Interleukin 1 production and accessory cell function of rat alveolar macrophages exposed to mineral dust particles

Immunostimulatory effects of respirable mineral dust particles on alveolar macrophages (AM) and T lymphocytes were tested in vitro. When rat AM were incubated with fibrogenic silica and asbestos particles, a significant interleukin 1 (IL-1) activity was generated into the culture supernatants, whereas neither AM alone nor AM incubated with non-fibrogenic titanium dioxide (TiO2) particles, however, produced a detectable amount of IL-1. Interleukin 2 (IL-2) activity, as tested with IL-2-dependent CTLL-2 assays, was not detectable from all of these cultures. It was also revealed that concanavalin A-induced proliferation of T lymphocytes was enhanced in autologous AM and nylon wool-passed spleen cell co-cultures incubated only with fibrogenic particles, but not with non-fibrogenic particles. Higher IL-1 activity was detected only from co-cultures exposed to fibrogenic particles, whereas IL-2 activity was almost similar among these co-cultures. These results indicate the differences in immunostimulatory effects on pulmonary macrophages and T lymphocytes among a variety of mineral dust particles.     

Fluorescence demonstration of cathepsin B activity in fractionated alveolar macrophages.

Histochemical localization of cathepsin B in alveolar macrophages (AM) that separated into four different density fractions (I, II, III and IV) by discontinuous Percoll gradient centrifugation was demonstrated in fluorescence microscope using CBZ-Arg-Arg-4-methoxy-2- naphthylamide as a substrate and 5-nitrosalicylaldehyde as a coupling reagent. The least dense AM (fraction I) was found numerous bright yellow fluorescing particles with high intensity in small granules distributed throughout the cytoplasm when compared to the most dense cells (fraction IV). The different localization of cathepsin B activity in the fractionated cells suggested differentiation of lysosomal system and existence of maturational (or aging) sequence in rat AM.     

Changes in chromatin structure during the aging of cell cultures as revealed by differential scanning calorimetry

Nuclei from cultured human cells were examined by differential scanning calorimetry. Their melting profiles revealed four structural transitions at 60, 76, 88, and 105 degrees C (transitions I-IV, respectively). In immortalized (i.e., tumor) cell cultures and in normal cell cultures of low passage number, melting profiles were dominated by the 105 degrees C transition (transition IV), but in vitro aging of normal and Werner syndrome cells was associated with a marked decrease in transition IV followed by an increase in transition III at the expense of transition IV. At intermediate times in the aging process, much DNA melted at a temperature range (95-102 degrees C) intermediate between transitions III and IV, and this is consistent with the notion that aging of cell cultures is accompanied by an increase in single-strand character of the DNA. Calorimetric changes were observed in the melting profile of nuclei from UV-irradiated tumor cells that resembled the age-induced intermediate melting of chromatin. It is suggested that aging is accompanied by an increase in single-stranded character of the DNA in chromatin, which lowers its melting temperature, followed by strand breaks in the DNA that destroy its supercoiling potential.     

Abnormal activation and loss of suppressor T cells in the spontaneously hypertensive rat.

Suppressor T cell function in the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rats was analyzed using syngeneic mixed lymphocyte reaction (SMLR) and concanavalin A (Con A) activation. A depressed SMLR was found in adult SHR but not in adult WKY. IL-2 synthesized by SHR was 40-fold lower than that of WKY, and the suppressor T cells generated in the SMLR were incapable of suppressing IgG synthesis. Precursors of cells that can be activated by Con A to become functional suppressor cells are reduced in adult SHR. Supernatant fluids derived from Con A-activated spleen cells from adult SHR failed to significantly inhibit IgG synthesis by cultures of syngeneic spleen cells compared to supernatant fluids from young SHR or WKY Con A-activated spleen cells. However, spleen cells from both adult SHR and WKY proliferated strongly and released equivalent amounts of IL-2 in response to Con A. Addition of exogenous IL-2 to the SMLR cultures in vitro restored the ability of SHR T cells to respond in the SMLR, with generation of cells capable of suppressing IgG synthesis. Administration of SHR with IL-2 in vivo also restored the suppressor T cell function in the SMLR. These results suggest a defective suppressor T cell activation and loss of suppressor T cell activity as the SHR age.     

Nickel allergy in mice: enhanced sensitization capacity of nickel at higher oxidation states.

Attempts to induce contact hypersensitivity to nickel in mice using, e.g., Ni(II)Cl2 often failed. Here, we report that sensitization was achieved by injecting Ni(II)Cl2 in combination with either CFA or an irritant, such as SDS and PMA, or IL-12, or by administering nickel at higher oxidation states, i.e., Ni(III) and Ni(IV). Although Ni(II), given alone, was ineffective in T cell priming, it sufficed for eliciting recall responses in vivo and in vitro, suggesting that Ni(II) is able to provide an effective signal 1 for T cell activation, but is unable to provide an adequate signal 2 for priming. Immunization of mice with nickel-binding proteins pretreated with Ni(IV), but not with Ni(II), allowed them to generate nickel-specific CD4+ T cell hybridomas. Ni(II) sufficed for restimulation of T cell hybridomas; in this and other aspects as well, the hybridomas resembled the nickel-specific human T cell clones reported in the literature. Interestingly, restimulation of hybridomas did not require the original Ni(IV)-protein complex used for priming, suggesting either that the nickel ions underwent ligand exchange toward unknown self proteins or peptides or that nickel recognition by the TCR is carrier-independent. In conclusion, we found that Ni(III) and Ni(IV), but not Ni(II) alone, were able to sensitize naive T cells. Since both Ni(III) and Ni(IV) can be generated from Ni(II) by reactive oxygen species, released during inflammation, our findings might explain why in humans nickel contact dermatitis develops much more readily in irritated than in normal skin.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

Characterization of apolipoprotein B-containing lipoproteins separated by preparative free flow isotachophoresis

Preparative free flow isotachophoresis (ITP) was used for the fractionation of apoB-containing lipoproteins (d less than 1.063 g/ml) from fasting and postprandial sera derived from normolipidemic individuals. According to their net electric mobility, four major particle groups (I-IV) have been recognized. The fast-migrating particles in group I, which correspond predominantly to very low density lipoproteins (VLDL), are rich in triglycerides, free cholesterol, phosphatidylcholine, and apoE and C apolipoproteins. This group expresses nonspecific binding to fibroblasts but binds to HepG2 cells with high affinity (KD = 3.6 micrograms/ml, Bmax = 37 ng) to a single class of binding sites. The particles migrating in group II, which are related to intermediate density lipoproteins (IDL), are richer in cholesteryl esters and apoB than those in group I. They interact specifically with a single site on fibroblasts (KD = 7.8 micrograms/ml, Bmax = 54 ng) while on HepG2 cells two binding sites, one with a higher (KD = 3.5 micrograms/ml, Bmax = 22 ng) and one with a lower affinity component (KD = 16.9 micrograms/ml, Bmax = 53 ng), are involved. The particles migrating in groups III and IV correspond to low density lipoproteins (LDL). The protein moiety of both fractions consists almost exclusively of apoB. Group III represents cholesteryl ester-rich LDL particles, while the particles in group IV contain smaller amounts of cholesteryl esters. The lipoproteins of both groups are ligands for apoB,E-receptors. However, the particles in group IV interact with fibroblasts with the highest affinity (KD = 2.3 micrograms/ml, Bmax = 58 ng) and with the biphasic HepG2 cell binding sites with the lowest affinity of all analyzed groups (KD1 = 11.2 micrograms/ml, Bmax1 = 58 ng, KD2 = 68 micrograms/ml, Bmax2 = 170 ng). When apoB-containing lipoproteins were isolated from postprandial sera of the same individuals, significant changes in the lipid composition were observed only in particle groups I and II, where the triglyceride and phospholipid content was enhanced. Group I particles from postprandial serum bind to HepG2 cells with a higher affinity (KD = 2.5 micrograms/ml) than group I particles from fasting serum. Postprandial group II particles bind with the same affinity to the biphasic HepG2 cell receptor as fasting group II particles, while the affinities of postprandial group III (KD1 = 4.1 micrograms/ml, KD1 = 47 micrograms/ml) and group IV particles (KD1 = 3.9 micrograms/ml, KD2 = 38 micrograms/ml) to the high affinity binding site of the biphasic receptor are enhanced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Suppression of mitogen- and tumor-cell-induced lymphocyte stimulation by tumor-associated fetal antigens

The ability of tumor-associated fetal antigens (TAFA) to suppress mitogen and tumor-cell-induced blastogenesis was investigated. Three different TAFA (I, III, and IV) were tested in PHA and Con A lymphocyte proliferation assays. Spleen cells from New Zealand black rats (NBR) were fractionated over nylon wool; and nonadherent (NA) and adherent (AD) cells were compared with unfractionated (UF) cell responses. Preincubation of NA cells with TAFA-I was followed by addition of phytohemagglutinin (PHA) elicited suppression in a 3-to 4-day assay. UF cells were unresponsive to TAFA and/or PHA at all concentrations tested. TAFA dose—response titration curves in Con A proliferation assays revealed that all TAFA tested (TAFA I, -III, and -IV from fibrosarcomas; TAFA-I and -III from osteosarcomas) were suppressive. For some TAFA, nanogram quantities produced significant suppression. In mixed leukocyte tumor cell assays (MLTC) both UF and NA normal rat spleen cells were tested for proliferative responses to syngeneic tumor cells. Four distinct TAFA, purified by high-pressure liquid chromatography, suppressed lymphocyte proliferation when added to MLTC cultures in 5-day assays. Specificity experiments demonstrated that trinitrophenol-bovine serum albumin did not produce similar immunosuppression. TAFA did not block recognition of tumor antigen nor produce nonspecific cytotoxicity of the spleen cells. Significant suppression of DNA synthesis was produced by TAFA-1 following cocultivation with spleen and tumor cells for 1, 2, and 3 days, compared to no suppression when spleen and tumor cells were washed free of TAFA-I prior to tumor cell addition at Day 0. Similar experiments using rat embryo fibroblasts (REF) as stimulators demonstrated that pre-REF cocultivation treatment of lymphocytes with TAFA-I significantly reduced subsequent blastogenic responses. This effect was not reversible; however, if TAFA-I was added to responders previously stimulated by REF, a suppressive response was not seen. Experiments were also carried out to determine the reversibility of TAFA-I effects. Cells were treated with TAFA-I from 1 to 5 days, followed by determination of lymphocyte blastogenesis. TAFA-I effects are reversible and antigen presence is required to completely suppress (or inhibit) stimulation by tumor cells.     

Immunosuppressive analogues of hexapeptide Tyr-Val-Pro-Leu-Phe-Pro, an immune system stimulant

It was found that substitution of Val2 and/or Leu4 residue in a hexapeptide Tyr-Val-Pro-Leu-Phe-Pro (I) transforms this peptide immunostimulant into analogues possessing the immunosuppressor activity--Tyr-Gly-Pro-Leu-Phe-Pro (II), Tyr-Val-Pro-Gly-Phe-Pro (III), and Tyr-Gly-Pro-Gly-Phe-Pro (IV). Biological effects of peptides I-IV were studied using PFC (plaque forming cell) test, GvH (graft vs host) reaction in mice, and ARFC (autologous rosette forming cell) test. The strongest immunosuppressor activity was observed for III--in this case the immunosuppressor effect was observed even in PFC test in vitro in which II and IV showed no such activity. These results suggest that simultaneous presence of both Val2 and Leu4 residues is necessary for the generation of immunostimulation. The CD study of I-IV in methanol solution suggests that the conformational preferences of II-IV change towards stabilization of the beta-turn structure, whereas in the case of I the gamma-turn on Leu4 and cis' orientation of the Pro3 carbonyl (distorted beta-turn of type I) was found as the preferred conformation. Competition in biological effects observed for I and III in PFC in vitro test suggests that these analogues may interact with the same cellular receptor. Drastic changes in the activity which accompany changes in the sequence are discussed in the terms of our stereochemical hypothesis.     

Both cloned interleukin 2 and purified interleukin 1 are required for optimal growth of purified L3T4+ and Lyt 2+ lymphocytes initiated by concanavalin A

Concanavalin A (Con A), cloned interleukin 2 (IL-2), purified interleukin 1 (IL-1) or two different crude preparations containing IL-1 activity alone, did not induce proliferation of rigorously accessory cell (AC)-depleted splenic L3T4+ or Lyt 2+ lymphocytes. Con A together with saturating concentrations of cloned IL-2 (100 U/ml) promoted less than 40% of the proliferative responses observed in AC-supplemented L3T4+ and Lyt 2+ T-cell cultures. The three preparations of IL-1 used supported minimal proliferation of Con A-treated purified L3T4+ or Lyt 2+ lymphocytes. However, all these IL-1 preparations promoted significant growth of the T-cell populations if AC (1%) were included in the cultures. Cloned IL-2 combined with purified IL-1 promoted proliferation of Con A-treated L3T4+ and Lyt 2+ lymphocytes achieving approximately 75% of the responses observed in AC-supplemented T-cell cultures. The additive effect of IL-1 was apparent in the presence of saturating concentrations of cloned IL-2. Finally, Con A alone induced a detectable number of both L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors as determined with the anti-mouse IL-2 receptor antibody 7D4 by immunofluorescence and FACS analysis. Purified IL-1 neither induced detectable number of L3T4+ or Lyt 2+ T cells to express IL-2 receptors nor increased the number of Con A-treated T cells bearing IL-2 receptors. We have interpreted these findings to indicate the following: Con A alone is sufficient to induce highly purified L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors. Cloned IL-2 and purified IL-1 are required for optimal growth of L3T4+ and Lyt 2+ lymphocytes and these cytokines together efficiently replace AC in growth of T cells initiated by Con A. IL-1 alone does not replace AC in Con A-induced activation of mouse T cells. IL-1 exerts potentiation on IL-2-driven growth of Con A-treated L3T4+ and Lyt 2+ lymphocytes. The additive activity of IL-1 on growth of normal T cells is not due to increased production of IL-2 in the cultures or induction of normal T cells to expression of IL-2 receptors by IL-1. We propose that IL-1 optimizes the action and/or interaction of IL-2 with its receptors on the T-cell membrane (by, i.e., increasing affinity of the IL-2 receptor for its ligand and/or stabilizing the IL-2 receptor).     

Defective activation of T suppressor cell function in nonobese diabetic mice. Potential relation to cytokine deficiencies

Nonobese diabetic (NOD) is an inbred mouse strain susceptible to development of T cell-mediated autoimmune diabetes. The strain is characterized by high percentages of T lymphocytes in lymphoid organs. The syngeneic mixed lymphocyte reaction (SMLR), a T cell response to self MHC class II Ag, is reportedly involved in the generation of a number of immunoregulatory cells, including suppressor inducers. A severely depressed SMLR characteristic of certain other autoimmune strains was found in NOD but not in nonautoimmune SWR/Bm mice. Moreover, IL-2 produced by NOD T cells at day 6 in an SMLR was at least one hundredfold reduced compared with SWR, and NOD T cells harvested from an SMLR at day 6 were functionally defective when tested for ability to induce suppression of an allogeneic MLR. However, functionally competent suppressor T cells were generated in NOD splenic leukocyte cultures in response to Con A, and IL-2 release from these was equivalent to that released by Con A-stimulated SWR splenocytes. A deficiency in cytokine release was not limited to IL-2, because peritoneal exudate cells from NOD exhibited a greatly diminished sensitivity to LPS-stimulated IL-1 release in comparison to SWR mice. IL-2 supplementation both in vitro and in vivo restored the ability of NOD T cells to respond in a SMLR, with production of cells capable of inducing suppression. Like SMLR-activated T cells from untreated SWR controls, SMLR blasts from IL-2-treated NOD mice were enriched for the L3T4 phenotype. IL-1 supplementation in vitro resulted in partial restoration of T suppressor activation in a SMLR. The depressed SMLR exhibited by NOD mice was apparently a stimulator cell dysfunction, because NOD stimulator cells failed to activate T cells from (SWR x NOD)F1 mice, whereas stimulators from SWR or F1 mice were capable of doing so. Collectively, these results suggest a defect in suppressor cell activation rather than an absence of this immunoregulatory cell population.     

Morphological heterogeneity among fractionated alveolar macrophages in their release of lysosomal enzymes

When coupled with separation of alveolar macrophages (AM) into four different density fractions (I, II, III and IV) by discontinuous Percoll gradient centrifugation, ultrastructural heterogeneity was evident in secreting process of lysosomal enzymes. Lower dense AM (I and II) released high levels of acid phosphatase and cathepsin B, whereas higher dense ones (III and IV) did not. Ultrastructurally, there were multiple ruffling and active extension of long cytoplasmic processes from one pole or around the cell surface of AM obtained from the higher density fractions. In contrast, AM from lower dense fractions had much less cytoplasmic processes and contained more cytoplasmic vacuoles showing positive reactions of acid phosphatase. These cells featured more frequently round or ovoid knobs with acid phosphatase activity along and from the tips of the cytoplasmic processes, suggestive of exocytosis. It was suggested that these ultrastructural changes linked to the maturation process and release of lysosomal enzymes from differentiated AM.     

Heterogeneity of immunohistochemical staining with pulmonary surfactant protein A among fractionated alveolar macrophages which involves metabolism of pulmonary surfactant.

Alveolar macrophages (AM) which are separated into four fractionated subpopulations (I, II, III and IV), represented differential immunohistochemical staining with antibody against pulmonary surfactant protein A (SP-A). In light microscopy, the least dense AM (fraction I) were intensely stained with antibody to SP-A in numerous granules of the cytoplasm, whereas the most dense cells (fraction IV) showed immuno-reactivity with the antibody in several granules distributed in the spreading and elongating cytosol. By Western blot analysis, antibody to SP-A recognized a triplet of nature molecules of SP-A in AM lysate. However, the antigen of the AM lysate almost disappeared when the cells were cultured for more than two days, which indicate that AM do not synthesize SP-A and have digested intracellular SP-A during the cultivation. Immunoelectron microscopically, AM of fraction IV sometimes had very large vacuoles including lamellar body-like structures, probably pulmonary surfactant immediately after taken up from the alveolar lumen by them, which were heavily deposited with gold particles indicating antigenic site of SP-A. Whereas cells of fraction I contained numerous cytoplasmic vacuoles that were frequently labelled with the immuno-gold particles and were not associated with lamellar body-like structures, which may indicate that the materials in the vacuoles are digesting. The results of this experiments suggest that pulmonary surfactant, layered on the alveolar epithelium, is in part taken up by higher dense AM and is digested during a process of their maturation in the direction of lower dense cells, which undergo an important role of metabolism of pulmonary surfactant by AM subpopulations.     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

Lymphokine production by mitogen and antigen activated mouse intraepithelial lymphocytes

Although most intraepithelial lymphocytes (IEL) in mouse small intestine bear surface markers classically associated with T lymphocytes, the T-cell nature of these cells remains controversial. In the present study IEL from normal mice, or from mice infected with the gut nematode Trichinella spiralis, were therefore tested for their ability to produce T-cell-derived lymphokines in response to in vitro stimulation with concanavalin A (Con A) or with specific worm antigens. The data show that Con A-stimulated IEL produce minimal amounts of IL-2, and intermediate levels of IFN-gamma and IL-3 in comparison to the levels produced by spleen T cells. The FDC-P2 cell line, which proliferates in response to both IL-3 and GM-CSF, was identified as the most sensitive and reproducible indicator of lymphokine activity in supernatants from mitogen-stimulated IEL from normal mice. IEL isolated from mice infected with T. spiralis also produced high levels of FDC-P2 growth factors when challenged in vitro with Trichinella-derived antigens; however, normal IEL did not respond to this stimulus. The data thus provide evidence that antigen-sensitive T cells can arise in (or migrate to) the gut epithelium during gut infection.     

Interleukin-2 mRNA expression, lymphokine production and DNA synthesis in glutathione-depleted T cells

The stimulation of DNA synthesis in lymphocyte populations was previously shown to depend strongly on the intracellular glutathione (GSH) level. Since T cell growth is known to depend on interleukin 2 (IL-2), the experiments in this report were designed to determine whether intracellular GSH depletion may inhibit IL-2 production or the IL-2 dependent DNA synthesis. Our experiments revealed that IL-2 production and DNA synthesis of mitogenically stimulated splenic T cells have indeed different requirements for GSH. The addition of relatively high concentrations of GSH (5 mM) to cultures of concanavalin A (Con A)-stimulated splenic T cells was found to augment strongly the DNA synthesis but inhibited the production of IL-2. Moderate intracellular GSH levels, however, are apparently not inhibitory for IL-2 production, since intracellular GSH depletion by cysteine starvation or by graded concentrations of DL-buthionine sulfoximine (BSO) had virtually no effect on IL-2-specific mRNA expression and the production of T cell growth factor (TCGF). The DNA synthesis activity, in contrast, was strongly suppressed after GSH depletion with either method. As in cultures of splenic T cells, GSH depletion had no substantial effect on the induction of IL-2 mRNA and TCGF production in several mitogenically stimulated T cell clones. Taken together, our experiments suggest that complex immune response may operate best at intermediate GSH levels that are not too high to inhibit IL-2 production but sufficient to support DNA synthesis.     

Regulation of a cytotoxic response toward alloantigens by T-amplifier and non-T-suppressor cells.

A system is described by which it is possible to generate in vitro non-specific T-amplifier and non-T-suppressor cells without accompanying formation of CTL. Such cells could be separated and tested for their capacity to regulate generation of CTL towards H-2 alloantigen. The experimental setup was as follows: M-locus or syngeneically activated spleen cells were fractionated on BSA-gradients and added to fresh, nylon wool purified (T) precursors. These cultures were stimulated during a second stage with H-2 alloantigen and lytic activities monitored with the chromium release assay. It was found that M-locus activated cells contained two separable subpopulations: 1) an anti-Thy 1 sensitive, non-adherent amplifier cell of intermediate density and 2) a blast cell which was insensitive to anti-Thy 1 as well as RaMIg treatment (i.e. a “null” cell). This cell was adherent, but non-phagocytosing and had strongly suppressive effects on the generation of CTL. It also suppressed—at least partially—the activity of fully differentiated CTL. In syngeneically stimulated cultures strongly enhancing T amplifier cells with blast cell characteristics were found.