Glutathione modulates activation-dependent proliferation of human peripheral blood lymphocyte populations without regulating their activated function

L-Buthionine-(S,R)-sulfoximine (BSO) specifically depletes GSH synthesis by inactivating gamma-glutamylcysteine synthetase, whereas 2-ME augments intracellular GSH concentration. These reagents were used to examine GSH regulation of the proliferation and function of human PBL in response to IL-2 or OKT-3 mAb directed at the CD3 T cell Ag. 2-ME enhanced both IL-2-induced proliferation of PBL and CD3- large granular lymphocytes (LGL) and OKT-3 mAb-induced proliferation of CD3+ T cells. BSO partially suppressed activation-induced proliferation in CD3- LGL and CD3+ T cells and totally inhibited the positive co-proliferative regulation by 2-ME in these cells. By contrast, neither BSO nor 2-ME appeared to affect the activation-dependent differentiation of cytotoxic lymphocytes. The absence of effect of 2-ME or BSO on activation-induced PBL NK activity and T cell cytotoxic potential was supported by their negligible effect on the induction of two different markers of activated cytotoxic lymphocytes, namely pore-forming protein gene expression and benzoyloxycarbonyl-1-L-lysine thiobenzylester-esterase activity. BSO inhibition of CD3- LGL proliferation accounted for the inhibitory effects of BSO on both IFN-gamma production in IL-2-stimulated PBL cultures and IL-2-induced PBL lymphokine activated killer activity. The modulatory effects of 2-ME and BSO on lymphocyte proliferation regardless of phenotype (LGL vs T cell) or stimulation (IL-2, via CD3, lectin, etc.) and the functional differentiation of cytotoxic lymphocytes independent of proliferation suggests that these cells share a common site of GSH regulation close to or at the level of DNA synthesis.     

Molecular signals in B cell activation. II. IL-2-mediated signals are required in late G1 for transition to S phase after ionomycin and PMA treatment

We report that sustained increase of intracellular calcium ion concentration and protein kinase C (PKC) activation maintained throughout the G1 phase of cell cycle do not provide sufficient signals to cause S-phase entry in rabbit B cells, and that additional signals transduced by IL-2 and IL-2 receptor interaction are essential for G1 to S transition. We have shown earlier that rabbit B cells can be activated to produce IL-2 and express functional IL-2 receptors after treatment with ionomycin and PMA. Herein we have compared the response of rabbit PBLs, which contain about 50% T cells, with those of purified B cells. After activation with ionomycin or PMA, comparable numbers of PBLs and B cells entered the cell cycle; but DNA synthesis by the PBL cultures was three to four times higher than that of cultures of purified B cells. Interestingly, IL-2 production by the PBL cultures was also three to four times higher than in B cell cultures, suggesting an involvement of IL-2 in inducing DNA synthesis in these cells. The hypothesis that IL-2, which is produced in early G1, acts in late G1 and is required for G1 to S transition in B cells was supported by the following observations: (i) IL-2 production by B cells was detected as early as 6 hr after activation and preceded DNA synthesis by at least 24 hr. (ii) B cell blasts in G1 (produced by treatment of resting B cells with ionomycin and PMA) showed DNA synthesis in response to IL-2, but showed very little DNA synthesis in response to restimulation with ionomycin and PMA. (iii) A polyclonal rabbit anti-human IL-2 antibody caused nearly complete inhibition of DNA synthesis by B cells activated by ionomycin and PMA. (iv) A PKC inhibitor, K252b, inhibited DNA synthesis in ionomycin and PMA-stimulated cells if added at the beginning of culture but was not inhibitory if added 16 hr later. We conclude that increased [Ca2+]i and PKC activation are not sufficient signals for G1 to S transition in B cells; entry into S is signaled by IL-2, and IL-2-mediated signal transduction probably does not involve increased [Ca2+]i or PKC activation.     

IL-4 inhibits IL-2 receptor expression and IL-2-dependent proliferation of human T cells

Recent studies have shown that IL-4 can affect lymphocyte responses to IL-2. To evaluate the effects of IL-4 on T cell responses to physiologically relevant stimuli, we studied normal human T cells cultured with a low concentration of anti-CD3 mAb and IL-2 in the presence and absence of added IL-4. The addition of IL-4 to cultures of T cells stimulated with anti-CD3 mAb and IL-2 reduced the proliferative response by 49 to 59%. The inhibitory effect was observed in 3-, 5-, and 7-day cultures. Inhibition was dose-dependent with maximal inhibition at concentrations greater than or equal to 5 to 10 U/ml IL-4. IL-4-mediated inhibition occurred early during the T cell response, inasmuch as addition of IL-4 after stimulation for 24 h did not result in significant inhibition. Phenotypic analyses of cells cultured in the presence of anti-CD3 mAb, IL-2, and IL-4 suggested that the mechanism of regulation by IL-4 involves the inhibition of IL-2R expression. The proportion of both CD4+ and CD8+ cells that expressed IL-2R in response to IL-2 was diminished in the presence of IL-4, although HLA-DR levels were unaffected. Soluble IL-2R was also reduced in supernatants of cultures stimulated with anti-CD3 mAb, IL-2, and IL-4 as compared to cultures stimulated with anti-CD3 mAb and IL-2. These findings indicate that when normal human T cells are stimulated in vitro in a manner that approximates a physiologic interaction with Ag in vivo, rIL-4 provides a potent inhibitory signal to IL-2 responsive cells that is likely mediated by IL-4-induced inhibition of IL-2R expression.     

Rapamycin blocks cell cycle progression of activated T cells prior to events characteristic of the middle to late G1 phase of the cycle

The effects of rapamycin (RAP) on cell cycle progression of human T cells stimulated with PHA were examined. Cell cycle analysis showed that the RNA content of cells stimulated with PHA in the presence of RAP was similar to that of control T cells stimulated with PHA for 12–24 hr in the absence of the drug. This level was substantially higher than that seen in cells stimulated in the presence of cyclosporin A (CsA), an immunosuppressant known to block cell cycle progression at an early point in the cycle. However, the point in the cell cycle at which RAP acted appeared to be well before the G1/S transition, which occurs about 30–36 hr after stimulation with PHA. In an attempt to further localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary were examined, including p110^Rb phosphorylation, which occurred at least 6 hr prior to DNA synthesis, p34^cdc2 synthesis, and cyclin A synthesis. In control cultures, p110^Rb phosphorylation was detected within 24 hr of PHA stimulation; p34^cdc2 and cyclin A synthesis were detected within 30 hr. Addition of RAP to the cultures inhibited each of these events. In contrast, early events, including c-fos, IL-2, and IL-4 mRNAs expression, and IL-2 receptor (p55) expression, were only marginally affected, if at all, in PHA-stimulated T cells. Furthermore, the inhibition of cell proliferation by RAP could not be overcome by addition of exogenous IL-2. These results indicate that RAP blocks cell cycle progression of activated T cells after IL-2/IL-2 receptor interaction but prior to p110^Rb phosphorylation and other key regulatory events signaling G₁/S transition. © 1993 Wiley-Liss, Inc.     

Gangliosides suppress the proliferation of autoreactive cells in experimental allergic encephalomyelitis: ganglioside effects on IL-2 activity

Gangliosides have been shown to suppress human and murine lymphocyte proliferative responses in vitro. We tested the suppressive effects of gangliosides on the proliferation of autoreactive lymphoid cells obtained from Lewis rats with experimental allergic encephalomyelitis (EAE). Exogenous rat brain gangliosides inhibited both antigen- and mitogen-induced proliferation by as much as 79 and 93%, respectively. Gangliosides similarly inhibited the antigen-induced proliferation of a myelin basic protein (MBP)-reactive T-cell line which is able to passively induce EAE. Suppression was greatest when gangliosides were added at the initiation of culture, and was not abrogated by supraoptimal antigen concentration. Interleukin 2 (IL-2) activity in culture supernatants was not diminished by the addition of gangliosides. Gangliosides did not inhibit the IL-2-induced proliferation of a murine IL-2-dependent cell line, CTLL-20, unless the IL-2 was first preincubated with gangliosides before the addition of CTLL-20. Preincubation of CTLL-20 with gangliosides resulted in no inhibition of the subsequent responses to IL-2. Exogenous gangliosides did not decrease the binding of a monoclonal antibody directed against the rat cell surface IL-2 receptor. Addition of exogenous IL-2 to ganglioside-suppressed cultures had no effect or only partially restored the proliferative responses. Therefore, gangliosides were shown to inhibit the proliferation of autoreactive lymphoid cells without affecting IL-2 production or IL-2 receptor expression.     

Cell cycle progression of glutathione-depleted human peripheral blood mononuclear cells is inhibited at S phase

Glutathione (GSH) the most abundant nonprotein thiol, is involved in the maintenance of the cellular redox state. In this capacity it may influence lymphocyte responsiveness to various stimuli. We have investigated the requirement of GSH during the activation and proliferation of PBMC. The intracellular GSH content of PBMC was altered by continuous culture or pretreatment with buthionine-S,R-sulfoximine (BSO), a specific and irreversible inhibitor of GSH synthesis. Initial experiments demonstrated that the addition of BSO at the initiation of culture, or shortly thereafter (6 hr), inhibited DNA synthesis and produced a simultaneous decrease in intracellular GSH. It was necessary that the BSO be present in the culture for at least 24 hr prior to the initiation of DNA synthesis for maximal inhibition. Cell cycle analysis revealed that BSO did not affect the entry and progression of PBMC through G1 of the cell cycle, however, entry into S-phase was inhibited in a dose-dependent fashion. These results were further substantiated by the inability of BSO to inhibit IL-2 production and expression of the IL-2R. In addition the timely expression of the transferrin receptor by BSO-treated cells indicated that the block occurred at the G1/S transition. The influence of GSH on early activation events was determined by BSO pretreatments. Lowering the intracellular GSH level of PBMC to less than 10% of the initial content prior to mitogenic stimulation did not impair the ability of these cells to produce IL-2 and express IL-2R, indicating that GSH may not be involved in the generation and response to early activation signals. Furthermore, the removal of BSO from these cultures rapidly reversed its inhibitory effects on DNA and GSH synthesis. In the course of these studies we also observed a modest (17%) albeit consistent increase during activation in the total thiol levels of GSH-depleted PBMC. These thiols may have a key role in the activation process. These data support our hypothesis that GSH is required for lymphocyte proliferation and that additional thiols are involved during the activation process.     

Effect of L-ornithine on proliferative and cytotoxic T-cell responses in allogeneic and syngeneic mixed leukocyte cultures

The effect of L-ornithine on cytotoxic and proliferative responses in mixed leukocyte cultures has been analyzed. The activation of cytotoxic T lymphocytes (CTL) was strongly inhibited when 9 X 10(-3) M L-ornithine was added at the initiation of the cultures. The CTL precursor cells were not completely and irreversibly inactivated, however, since the cells generated normal cytotoxic activity if resuspended after 6 days in fresh culture medium together with a fresh set of stimulator cells. Experiments in microcultures with nylon-wool-nonadherent T-cell-enriched spleen cells as responder cells and "plastic adherent cells" as stimulator cells revealed that the cytotoxic responses were almost completely suppressed if ornithine was added within the first 20 hr but were only partially suppressed if ornithine was added after 48 hr. Also, ornithine had only a mild suppressive effect on proliferative responses in allogeneic and syngeneic mixed leukocyte cultures. The strong suppressive effect of the cytotoxic response was therefore not explained by a general toxic effect of L-ornithine on the responding cells in the culture. The addition of interleukin 2 (IL-2)-containing EL-4 supernatants did not prevent but rather enhanced the suppressive effect of L-ornithine. This indicated that the inhibitory effect was not (exclusively) expressed at the level of the IL-2-producing helper T cells. Since activated macrophages have been reported to secrete arginase, it appears that L-ornithine may be part of a regulatory circuit that selectively regulates the development of cytotoxic effector T cells.     

Cytokine regulation of localized inflammation. Induction of activated B cells and IL-6-mediated polyclonal IgG and IgA synthesis in inflamed human gingiva

It is well established that increased numbers of plasma cells occur in the localized tissues of chronic inflammatory diseases such as adult periodontitis, and enzymatic isolation has shown that most B lineage cells produce IgG-subclass with some IgA-subclass responses. It would be of importance to determine if excess production of cytokines in the localized lesion account for these responses and in the present study we have assessed gingival mononuclear cell (GMC) supernatants for cytokines that activate B cells including IL-6R expression and for levels of IL-6 present. Inasmuch as limited numbers (approximately 1 to 3 x 10(6) cells) of GMC were obtained from surgically removed tissues (approximately 400 mg), we have focused on the analysis of IL-6 production by GMC in this study. Further, initial evidence of additional cytokines that are produced by GMC and induce expression of IL-6R on resting B cells has been obtained. The GMC and PBMC from individual patients were cultured in the presence (or absence) of Con A. Higher levels of IL-6 were produced spontaneously by GMC when compared with Con A-stimulated PBMC. When PBMC cultures were supplemented with GMC supernatants obtained from the same patient, high numbers of spot-forming cells (SFC), mainly of IgG followed by IgA isotype, were seen. The induction of SFC by GMC supernatants was inhibited by incubation with a goat anti-human IL-6 antibody. When the effect of GMC supernatants on subclasses of PBMC SFC was determined, the response was IgG1 greater than IgG2 greater than IgG3 = IgG4 and IgA1 greater than IgA2, a pattern remarkably similar to the distribution of plasma cells in the GMC itself. To assess for cytokines in GMC supernatants that mediated B cell activation, supernatants containing anti-IL-6 were cultured with PBMC or purified B cells for 72 h. This treatment induced small proliferative B cell responses and elevated expression of IL-6R on B cells, but did not induce SFC responses. Further, incubation of B cells with GMC supernatants induced resting B cells (G0/G1) to enter the cell cycle (S and G2/M). Addition of human rIL-6 to these cultures on day 3 restored IgG- and IgA-subclass SFC responses by day 7. Cytokine-induced IL-6R expression also occurred in vivo because freshly isolated GMC expressed high levels of this receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Further evidence for a human B cell activating factor distinct from IL-4

Supernatants from activated human T cell clones were previously shown to contain B cell-activating factor (BCAF), an activity which results in polyclonal resting B cell stimulation. In the present study, we investigate the relationship between this activity and human interleukin-4 which was also shown to act on resting B cells. The supernatant of the T cell clone TT9 contains IL-4 but anti-IL-4 antiserum does not affect the response of B cells as measured by thymidine uptake or cell volume increase. Furthermore, IL-4 induces Fc epsilon-receptor (CD23) expression on 30% of unstimulated human B cells, whereas BCAF-containing supernatants from clone P2, that do not contain detectable amounts of IL-4, promote B cell proliferation without inducing CD23 expression. Our results therefore establish that IL-4 and BCAF are distinct activities and suggest that they trigger different activation pathways in human B cells. In addition, culture of B cells with T cell supernatants for 72 hr induces a three- to fourfold increase in the expression of HLA-DR, -DP, and -DQ antigens in 50% of B cells. The addition of inhibiting concentrations of anti-IFN-gamma, LT, or IL-4 antisera to the cultures does not change these results. Finally, 30% of B cells cultured with T cell supernatants leave the G1 phase of the cell cycle and 20% reach mitosis. Taken together, our findings further support the existence of a B cell-activating factor responsible for the activation of resting human B cells.     

Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

Lymphokine regulation of activated (G1) lymphocytes. I. Prostaglandin E2-induced inhibition of interleukin 2 production

PGE2-induced inhibition of the proliferatory response of PHA-stimulated human PBL and Con A-stimulated murine thymocytes was analyzed by flow cytometry. It was found that the activation process (G0-G1a transition) was not influenced by PGE2 over a wide range of concentrations (10(-10) to 10(-6) M), nor was the formation of IL 2 receptors inhibited. Similarly, the viability of human lymphocytes was practically unaltered. In contrast, the IL 2-dependent cell cycle event (G1a-G1b transition), which is required for proliferation, was inhibited in a dose-dependent fashion. The addition of IL 2-containing supernatants to such cultures prevented the PGE2-mediated block in the G1a phase and reconstituted a normal lymphocyte proliferation. Furthermore, lower IL 2 titers were measured in supernatants from PHA-stimulated human PBL treated with PGE2. These findings strongly suggest that PGE2 primarily exerts its inhibitory effect on lymphocyte proliferation through an inhibition of IL 2 production.     

Development and characterization of interleukin-2-independent antigen-specific human T cell clones that produce multiple lymphokines

The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of T-cell recognition of and response to foreign antigens. In the present study, human antigen-specific helper T cell lines and clones which are completely independent of exogenous interleukin-2 (IL-2) have been developed by cyclic restimulation with the soluble antigen keyhole limpet hemocyanin (KLH) to which the T cell donor had previously been immunized. These T cells uniformly bear the OKT4 phenotype and were shown to require both histocompatible antigen-presenting cells (APC) and antigen for optimal proliferation. The T cell line was composed of a highly antigen-specific and clonable T cell population. Following four cycles of antigen stimulation, limiting dilution cloning analysis showed a Poisson distribution of clonable T cells with a precursor frequency of 0.62, and from 88 to 92% of viable clones were specific for the stimulating antigen. Individual clones were obtained which recognized KLH with either DR 1 (one parental Ia haplotype of the donor) or DR 2 (the other parental Ia haplotype) allogeneic APC, but not both. Following stimulation with KLH, the T cell clones produced IL-2. Peak amounts of IL-2 were assayable in the first 6 to 24 hr after stimulation. In contrast, virtually no IL-2 was detectable in supernatants at 72 to 96 hr, suggesting autoutilization by the proliferating T cells. In addition, some clones were also capable of producing both B cell growth factor and IL-2 following KLH stimulation. These IL-2-independent T cells appeared to be derived from a discrete Leu 8-negative subclass of T4⁺ cells and expressed the full complement of Ia antigen of the donor. Thus, soluble antigen-specific human helper T cell clones have been produced which can be maintained in the absence of exogenous IL-2, elaborate their own growth factors and other immunoregulatory lymphokines, and show fine DR-related restriction to either one or the other parental DR haplotypes in antigen-stimulated proliferative responses.     

Interaction between cytokines and 8-mercaptoguanosine in humoral immunity: synergy with interferon

In previous studies it has been demonstrated that a T cell-like differentiation signal is transmitted by C8-substituted guanine ribonucleosides such as 8-mercaptoguanosine (8MGuo) to antigen-stimulated B cells. A large subset of potentially reactive B cells remains unresponsive to antigen even in the presence of signals provided by these nucleosides except when this signal is preceded by a soluble activity present in mixed lymphocyte culture supernatants. Studies with purified preparations of interleukin (IL)-1, IL-2, IL-3, granulocyte-macrophage colony stimulating factor, B cell stimulatory factor 1 (IL-4), and B cell growth factor II (IL-5) indicated that none of these activities is capable of synergizing with 8MGuo to augment B cell responsiveness to antigen. Therefore, supernatants from a number of cloned cell lines were examined for activity that could synergize with 8MGuo, in order to determine the cellular source of this activity. Soluble products secreted by cloned 24/G1 T cells act synergistically with 8MGuo to evoke enhanced antibody responses to specific antigen in populations of purified B cells. Because concanavalin (Con) A-activated 24/G1 cells produce large quantities of interferon-gamma (IFN-gamma), the possibility that interferons might mediate synergy with 8MGuo was investigated. Purified murine IFN-gamma is unable to interact synergistically with 8MGuo; moreover, treatment of active 24/G1 supernatants with monoclonal anti-IFN-gamma antibodies or at pH 2 fails to abrogate their ability to synergize. In contrast to IFN-gamma, when B cells were supplemented with either IFN-alpha or IFN-beta, antigen-dependent synergy with 8MGuo was observed. However, abrogation of IFN-alpha and IFN-beta activity with specific antibodies fails to interfere with synergy between 8MGuo and mixed lymphocyte culture or Con A supernatants. Therefore, it appears that although IFN-alpha and IFN-beta are not responsible for the synergizing activity present in activated T cell supernatants, they nonetheless represent a previously unrecognized source of synergizing activity.     

Effect of anti-beta2-microglobulin on antigen and allogeneic lymphocyte-induced proliferation of human lymphocytes.

The effect of anti-beta2-microglobulin (beta2m) on the mixed lymphocyte reaction (MLR), and an antigen-induced proliferative response was studied. Anti-beta2m IgG and Fab' fragments completely inhibited the MLR. Preincubation of stimulator or responder cells with anti-beta2m suggested that the major effect of anti-beta2m may be on the responder cell population. A clear-cut effect on responder cells was demonstrated by showing that anti-beta2m completely inhibited a MLR in which the stimulator population was a beta2m negative lymphoblastoid cell line. Anti-beta2m also inhibited PPD-induced proliferation of sensitized lymphocytes. The kinetics of this inhibition indicated that anti-beta2m added within the first 18 hr of stimulation was effective in inhibiting the proliferative response. These data are discussed in light of the hypothesis that beta2m may be a subunit of an antigen receptor on T cells.     

Neoplastic cells obtained from Hodgkin's disease are potent stimulators of human primary mixed lymphocyte cultures

Neoplastic cells obtained from the pleural effusion of a patient with Hodgkin's disease have been maintained in culture since 1978. These tumor cells have been shown to have the cytologic features, cytochemical staining, and cell surface markers of Reed-Sternberg cells. In this study we demonstrate that the cell line termed L428 is a potent stimulator of the primary human mixed lymphocyte reaction. Significant proliferation occurred when mononuclear leukocytes obtained from normal donors were stimulated with radiated L428 cells at responder:stimulator ratios varying from 200:1 to 20:1. Proliferative responses occurred between days 3 and 6 of the cultures with maximal proliferation on day 5. Under optimal culture conditions, mean net proliferative response of 14 normal donors was 51,000 +/- 10,600 dpm. The mixed lymphocyte response was totally blocked by concentrations of monoclonal anti-Ia antibody that had no effect on concanavalin A-induced proliferation. However, the mixed lymphocyte response was not blocked by an anti-K562 cell monoclonal antibody of the same immunoglobulin subclass that binds to the L428 cells. Antigen processing by responder monocytes or Ia-positive cells was not required for the MLC. When responder T cells from two normals were depleted of Ia-bearing cells and monocytes, the mixed lymphocyte reaction between the two normals was eliminated, yet the stimulation of each normal by the L428 cells was not reduced. The cells that proliferated in response to stimulation by the L428 cells were T cells, primarily of the helper subset. No IL 1 activity could be detected in concentrated supernatants of L428 cultures after stimulation of L428 cells by mitogens, phorbol esters, or muramyl dipeptide, or in the MLC. All of these cultures contain fetal calf serum. However, the L428 cells are capable of producing IL 1, because IL 1 was detected when the L428 cells were stimulated with LPS in the absence of fetal calf serum. These neoplastic cells, obtained from Hodgkin's disease, have many similarities to the murine as well as human dendritic cells.     

Modulation of cytolytic T cell function by lectins

Human thymocytes cultured for 5 days in interleukin 2 containing supernatants (IL 2 Sup) virtually become a population of mature T cells (T3+, HTA-) that acquires strong cytotoxic activity against NK-sensitive and NK-resistant target cells. The addition of different lectins to the cultures abrogated the expression of the cytotoxic activity and enhanced thymocyte proliferation. The modulation of this cytotoxic activity by lectins has the following properties: a) inhibition of cytotoxicity is related to the concentration of lectins added, but does not correlate with their mitogenic properties, because either strong mitogens such as PHA or weak mitogens such as wheat germ agglutinin (WGA) are both able to strongly decrease cytotoxicity; b) lectin presence is not required at the onset of the culture but is required during the last 24 hr of the 5-day incubation period; c) reversion of inhibition with full expression of cytotoxic activity can be obtained after removal of the lectin and subsequent culture in lectin-free conditions for at least an 18 to 24 hr period; d) lack of cytotoxicity is observed regardless of target cell specificities and cannot be overcome in a lectin-dependent cytotoxicity assay (LDCC); and e) abrogation of cytotoxicity is not restricted to thymocyte cultures because it can also be observed in peripheral lymphocytes. These results cannot be explained by a simple steric blockade, the overgrowth of a distinctive noncytotoxic lymphocyte population, autologous killing, or a failure in the recognition phase of the lytic event. Modulation by lectins of function-associated cell surface structures implicated in cytolysis is discussed as an alternative hypothesis that might account for the observed phenomenon.     

Aggregated immunoglobulin and Fc fragment of IgG induce IL-6 release from human monocytes

The Fc fragment of immunoglobulin (Ig) has been shown to play an important role in the regulation of humoral immunity, cellular immunity, lymphocyte and monocyte activation, and immune mediator secretion. We wished to determine if Ig or Fc fragments would induce IL-6 production from monocytes. Incubation of monocytes purified from human peripheral blood mononuclear cells with aggregated Ig or Fc fragments of Ig induced interleukin-6 (IL-6) activity in the supernatants. Monomeric Ig taken from an intravenous preparation of Ig, from which all aggregated Ig are removed, would not induce IL-6 production from monocytes whereas as a heat-treated aliquot, presumably containing aggregates, did induce IL-6. The supernatants were assayed according to their ability to induce growth in a murine hybridoma cell line B9, or enhance Ig secretion of B cells stimulated with Staphylococcus aureus Cowan 1 (SAC). The IL-6 activity in the supernatants could be neutralized by a polyclonal rabbit anti-human IL-6 antiserum in both assays of IL-6 activity. Exposure of T-enriched or B-enriched lymphocyte subpopulations to Fc fragments did not induce the release of any IL-6 after 12 hr of incubation, but small amounts of IL-6 were produced by B-enriched cells after 60 hr of exposure to Fc fragments. Hence Fc fragments and aggregated Ig induce peripheral blood monocytes to rapidly secrete large quantities of interleukin-6.     

Production of IL 2 and IFN-gamma by T cells from malaria patients in response to Plasmodium falciparum or erythrocyte antigens in vitro

T cells from patients acutely infected with malaria exhibit a disease-related stimulation of DNA synthesis in response to Plasmodium falciparum antigen in vitro. This response is weak and short-lived, suggestive of induction of suppressor mechanisms. Exogenous T cell growth factor (IL 2) that was added to antigen-stimulated T cell cultures enhanced proliferation in antigen-responsive cultures, indicating that the lymphocytes expressed IL 2 receptors. In contrast, the addition of IL 2 to cultures that did not respond to antigen had no effect. Antigen-responsive cultures contained endogenous IL 2 as well, and the antigen-induced lymphocyte proliferation was correlated with IL 2 production. However, the results suggested that IL 2 production by the patients' T cells was insufficient or actively shut off, and that this was responsible for the premature cessation of their DNA synthesis. Supernatants from 60% of the T cell cultures treated with malaria antigen and from 30% treated with RBC ghost antigen contained interferon-gamma (IFN-gamma), as determined by a cytopathic effect inhibition assay combined with acid treatment and antibody neutralization or by an IFN-gamma-specific ELISA. There was no obvious correlation between antigen-induced lymphocyte proliferation and the presence of IFN-gamma in the culture supernatants. A high IFN-gamma activity was also seen in antigen-treated cultures from P. falciparum-immune donors living in highly endemic malaria areas. In contrast, no IFN-gamma was found in supernatants of antigen-treated T cells from healthy donors or patients with Plasmodium vivax malaria. Thus, the IFN-gamma activity of these cultures appears to reflect the presence of antigen-reactive T cells and may be useful as a sensitive indicator of cellular immunity in P. falciparum malaria.     

Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor beta and gamma expression and their JAK-STAT signaling pathway

Deficient T cell immune function and intracellular signaling in cancer patients may result from effects of tumors or their products on lymphocytes. Recently, it was demonstrated that several ovarian carcinoma cell lines could produce soluble factors that inhibited T cell proliferation. The aim of this study is to assess the effect of supernatants from 3 ovarian carcinoma cell lines (OVCAR3, CAOV3, SKOV3) on signal transduction elements that are linked to the IL-2R and its JAK-STAT pathway. A profound inhibition of proliferation, lower level of IFN-gamma and higher level of IL-10 gene expression were observed when CD8+ T cells were co-cultured with supernatants from 3 ovarian carcinoma cell lines. Cell cycle studies on inhibited CD8+ T cells showed most of them were growth arrested in G0/G1 phase. Western blot analysis showed that tumor supernatants suppressed expression of JAK3 and tyrosine phosphorylation of STAT5. JAK1 was not altered and the inhibition of STAT3 only appeared in OVCAR3 cells. Tumor supernatants also partially blocked induction of IL-2R beta and gamma chains expression. These findings suggest that ovarian carcinoma cells may suppress T cell proliferation through inhibition IL-2 dependent signaling pathways, which may be a mechanism of ovarian carcinoma induced immunosuppression.     

Regenerative proliferation of mouse epidermal cells following adhesive tape stripping. Micro-flow fluorometry of isolated epidermal basal cells combined with 3H-TdR incorporation and a stathmokinetic method (colcemid).

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) Laerum, 1969) and brought into a monodisperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G1, S and (G1 + M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. 3H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G2 cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G2 pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G2 phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G2 phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the G1 phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.