Suppression of delayed-type hypersensitivity reactions and lymphokine production by cyclosporin A in the mouse.

Two consecutive daily i.m. injections of cyclosporin A (Cs A) (greater than 50 mg/kg) inhibited delayed type hypersensitivity (DTH) responses in mice immunized with SRBC. Maximal suppression was observed when Cs A was administered 24 and 48 h after sensitization. Culture of spleen cells from these animals with antigen, insoluble concanavalin A (iCon A) or PHA revealed inhibition of the production of two lymphokines: that inducing macrophage procoagulant activity (MPCA) and macrophage chemotactic factor (LDCF). The inhibitory effect on lymphokine production was not due to depletion of T cells. In vitro, 25 ng/ml Cs A suppressed T cell proliferative responses to antigen and mitogen but much higher doses were required to impair the response to LPS. Similar doses of Cs A also suppressed lymphokine production, but the responses of macrophages to these lymphokines was unaffected, even at doses which totally inhibited lymphokine production. Production of interleukin 1 by LPS stimulated macrophages was inhibited by Cs A only at concentrations much greater than those required to suppress lymphokine production.     

Cyclosporin A inhibits lymphokine production but not the responses of macrophages to lymphokines.

Cyclosporin A (Cs A) exerted a dose-related inhibitory effect on antigen (ovalbumin, OVA) and phytohaemagglutinin (PHA)-induced transformation of guinea-pig lymph node cells (LNC). Whereas 0.05 micrograms/ml was sufficient to depress these responses markedly, it required 100-fold this concentration of Cs A to inhibit the production of lymphocyte activating factor (LAF) by lipopolysaccharide (LPS) stimulated peritoneal macrophages. Addition of Cs A together with insoluble concanavalin A (iCon A) to LNC cultures resulted in suppressed lymphokine production, as assessed by measurement of migration inhibition factor (MIF), the generation of macrophage procoagulant activity (MPCA) and the release of lymphocyte-derived-macrophage chemotactic factor (LDCF). Cs A also inhibited MIF and procoagulant production by sensitized peritoneal exudate cells in response to antigen, at the same concentrations which blocked lymphocyte transformation. In contrast, Cs A had no direct effect on the migration of peritoneal cells from capillary tubes, or on the responses of macrophages to preformed MIF, the lymphokine inducing MPCA or LDCF. Overnight incubation of macrophages with Cs A did, however, result in mild inhibition of their basal level of procoagulant activity.     

Ontogeny of mitogenic responsiveness to PHA and sheep erythrocytes and lymphokine production in foetal guinea-pigs.

Pre-culture of spleen cells for 24 h before addition of activator greatly improved the mitogenic response to phytohaemagglutinin (PHA) and sheep erythrocytes (SRBC). Spleen cells were separated into plastic-adherent and non-adherent populations and in some experiments purified further to 'macrophages' and 'lymphocytes' respectively. A critical mixture of lymphocytes and macrophages (10:1) gave twice the mitogenic response of lymphocytes alone to PHA and SRBC in cultures of foetal, juvenile and adult guinea-pig spleen cells. Vigorous mitogenic responses to PHA and SRBC were found at 36 days of gestation. The mitogenic response increased from 46-56 days of gestation to above adult level. Lipopolysaccharide (LPS) obtained by the Westphal method was a superior mitogen to LPS extracted by the Boivin technique. Plaque-forming cells (PFC) could not be induced in vitro at any age even with the addition of LPS. Evidence was obtained for mitogenic and blastogenic lymphokines produced by lymphocytes activated with PHA or produced by lymphocytes activated with PHA or SRBC (but not LPS). These lymphokines were produced by activated splenic lymphocytes of a 40-day-old foetus; older animals showed no evidence for a quantitative increase in lymphokine production. The onset and maturation of mitogenic responsiveness in the guinea-pig and human foetus is compared by age-equivalence.     

Interactions of human T cell subsets during the induction of cytotoxic T lymphocytes: the role of interleukins.

In this work we study the role of subsets of human T cells, detectable by the OKT series of monoclonal antibodies, in the production of and the response to the lymphokine interleukin-2 (Il-2) during the course of an allogeneic cytotoxic T lymphocyte response in vitro. The results obtained establish that the Il-2 producer cells reside within the OKT4 positive T cell subset. Once produced, Il-2 mediates the clonal expansion of alloantigen-activated cytotoxic T killer cells which reside in the OKT8 positive T cell subset. Il-2 appears to have no mitogenic activity on the activated OKT4 positive T cells which produce the lymphokine. In order to release Il-2, the OKT4 positive T cell requires a stimulus, such as allogeneic cells or the lectin phytohaemagglutinin A (PHA). Macrophages are also required for Il-2 production, but the macrophage requirement can be bypassed by a soluble macrophage product as found in supernatants of lymphocyte cultures stimulated with lipopolysaccharide (LPS), the biological activity presumably representing Interleukin-1 (Il-1).     

Leucocyte migration inhibitory activity in supernatants of cultured human mono-nuclear cells stimulated with mitogens or C3- and Fc-receptor reactions.

We studied whether reactions at the lymphocyte membrane receptors for complement, immunoglobulin or mitogens would induce lymphokine production. Human peripheral blood mono-nuclear cells were stimulated by C3- or Fc-receptor reactions and with phytohaemagglutinin (PHA) or lipopolysaccharide (LPS) and cultured for 3 or 4 days. C3- and Fc-receptor reactions were brought about by rosette formation with red cells coated with human complement or antibody. Culture supernatants were assayed for migration inhibitory activity with human leucocytes by the leucocyte-migration-in-agarose test. On the average, no migration inhibitory activity could be detected in cultures stimulated by C3- or Fc-rosette formation, mean migration indices (MI) being 99-2 and 96-1, respectively. Of the mitogens, PHA induced distinct lymphokine synthesis (mean MI 60-2) while the mean MI with LPS varied from 97-9-69-8, depending on the mitogen preparation used and the conditions of culture. We conclude that PHA and LPS are able to activate human lymphocytes into elaboration of migration inhibitory factors whilst reactions at C3- or Fc-receptors fail to do so.     

Differential in vitro effects of etretinate and retinoic acid on the PHA and con A induced lymphocyte transformation, suppressor cell induction and leukocyte migration inhibitory factor (LMIF) production

The in vitro influence of two retinoids, etretinate and retinoic acid, on the human lymphocyte transformation, on the induction of suppressor cells and on leukocyte migration inhibitory factor (LMIF) production were investigated. Nontoxic concentration of retinoic acid increased significantly the PHA response to suboptimal mitogen concentrations, but had no effect on the Con A response; it also abolished the PHA induced LMIF production. In corresponding assays etretinate was without effect. Etretinate augmented the PHA induced suppressor cell activity, while retinoic acid was ineffective. No effect was observed on Con A induced suppressor cells by either retinoid. The findings extend the information about the immunomodulatory effects of retinoids and demonstrate that retinoic acid and etretinate have different effects on PHA and Con A induced immune responses. The mode of action of retinoids is discussed.     

Cytomegalovirus (CMV): specific lymphokine production in congenital CMV infection.

The studies reported here were designed to examine cytomegalovirus (CMV)-specific lymphokine production in infants with congenital CMV infection to elucidate the role of the efferent limb of the immune response to this virus. Mononuclear cells (MNC) from adult control donors and congenitally infected infants were stimulated with mitogen (PHA) or antigens (CMV, mumps), and the supernatants were assayed for production of migration inhibitory factor (MIF) and interferon (IFN). Concordance was observed between lymphocyte proliferative responses to CMV antigen and production of MIF in both control donors and congenitally infected infants. PHA-stimulated cultures from both controls and patients resulted in immune-specific IFN-gamma production in all cases. CMV-stimulated MNC from controls produced IFN when lymphocyte proliferation responses to the antigen were present, whereas those with absent proliferation did not produce IFN. CMV-induced IFN was detected in several patients who had reduced lymphocyte proliferative responses to CMV. The predominant species of IFN stimulated by CMV was neutralized by anti-IFN-gamma, suggesting that CMV induced primarily immune-specific IFN-gamma. In some cases, IFN activity was also reduced to a lesser degree by anti-IFN-alpha, suggesting either the presence of IFN-alpha or of cross-reacting antigenic determinants shared by the two species of IFN. Mumps viral antigen induced primarily IFN-alpha regardless of the immunological status of the donor. We conclude that lymphokine production in congenital CMV reflects the state of activation and/or proliferation of CMV-specific T helper cells rather than an intrinsic defect in the efferent limb of the immune response.     

Beta-lactam antibiotics and human lymphocyte function: The in vitro effect on blastogenesis,lymphokine production and suppressor cell functions

The effects of penicillin, cephalothin and cefoxitin on lymphokine production and mitogen stimulation of human peripheral blood lymphocytes and suppressor cell functions in man were studied with concentrations achievable in serum. Penicillin (50 and 100 μg/ml) and cephalosporins (50 and 100 μg/ml) directly stimulated production of the lymphokine leukocyte aggregating factor (LAgF) by unstimulated lymphocytes and enhanced mitogen-stimulated production of this lymphokine. Neither β-lactam derivative interfered directly with neutrophil aggregation. Using the same concentration, penicillin did not suppress but had either no effect on or slightly increased the unstimulated and mitogen-stimulated lymphocyte transformation responses, whereas cephalosporins significantly suppressed both responses. Suppression of blastogenesis by the latter could be mediated through suppressor cell enhancement as indicated by the significant suppressed lymphocyte transformation observed while using the supernatant of cephalosporin-stimulated and cultured lymphocytes. This suppressive activity was more pronounced with cephalothin than with cefoxitin while it was not observed with penicillin which rather stimulated thymidine uptake. These findings suggest that enhancement of lymphokine production and suppression of lymphocyte transformation by cephalosporins are not contradictory and reflect a stimulating effect on two different lymphocyte subsets. The enhancement of suppressor function might account for the lower incidence of late hypersensitivity reactions observed in patients treated with the cephalosporins compared with those treated with penicillins.     

Influence of growth hormone-releasing hormone (GHRH) on phytohemagglutinin-induced lymphocyte activation: comparison of two synthetic forms. GHRH and PHA-induced lymphocyte activation.

The in vitro effect of synthetic human growth hormone-releasing hormone (GHRH) on mitogen-induced lymphocyte proliferation and lymphokine secretion was investigated. Peripheral blood mononuclear cells (PBMC) of healthy adults were incubated in the presence and absence of increasing concentrations (from 0.006 to 50 micrograms/ml) of two forms of GHRH differing in amino-acid sequence (GHRH 1-44 and GHRH 1-29) or of increasing concentrations (from 0.0012 to 20 U/ml) of recombinant human insulin (rh-insulin). Low concentrations of GHRH 1-29 increased phytoemoagglutinin (PHA)-induced lymphoproliferation, while high concentrations inhibited lymphocyte response, interleukin-2 (IL-2) secretion and IL-2 receptor expression on activated cells. A toxic effect was excluded since no differences in cell viability were observed between cells cultured with and without hormone. GHRH 1-44 did not affect PHA-induced lymphoproliferation, IL-2 production and IL-2 receptor expression. Low concentrations of rh-insulin increased PHA-elicited lymphoproliferation, while high concentrations did not decrease lymphocyte response. The present study suggests that GHRH modulates in vitro human T lymphocyte functions.     

Perturbation of the human immune system by lithium.

The effect of lithium on the human immune system is unknown. We studied lymphocyte responses to mitogens and their ability to produce lymphokines in four patients before and during lithium therapy. We found that with time, lithium increased lymphocyte responses to mitogens (PHA and PWM) and lymphokine production was altered from migration stimulation factor to migration inhibition factor. Although the exact mechanism is unknown, we propose that lithium has an effect on the suppressor cell system.     

Inhibition of cytokine production by a tumor cell product.

Supernatants from cultured mouse and human tumour cells, but not mouse or guinea-pig fibroblasts, inhibited the production of a lymphokine, macrophage chemotactic factor, by PHA-stimulated mouse spleen cells. The supernatants affected spleen cells from old, but not young, mice. They were most active if added at the start of the spleen cell culture and did not act by binding phytohaemagglutinin (PHA). The active material had an approximate molecular weight, on membrane filtration, of 1000-10,000 and could be bound to and eluted from Con A-Sepharose. Tumour supernatant factor(s) of similar molecular weight inhibited the production of interleukin 1 (lymphocyte activating factor) in response to lipopolysaccharide by stimulated thioglycollate-induced peritoneal exudate macrophages, but not by Corynebacterium parvum-activated macrophages. Similar tumour-produced material has been found to inhibit the early phase of delayed-type hypersensitivity reactions in older mice. It is suggested that this effect is due, at least in part, to inhibition of interleukin 1 production leading to inhibition of lymphokine production.     

Prolonged desipramine treatment increases the production of interleukin-10, an anti-inflammatory cytokine, in C57BL/6 mice subjected to the chronic mild stress model of depression

BACKGROUND: Depression is associated with activation of the inflammatory response system (IRS). In humans, antidepressants significantly increase the production of interleukin-10 (IL-10), a negative immunoregulatory cytokine. The aims of the present study were to examine the effects of desipramine, a tricyclic antidepressant, on the IRS in C57BL/6 mice with and without exposure to chronic mild stress (CMS). METHODS: We examined the effects of desipramine on the cytotoxic activity of natural killer (NK) cells, the proliferative responses of lymphocytes after stimulation with IL-1, IL-2, lipopolysaccharide (LPS), concanavaline-A (Con-A), phytohaemagglutinin-P (PHA), pokeweed mitogen (PWM), and anti-CD3 monoclonal antibodies, the production of IL-2, IL-4, IL-10 and interferon-gamma (IFNgamma) by T lymphocytes and the ability of B cells to proliferate after stimulation by lipopolysaccharide (LPS). RESULTS: Prolonged treatment of C57BL/6 mice subjected to CMS with desipramine increases the ability of T cells to produce IL-10 and the ability of B cells to proliferate after stimulation with LPS; and significantly decreases the cytotoxic activity of NK cells and the proliferative responses of lymphocytes after stimulation with Con-A, PHA and anti-CD3 monoclonal antibodies. Repeated administration of desipramine to non-stressed mice increases the activity of T lymphocytes, lowers that of B lymphocytes, increases the production of IL-10 by T cells and has no significant effect on the activity of NK cells. CONCLUSION: Prolonged desipramine treatment of stressed and non-stressed C57BL/6 mice induces an increase in the production of IL-10, an anti-inflammatory cytokine.     

Immune function in aged mice. III. Role of macrophages and effect of 2-mercaptoethanol in the response of spleen cells from old mice to phytohemagglutinin, lipopolysaccharide and allogeneic cells.

Spleen cells from old mice responded less well to phytohemagglutinin (PHA), lipopolysaccharide (LPS), lipid A and allogeneic cells than those from young mice. The defect in each case resided with the nonadherent responding lymphocyte population rather than adherent accessary cells (macrophages). This conclusion was reached by showing that macrophages from old mice functioned normally in each of their known roles in lymphocyte responses in vitro; namely, presentation of antigen or mitogen to responding lymphocytes, support of lymphocyte responses and regulation of responses to mitogens and antigens. Normal presentation function was demonstrated by the comparable ability of peritoneal exudate cells from old and young mice to furnish the macrophage requirement for PHA responsiveness in vitro. This conclusion was confirmed by the finding that old, nonadherent cells reconstituted with macrophages from young mice, responded to PHA like unfrationated old cells. Old macrophages were also shown to be normal in their ability to provide the 2-mercaptoethanol (2ME) replaceable support function to young lymphocytes in vitro. Furthermore, replacement of macrophage supportive activity with 2ME did not rejuvenate the response of old cells to PHA, LPS, lipid A or alloantigens. Finally, no evidence of adherent cell suppression of responses of old cells could be obtained by two different approaches. First, although depletion of adherent cells increased, to a similar degree, the responses of both old and young cells to allogeneic stimulation, this procedure did not reduce the difference in responsiveness between them. That is, the decreased response of old cells in a mixed lymphocyte culture (MLC) could not be explained by excessive adherent cell suppression. Second, no evidence for suppression could be obtained in co-cultures of old and young cells responding to PHA, LPS or allogeneic cells. Taken together, these results suggest that macrophage function, in contrast to lymphocyte function, does not decline with age. It is suggested that this difference may result from the different half-lives of macrophages and lymphocytes in the intact animal. In contrast to the above results, old spleen cells acting as stimulators in an MLC were not always less effective than young cells. Furthermore, any difference that did exist between young and old cells was largely ablated by depletion of adherent cells. This result suggests that adherent cells from old mice may determine the ability of old cells to stimulate allogeneic lymphocytes in an MLC. It is not known whether this adherent cell is a macrophage or some other adherent (perhaps regulatory) cell.     

Failure of exogenous interleukin 1 and interleukin 2 to correct decreased lymphocyte transformation in chronic hepatitis B virus carriers.

To test the hypothesis that reduced lymphocyte transformation in response to PHA in chronic hepatitis B virus infection might be due to deficient lymphokine production, lymphocyte transformation was measured in the presence or absence of exogenous interleukin 1, interleukin 2 or both, or, as a source of mixed lymphokines, supernatants from mixed lymphocyte reactions. The response to PHA was significantly impaired in patients compared to controls, but was not corrected by interleukin 1, interleukin 2 or supernatant from mixed lymphocyte reactions over a wide range of concentrations. Variation of the proportion of monocytes in culture or the addition of indomethacin had no effect on lymphocyte transformation. Thus, reduced lymphocyte proliferation in response to PHA in patients with chronic hepatitis B virus infection cannot be attributed to deficient lymphokine production or to active suppression by monocytes or prostaglandins and a direct role for the hepatitis B virus or a viral product is under investigation.     

An inhibitor of lymphocyte proliferation and lymphokine production released by unstimulated foetal monocytes.

Supernatants obtained from 3-day cultures of cord blood monocytes inhibited normal lymphocyte activation by either PHA or in a two-way MLC. Both lymphocyte transformation and lymphokine production was significantly inhibited by these supernatants but not by those derived from adult mononuclear cell cultures. The inhibitory material produced by foetal monocytes was dialysable and was non-cytotoxic to target cells. It is postulated that this factor contributes to the depressed maternal cell-mediated immune response observed in pregnancy.     

Effects of supernatants of polymorphonuclear neutrophils recruited by different inflammatory substances on mitogen responses of lymphocytes

Two different substances, glycogen and thioglycollate, were used to recruit early peritoneal exudate cells (4 h). In the acute phase of the inflammatory response the cellular infiltrate is large, and the predominant cell (>95%) is the polymorphonuclear neutrophil. Supernatant had differing effects on lymphocyte responses to the mitogens PHA and LPS, also carried out in serum-free media, depending on recruiting substance and time of culture. While glycogen-recruited PMN supernatant (GPMN-S) always enhanced splenocyte responses to PHA, thioglycollate-recruited cells (TPMN-S) did not produce an enhancing factor until the cells had been in culture for 24 h. Whereas GPMN-S enhanced the splenocyte response to LPS only after 1 or 4 h of culture, TPMN-S failed to have any significant effect. Thymocyte responses to PHA were facilitated by all supernatants. Dilution of the soluble PMN factors resulted in a suppressive effect on splenocyte responses to both PHA and LPS, regardless of whether PMN were recruited by the thioglycollate or glycogen or of the time of cell incubation. These results indicate that PMN-rich cell populations of different types of activity are recruited by glycogen and thioglycollate and that these cells produce factors capable of potentiating, enhancing, or suppressing responses to T- or B-cell mitogens by normal syngeneic lymphocytes.Supported in part by grant R01 DE 04677 from the National Institute of Dental Research and the Brigham Surgical Group Foundation.     

The functional association of Lyt antigens with lymphokine production.

The effects of anti-Lyt antibodies on secretion of lymphotoxin, macrophage-activating factor and interferon were studied. Anti-Lyt-1 antibodies added to primary mixed lymphocyte reactions (MLR) significantly enhanced lymphokine secretion, whereas anti-Lyt-2 antibodies had no effect in primary responses. However, when added to secondary MLR, anti-Lyt-1 antibodies manifested moderate enhancing effect on lymphokine levels, whereas anti-Lyt-2 antibodies markedly reduced lymphokine secretion. These results suggested that T cells of different subsets produced lymphokines during primary and secondary responses. The Lyt phenotype of T cells involved in lymphokine secretion and the effects of anti-Lyt antibodies on selected T-cell subsets were therefore studied. It was demonstrated that the majority of lymphokine producing cells during primary MLR reside within the Lyt-1+2- population, which was potentiated by anti-Lyt-1 antibodies, but unaffected by anti-Lyt-2 antibodies. However, during secondary MLR, a considerable proportion of lymphokines was produced by the Lyt-1-2+ population, which was blocked by anti-Lyt-2 antibodies. Lymphokine production induced by concanavalin A, in contrast to that induced by alloantigens, was unaffected by anti-Lyt-2 antibodies. The implication of these results on the role of Lyt molecules in T-cell functions is discussed.     

Mononuclear cell function in Mycobacterium tuberculosis infected guinea pigs

In this study mononuclear cell function was studied in the lymph glands, spleen, and peripheral blood of Mycobacterium tuberculosis infected guinea pigs. Adherent cells from draining lymph nodes and spleens of infected animals spontaneously produced a factor which inhibited normal lymphocyte proliferative responses. As it has previously been shown that this factor activates a population of suppressor T cells, resident lymphocytes in the lymph nodes and spleen were examined and were shown to inhibit normal lymphocyte functions. It is suggested that adherent cells ingesting M. tuberculosis spontaneously release a suppressor cell activating factor (SCAF) which locally activates lymphocytes to become suppressor cells. Even at a time of overwhelming infection, peripheral blood adherent cells could not be shown to release SCAF and peripheral blood suppressor cells could not be identified. Although peripheral blood lymphocyte proliferative responses to PHA were normal in infected animals, their ability to produce the lymphokine macrophage inhibition factor was considerably reduced after the second week of infection. This dissociation between lymphocyte proliferation and lymphokine production is similar to that previously described in humans overwhelming tuberculosis.     

Reduced Th1, but not Th2, cytokine production by lymphocytes after in vivo exposure of healthy subjects to endotoxin

Endotoxin (lipopolysaccharide [LPS]) tolerance is characterized by a reduced capacity of monocytes to produce proinflammatory cytokines upon restimulation in vitro. To determine whether LPS exposure induces a change in lymphocyte cytokine production and whether this results in a shift in the T-helper 1 (Th1)/Th2 balance, whole blood obtained from seven healthy subjects before and after an intravenous injection of LPS (4 ng/kg) was stimulated in vitro with the T-cell stimulus anti-CD3/CD28 or staphylococcal enterotoxin B. Whole-blood production of the Th1 cytokines gamma interferon (IFN-gamma) and interleukin-2 (IL-2) was markedly reduced at 3 and 6 h, while the production of the Th2 cytokines IL-4 and IL-5 was not influenced or was slightly increased. The IFN-gamma/IL-4 ratio was strongly decreased at 6 h. Serum obtained after LPS exposure could slightly inhibit the release of IFN-gamma but increased IL-4 production during stimulation of blood drawn from subjects not previously exposed to LPS. Normal serum also inhibited IFN-gamma production, albeit to a lesser extent. LPS exposure influences lymphocyte cytokine production, resulting in a shift toward a Th2 cytokine response, an effect that may be mediated in part by soluble factors present in serum after LPS administration in vivo.     

In vitro and in vivo suppressive effects of delta-9-tetrahydrocannabinol on interferon production by murine spleen cells

Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, has been reported to be suppressive on some immune functions. Since interferons (IFNs) are important immunomodulatory proteins, the effect of in vivo or in vitro administration of THC on induction of IFN by various mitogens was examined. Splenocytes from normal mice in the presence of THC produced significantly less IFN when stimulated by phytohemagglutinin (PHA), concanavalin A (Con A), or Escherichia coli lipopolysaccharide (LPS). Induction of IFN by a bacterial antigen, Legionella pneumophila bacterial cells, was also suppressed by THC. Also, splenocytes which were incubated up to 24 h in the presence of THC partially recovered responses to mitogens when cells were washed before stimulation. This suggested that THC must be present in order to mitigate IFN induction. Splenocyte cultures from mice which were chronically injected with THC for 6-8 weeks were also less responsive to induction of IFN by the various mitogens. These results suggest that at least part of the immunosuppressive effects of THC may be related to depressed IFN production by stimulated lymphocytes. Since Con A and PHA are T cell mitogens and LPS is considered to be a macrophage and B cell stimulator, suppression of IFN production by these classes of cells indicate a wide range of effects of THC.