte culture supernatant (not containing IL-2) to the PHA-stimulated PBLC cultures resulted in T cell proliferation. Although IL-1 could not restore the PHA-induced proliferative response of isolated T cells by itself, it would enhance the helper effect of Pro alpha. Moreover, a polyclonal goat anti-human IL-2R (Tac Ag) did block the proliferative response induced by combined rIL-1 and Pro alpha, suggesting that an IL-2-dependent pathway of T cell proliferation was involved.     

Requirements for stimulation of autoreactive T cells by thymic stroma

Thymic stromal cells are more efficient than similarly treated spleen cells for Ag presentation to Ag-specific, MHC-restricted T cell lines. Thymic stromal cells fail, however, to stimulate proliferation of autoreactive T cell lines. This failure to stimulate autoreactive T cells does not appear to be due to tolerance induction because thymic stroma does not interfere with subsequent stimulation by spleen cells. Moreover, the ability of thymic stromal cells to stimulate autoreactive T cells can be restored by addition of exogenous IL-1. This demonstrates that the specific self-determinants recognized by autoreactive T cells can be expressed on thymic stromal cells. Failure of stimulation by thymic stromal cells in the absence of exogenous IL-1 may reflect a difference in the physiologic requirements for activation of autoreactive T cells as compared to Ag-specific, MHC-restricted T cells.     

Stimulatory effect of thymic factor(s) on steroidogenesis in cultured rat granulosa cells.

Thymic cells from immature female rats were isolated and used for production of thymic cell culture conditioned medium (TCM). Granulosa cells were obtained from immature diethylstilbestrol (DES)-treated rats. TCM stimulated basal progesterone and estradiol secretion from the granulosa cells in a dose and time dependent manner. Maximal stimulation of progesterone production occurred at 48 hours of incubation, during which period TCM caused approximately 5 times more progesterone secretion than heart cell conditioned medium (HCM) or mock extract (ME). The maximum progesterone secretion by granulosa cells occurred when they were exposed to 48% TCM causing 7 times more progesterone secretion than controls. Under the same maximum stimulatory conditions, however, TCM only approximately doubled estradiol secretion compared to concentrations secreted in the presence of HCM or ME. Thus, the effect of TCM on progesterone secretion was more prominent than its effect on estradiol secretion. The stimulatory action of TCM was not mimicked by HCM, thymosin-alpha 1 or thymulin. Furthermore, the stimulatory action of TCM on steroidogenesis did not appear to be mediated by the cAMP system. The stimulatory factor(s) in TCM were heat, acid and acetone labile, but could not be sedimented by activated charcoal. Thus, the present study demonstrates that the secretory product(s) of thymic epithelial cells can stimulate steroidogenesis in cultured rat granulosa cells. Our data imply that thymic factor(s) may have a direct effect on ovarian function.     

The amphipathic membrane proteins associated with gangliosides: The Paul-Bunnell antigen is one of the gangliophilic proteins

Two amphipathic protein fractions soluble in organic solvents as well as in water have been isolated from the ganglioside fraction of bovine erythrocyte membranes by successive chromatography in chloroform-methanol mixture on DEAE-Sephadex, silicic acid, and α-hydroxypropylated Sephadex G50 (LH60) columns. These two fractions contained a similar low molecular weight protein but with distinctively different amino acid composition. One of these proteins has been characterized by having a strong Paul-Bunnell antigen activity and had a binding affinity to ganglioside. A similar protein without Paul-Bunnell antigen activity was isolated as the major ganglioside-associated protein.     

The chemistry and biology of thymosin. I. Isolation, characterization, and biological activities of thymosin alpha1 and polypeptide beta1 from calf thymus.

A partially purified extract from thymus tissue termed thymosin Fraction 5 has been shown to reconstitute immunological deficiencies resulting from the lack of thymic function in several animal models, as well as humans with primary and secondary immunodeficiency diseases. Thymosin Fraction 5 consists of a family of polypeptides with molecular weights ranging from 1,000 to 15,000. Several of these polypeptides contribute individually to the biological activity of the parent compound. Two polypeptide components of thymosin Fraction 5, termed thymosin alpha1 and polypeptide beta1, have been characterized chemically and biologically. Thymosin alpha1 is a highly acidic molecule composed of 28 amino acid residues. This polypeptide has potent biological activity and has been found to be 10 to 1,000 times as active as thymosin Fraction 5 in one in vivo and several in vitro bioassay systems designed to measure differentiation and function of thymus-dependent lymphocytes (T cells). Polypeptide beta1, in contrast, is inactive in our bioassay systems, suggesting that it is not involved in thymic hormone action. Sequence analysis and homology studies have indicated that polypeptide beta1, although present in Fraction 5, does not contribute to the biological activity of thymosin Fraction 5.     

IL-1 production by human thymic dendritic cells: studies on the interrelation with DC accessory function

Thymic dendritic cells (DC) have been proposed to play a critical role in the generation of immunocompetent T lymphocytes. Since IL-1 is widely considered to be an important second signal in T cell stimulation, we have studied the ability of isolated human thymic DC to produce IL-1. Using the EL4/CTLL conversion assay standardized with recombinant IL-1 beta (rIL-1 beta), we demonstrate that upon LPS-stimulation thymic DC produce small amounts of IL-1 as compared to peripheral blood monocytes (PBM). In contrast with PBM, DC IL-1 production is not influenced by indomethacin. IL-1 activity was detected in the supernatants of DC cultures from all thymuses tested, although quantitative variability was noted among individual thymic donors. The specificity of the active factor was confirmed by neutralization assays with anti-IL-1 beta mAb. On the other hand, we demonstrate that rIL-1 beta cannot substitute for nor amplify the accessory function of thymic DC and that anti-IL-1 beta mAb fails to block the DC accessory function. Thus we conclude that IL-1 beta might not be a major factor for the efficient DC accessory function toward mature thymocytes recently demonstrated in our laboratory. Of interest, IL-1 beta was also detected in the supernatants of DC-thymocyte cocultures in the absence of mitogenic factor, suggesting that thymocyte contacts can constitute a sufficient signal to induce DC to produce IL-1. These observations indicate that human thymic DC represent an intrathymic source of IL-1 whose role in thymocyte proliferation or maturation remains to be understood.     

Acquisition of non-MHC restricted cytotoxic function by IL 2 activated thymocytes with an "immature" antigenic phenotype

Culture of human thymocytes in interleukin 2 (IL 2) results in the generation of cytotoxic T lymphocytes (CTL) that kill tumor cell targets without major histocompatibility complex (MHC) restriction. Thymic non-MHC restricted CTL expressed Leu-19 antigen, but were generated from thymic precursor cells that lacked expression of Leu-19. In contrast, short term culture in Il 2 of peripheral blood lymphocytes depleted of Leu-19+ lymphocytes did not result in the generation of cytotoxic activity. IL 2 was necessary and sufficient for the generation of cytotoxic thymocytes and induction of Leu-19 antigen expression. Thymic non-MHC restricted CTL were generated from precursor cells expressing CD1, an antigen present on the majority of thymocytes. Furthermore, cytotoxic activity was detected in IL 2 cultured thymocyte populations with an "immature" antigenic phenotype, i.e. CD1+ and CD4+, CD8+. Upon subsequent culture, thymic non-MHC restricted CTL lost expression of CD1, and developed an antigenic phenotype similar to peripheral blood non-MHC-restricted CTL, suggesting that peripheral non-MHC-restricted CTL may originate from these thymic precursors.     

Trypanosoma gambiense: immunity with thymic cell transfer in mice.

This report deals with the enhanced agglutinin production and protection in thymectomized, lethally irradiated mice (TI-mice) with transferred thymic cells from mice immune to T. gambiense. Such mice, when sensitized with trypanosome antigen showed protection against experimental infection and also produced agglutinins. Thymic cells from cortisone-treated immune mice were able to induce the production of agglutinins in TI-mice subsequently injected with antigen. However, these agglutinin titers were very low. In bovine serum albumin gradient centrifugation experiments, agglutinin production could be efficiently induced by inoculation of TI-mice with a rather high density thymic cell subpopulation taken from immune mice. Fractionated by Sephadex G-200, the agglutinins displayed a division into two parts, a first and second peak. The main agglutination reaction was seen in the first or macroglobulin peak. In the fractionation of serum by DEAE-cellulose column chromatography, agglutinins were eluted in two parts, the 0.0175 M and 0.4 M effluents. The agglutination by the 0.4 M effluent was much stronger than that of the 0.0175 M effluent, in agreement with the gel filtration results. The sera containing agglutinins were able to enhance the phagocytosis of trypanosomes by cultured macrophages from the peritoneal cavity of normal and irradiated mice. Delay of parasitemia was evident in some of the TI-mice having detectable agglutinins. The delayed parasitemia resulted from antigenically altered trypanosomes which were able to withstand the lethal factors of TI-mice. Transplantation of thymic cells was considered to be responsible for agglutinins induced by the antigenic stimulation in TI-mice and for protection against experimental infection.     

Cellular regulation of ADP-ribosylation of proteins. III. Selective augmentation of in vitro ADP-ribosylation of histone H3 in murine thymic cells after in vivo emetine treatment

Thymic cells were isolated at intervals of between 0 and 144 h from mice that received one intraperitoneal injection of emetine (33 mg/kg), and thymus weight, incorporation of [14C]leucine into proteins and [3H]thymidine into DNA in intact thymic cells, as well as initial rates of protein ADP-ribosylation in permeabilized cells [A. Sóoki-Tóth, F. Asghari, E. Kirsten, and E. Kun (1987) Exp. Cell Res. 170, 93] were simultaneously monitored. The effect of emetine as an inhibitor of protein synthesis [F. Antoni, N. G. Luat, I. Csuka, I. Oláh, A. Sóoki-Tóth, and G. Bánfalvi (1987) Int. J. Immunopharmacol. 9, 333] corresponds to the induction of sequential cellular events, such as cell exit and remigration, by other antimitotic agents [C. Penit and F. Vasseur (1988) J. Immunol. 140, 3315] and produces an activation of proliferation of cells reentering into this organ. Proliferation, as demonstrated by a large increase in DNA synthesis and entrance into S phase, was kinetically related to an apparent increase in poly(ADP-ribose) polymerase activity in thymic cells and a highly significant in vitro ADP-ribosylation of histone H3. Since no DNA fragmentation occurred in thymic cells, as tested by a fluorometric technique [C. Birnboim and J. J. Jevac (1981) Cancer Res. 41, 1889], it is probable that a selective activation of poly(ADP-ribose) polymerase may have been induced in cells that undergo differentiation and proliferation while repopulating the thymus.     

Thymic hormones and prostaglandins. I. Lack of stimulation of prostaglandin production by thymic hormones

Prostaglandins (PGs) have been implicated as possible mediators of the biological activity of thymic hormones. It has been shown that type E-PGs are able to mimic the action of several thymic hormones and that indomethacin prevents in vivo or in vitro the appearance of Thy-1+ antigen induced by some of these factors. We thus investigated a possible role for PGs in the mechanism of action of different thymic extracts and peptides. Attempts to modulate prostaglandin production showed that neither thymosin fraction 5 (0.01-100 micrograms/ml), nor thymosin alpha 1 (1-10 micrograms/ml), thymulin (0.001-100 ng/ml), thymopoietin II (10-1000 ng/ml) or TP5 (10-1000 ng/ml) affect PGE2, 6-keto-PGF1 alpha, PGF2 alpha and TXB2 production by spleen cells from control and thymectomized mice. These results do not support the hypothesis that prostaglandins could act as mediators of thymic hormones.