In vitro induction of lymphocyte responsiveness by a Strongylus vulgaris-derived mitogen

Proliferation in vitro of peripheral blood lymphocytes both from horses infected with Strongylus vulgaris and from helminth-free ponies was observed in the presence of extracts of the fourth and fifth stage larvae and adults of S. vulgaris. In addition, S. vulgaris extracts induced transformation in cultures of peripheral blood lymphocytes from sheep and dogs and in mouse spleen cell cultures. Nylon wool non-adherent, T cell enriched fractions of lymphocytes from both mice and horses were stimulated by the S. vulgaris larval mitogen while no proliferation was observed in cultures containing nylon wool adherent, B cell enriched fractions. Macrophage co-operation appeared not to be necessary for S. vulgaris mitogen-induced transformation of spleen cells. The S. vulgaris mitogen stimulated a subpopulation of mouse spleen cells different from those responsive to PHA, Con A and LPS. These cells might be T helper cells since B cells were stimulated to proliferate in the presence of both T cells and S. vulgaris larval mitogen. In addition, the supernatant of in vitro cultured larvae of S. vulgaris induced slight, but significant transformation of equine peripheral blood lymphocytes. Therefore, it is possible that the S. vulgaris mitogen released by both viable parasites and degenerating larvae might induce T cell dependent production of immunoglobulin in vivo and account for the beta-globulinaemia, of which IgG(T) is a major component, in S vulgaris infected horses.     

Kinetics of lymphocyte stimulation in vitro by non-specific mitogens

The role of mitogens during lymphocyte activation was studied with kidney bean leucoagglutinin, concanavalin A and kidney bean phytohemagglutinin. The mitogens were removed by treatment with appropriate antisera, which was demonstrated to remove also mitogens already attached to the cells. The process of lymphocyte activation in vitro can be divided into four distinct steps, three of which are mitogen-dependent and the fourth is mitogen-independent. The first step consists of attachment of the stimulatory molecules to the cell membrane. The second step consists of reaction between mitogen and an activating system. During these two phases the cells become preactivated. The establishment of a preactivated state involves at least some synthesis of cytoplasmic RNA. The preactivated state is reversible and during the third step of lymphocyte activation the final result of preactivation is determined. Depending on the presence or absence of mitogen the cells may remain preactivated for over 60 h, they may return to the resting state or they may proceed through the final stages of the proliferation cycle and eventually divide. This fourth step is independent of the presence or absence of mitogen. A prolonged contact between cells and mitogen is required during the mitogen-dependent steps. The process of lymphocyte activation by mitogen is thus continuously being regulated by the stimulatory molecules on the lymphocyte membrane, which may be of considerable significance also for in vivo immunologicai reactions at the cellular level.     

In vitro lymphocyte cytotoxicity. I. Evidence of multiple cytotoxic molecules secreted by mitogen activated human lymphoid cells in vitro.

Multiple families of cytotoxic molecules [Lymphotoxin (LT)] have been identified in phytohemagglutinin (PHA-P) activated human lymphocyte supernatants and lymphocyte homogenates, using gel filtration chromatography on Sephadex G-150. These macromolecules have molecular weights of 80–90,000, 50,000, and 10–15,000 daltons and have been termed LT₂, LT₂ and LT₃, respectively. They are secreted by cells from a variety of lympboid tissues, i.e., tonsil, adenoid, and peripheral blood. The kinetics of appearance of the cytotoxins indicate that all three are present within 16 hr after lymphocyte activation. However, while LT₁ and LT₂ persist in these cultures through day 5, LT₃ is not detectable after day 3. These molecules can also be detected when either PHA or concanavalin A are employed as the stimulating agent. Moreover, the relative amounts of LT₁, LT₂ and LT₃ activity in a given supernatant vary dramatically from culture to culture. Extracellular levels of LT accumulate and peak by 4 to 5 days in culture, however, intracellular levels of LT reach a maximum on day 3 and decrease to very low levels on day 5. Mitogen-stimulated lymphocytes at 3 days contain intracellular levels of LT which are several logs higher than that detectable in unstimulated cells. This observation suggests that both the biosynthesis and secretion of lymphotoxin is governed by a regulatory control process(es).     

T cell surface antigen expression on lymphocytes of patients with AIDS during in vitro mitogen stimulation

Summary Surface marker expression on peripheral blood mononuclear cells (PBMC) was evaluated daily in PHA- and PWM-stimulated cultures of eight AIDS patients and eight normals. Before culture, the patients' cells showed the characteristic decrease in OKT 4+ cells (normals 40.4%, patients 22.3%; P<0.001), increase in OKT 8+ cells (normals 27.6%, AIDS 38.4%; P=0.002), increase in OKT 10+ cells (normals 15.5%, AIDS 42.8%; P=0.002), and increase in HLA-DR+ cells (normals 11.4%, AIDS 28.7%; P=0.01). The percentage of OKT 11+ cells remained unchanged, while the percentage of OKT 3+ cells dropped over the first 2 days in PHA but not in PWM cultures of both groups (PHA: normals 69.8% to 35.1%; P=0.001, AIDS 56.5 to 38.5%; P=0.001, PWM: normals 62.8%–65.9%, AIDS 66.8% to 63.9%), and recovered in both groups by day 5. In PWM cultures OKT 3+ cells increased significantly in normals but not in AIDS (normals 62.6%–77.7%; P=0.04, AIDS 61.8 to 48.7%). OKT 4 expression decreased in normal PHA cultures after 1 day (38.9% to 29.6%; P=0.05) and then recovered by day 5. Its expression increased in AIDS PHA cultures by day 5 (18.0%–41.1%; P<0.001). The final percentage of OKT 4+ cells in AIDS cultures was within the normal range (35.0%–49.0%). OKT 8 expression increased in both study groups after PHA stimulation (normals 29.5%–50.4%; P=0.002, AIDS 37.4%–50.7%; P=0.02) and in normals but not AIDS after PWM stimulation (normals 28.9%–35.5%; P=0.004, AIDS 38.5%–35.6%). Because of the relative changes in expression of OKT 4 and OKT 8, the 4/8 ratio declined in the normal PHA cultures (1.89 to 1.03; P=0.1) and increased in the AIDS cultures (0.68–1.18; P=0.09). Also, the sum of OKT 4+ and OKT 8+ cells in PHA cultures increased from 68% to 94% whist expression of OKT 11 remained unchanged, indicating co-expression of these antigens on individual cells. Both PHA- and PWM-stimulated normal cells showed an increase in OKT 10 (PHA 16.0%–53.4%; P=0.01, PWM 16.1%–33.9%; P=0.03) and HLA-DR (PHA 8.6%–27.3%; P=0.03, PWM 12.5%–26.6%; P=0.07). In AIDS PHA cultures this did not change, and in their PWM cultures OKT 10 expression declined (44.8 to 23.0%; P=0.05). The PHA- and PWM-stimulated cultures of AIDS patients showed a marked deficit in generation of Tac (PHA increased from 5.4% to 77.1% in normals and from 3.2% to 48.0% in AIDS; P=0.001; PWM increased from 6.1% to 35.3% in normals, and from 5.0% to 15.5% in AIDS; P=0.04). Analysis showed that this deficit was limited to a reduced expression on small lymphocytes and that those cells that did become lymphoblasts expressed Tac normally. These results indicate that the poor blastogenic responses in AIDS are related to failure of OKT 10, HLA-DR, and Tac to increase after stimulation.Abbreviations AIDS acquired immunodeficiency syndrome - PBMC peripheral blood mononuclear cells - PHA phytohemagglutinin - PWM pokeweed mitogen - Tac T cell activation antigen - ARC AIDS-related complex of symptoms - IL-2 interleukin 2 - GVHD graft-versus-host disease - HBSS Hank's balanced salt solution - RPMI 1640 Roswell Park Memorial Institute tissue culture medium 1640 - FITC fluorescein isothiocyanate     

Modulation of lymphocyte mitogen responses by cocultured fibroblasts

Immunological reactions in vivo occur in an environment rich in fibroblasts (FB) and other connective tissue cells. The possibility that FB might affect mononuclear cell proliferative responses to mitogens in vitro was examined in a microculture system. Human peripheral blood mononuclear cells (MC) were cocultured with mitomycin C-treated FB in the presence of phytohemagglutinin (PHA) or pokeweed mitogen (PWM). Cocultured FB (at a 1:100 to 1:10 FB:MC ratio) suppressed the response of MC to PHA (by as much as 35%) but did not significantly affect PWM responses. Cocultures of FB and MC were characterized by 10- to 30-fold increases in prostaglandin E₂ concentrations compared to MC or FB cultured alone. Inhibition of prostaglandin synthesis with indomethacin or mefenamic acid significantly reversed the FB-mediated suppression of lymphocyte PHA responses. Prostaglandin-dependent FB suppression of lymphocyte PHA responses was seen only when FB:MC coculture was initiated at the onset of exposure of MC to PHA. When FB were added to MC after 24 hr of culture with PHA, no effect was seen. Addition at 48 or 66 hr resulted in prostaglandin-independent enhancement of lymphocyte proliterative responses to PHA. Thus in cocultures of FB and MC, MC reactivity to PHA may be influenced in part via alterations in FB prostaglandin metabolism. The interaction between FB and MC may be important in the modulation of immune responses in inflammatory lesions.     

Short-term stimulation of lymphocyte proliferation by indomethacin in vitro and in vivo

The effect of short-term (up to 24 h) in vitro and in vivo treatment with indomethacin was studied on the blastogenesis of mouse spleen cells. Indomethacin in itself induced a strong proliferation of the lymphocytes starting after 6 h treatment both in vitro and in vivo. Besides, significantly enhanced the blastogenesis of splenocytes in response to various doses of PHA and Con A. The stimulation of lectin-induced lymphocyte proliferation occurred after indomethacin treatment both in vitro and in vivo. Indomethacin had no major effect on the distribution of Lyt-1+ and Lyt-2+ subsets within the spleen cell population. An important role of the prostaglandins in the early phase of lymphocyte activation is suggested.     

Steric factors in lymphocyte stimulation by organomercurials.

Resting human lymphocytes were stimulated to initiate DNA synthesis by divalent mercury ions or by the divalent organomercurial, 1,4-bismercury-3, 4-dihydroxybutane. Monovalent methylmercury was ineffective, as was mercury-substituted dextran which is a polyvalent compound where the mercury atoms are farther apart than in the divalent butane derivative. These findings suggest that for organomercurials to stimulate lymphocyte DNA synthesis, they must crosslink protein sulfhydryl groups and bring these groups into close proximity.     

Enhancement of intracellular glutathione promotes lymphocyte activation by mitogen

We have examined the effect of chemically modulating intracellular glutathione (GSH) levels on murine lymphocyte activation. Lymphocyte activation was determined by the induction of polyamine synthesis (ornithine decarboxylase (ODC) induction) and DNA synthesis ([3H]thymidine([3H]Tdr) incorporation). Intracellular GSH levels were enhanced using L-2-oxothiazolidine-4-carboxylate (OTC), which delivers cysteine intracellularly, and suppressed by buthionine sulfoximine (BSO), which inhibits gamma-glutamylcysteine synthetase. In addition, the thiol 2-mercaptoethanol (2-ME) was tested for its ability to augment intracellular GSH levels. Our results indicate that both OTC and 2-ME enhance GSH concentrations and [3H]Tdr incorporation in resting and mitogen (concanavalin A)-stimulated cells. The induction of ODC by concanavalin A (Con A) was augmented by the addition of OTC or 2-ME. The GSH concentration of Con A-stimulated cells was reduced when compared to resting cells; however, it was markedly enhanced by OTC or 2-ME. The stimulatory effects of 2-ME on GSH concentrations, [3H]Tdr incorporation, and ODC induction in both resting and Con A-stimulated cells were much more potent than those of OTC. In contrast, BSO suppressed intracellular GSH and [3H]Tdr incorporation in resting and Con A-stimulated cells. BSO also inhibited the promotion of intracellular GSH concentrations and [3H]Tdr uptake by OTC or 2-ME. However, BSO did not affect the induction of ODC by Con A or its enhancement by OTC or 2-ME. We conclude that enhancement of intracellular GSH concentration results in an increased lymphocyte response to mitogen stimulation.     

Periodate-induced lymphocyte transformation : II. Character of response and comparison with phytohemagglutinin and pokeweed mitogen stimulation

Sodium metaperiodate is mitogenic for human peripheral lymphocytes. Evidence of stimulation can be detected as increased thymidine incorporation at 72 h after only 10 sec of exposure to the IO₄⁻. The degree of response varies with lymphocytes from different donors, but maximum stimulation for the healthy donors studied was obtained at concentrations of IO₄⁻ between 10⁻³ M and 4 × 10⁻³ M. Concentrations of 8 × 10⁻³ M and above are non-stimulatory and toxic. Exposures to optimum concentrations for 1 h or longer result in essentially no stimulation and inincreased cell death. However, a significant response to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) remains. The kinetics of response over a 4 day culture period are similar for IO₄⁻, PHA and PWM. The morphology of the blast cells and the degree of response suggest that the IO₄⁻ responsive lymphocyte population may be more closely related to the PWM stimulated cells than the PHA responsive lymphocytes.     

Inhibitors of arachidonic acid lipoxygenase impair the stimulation of inositol phospholipid hydrolysis by the T lymphocyte mitogen phytohaemagglutinin

Piriprost and nordihydroguiaretic acid (NDGA), specific inhibitors of arachidonate lipoxygenase, inhibited phytohaemagglutinin (PHA)-stimulated breakdown of inositol lipids in human T lymphocytes. The dual inhibitors eicosatetraynoic acid (ETYA) and BW 755C, which inhibit both lipoxygenase and cyclooxygenase, also had similar actions, whereas indomethacin and acetylsalicyclic acid, which inhibit cyclooxygenase alone, did not. The effects of lipoxygenase inhibitors and dual inhibitors were reversible. These agents did not inhibit phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) in vitro. Bromophenacyl bromide, and irreversible inhibitor of phospholipase A2, also abolished PHA-stimulated inositol lipid breakdown without affecting PIP2-PLC in vitro. The results are consistent with a role for the PHA-stimulated generation of arachidonic acid and its conversion to lipoxygenase metabolites (e.g. leukotrienes and/or hydroxyeicosatetraenoic acids) as intermediate steps in the signal transduction pathway between cell-surface mitogen receptors and the stimulation of PIP2-PLC in lymphocytes.     

Mastocytoma-medicated suppression of mixed lymphocyte culture and mitogen responsiveness.

Murine spleen cells, stimulated in vitro by allogeneic spleen, display a strong proliferative response with the subsequent development of cytotoxic cells. This proliferation and sensitization can be abrogated by the addition of mitomycin-treated or X-irradiated murine DBA/2 mastocytoma cells (P-815). The substance required for this depression of lymphocyte responsiveness is present in the cell-free supernatant fluids of P-815 cultures. The suppression appears to be due to interference with cell proliferation in the mixed lymphocyte culture, because the P-815 also prevents spleen cells from proliferating in response to the mitogens concanavalin A (Con A), lipopolysaccharide (LPS), and phytohemagglutinin (PHA). The significance of these findings is discussed.     

Effects of adrenoreceptor stimulation on in vitro spontaneous lymphocyte adhesion

The influence of adrenergic agents: alpha, beta-agonists adrenalin and noradrenaline, alpha-agonist mesaton (phenylephrine) and beta-agonist isadrin on spontaneous adhesion of lymphocytes of healthy donors in vitro was studied. It was established, that the influence on lymphocyte alpha-adrenoreceptors caused stimulation, and on beta-adrenoreceptors--inhibition of the lymphocyte spontaneous adhesion. With the help of alpha-adrenoblocking agent phentolamine and beta-adrenoblocking agent propranolol it was demonstrated, that the adrenergic effects revealed were pharmacologically specific.     

Effect of cholinergic stimulation on spontaneous lymphocyte adhesion in vitro

With the help of spontaneous lymphocyte adhesion test it was demonstrated that acetylcholine and carbacholine enhanced the adhesion of lymphocytes. The enhancement revealed was completely abolished by a specific M-cholinergic blocker--atropine, but not N-cholinergic blocker--hexonium. Thus, the enhancement of spontaneous human lymphocyte adhesion by cholinergic stimulation is mediated by M-cholinoreceptors. It is suggested that the phenomenon revealed is mediated by the enhancement of cGMP intracellular levels after cholinergic stimulation.     

Studies on rabbit lymphocytes in vitro : XX. Demonstration of cells reactive to more than one mitogen by mixed sequential stimulation

Rabbit peripheral blood lymphocyte (PBL) cultures stimulated by ConA and then blocked by the addition of competing sugar or antiserum after 6–15 h of ConA prestimulation respond to restimulation by PHA or PWM to a much greater extent than to continuous stimulation or delayed stimulation with PHA or PWM. This effect of mixed lectin sequential stimulation indicates that many of the same PBLs will respond to more than one mitogen, but that some cells require preactivation by one mitogen in order to respond fully to another mitogen. Thus, the number of PBLs which respond to PHA or PWM alone is much less than the number which respond following pretreatment with ConA when the pretreatment effect of ConA alone is blocked. Rabbit PBLs do not respond to LPS and preactivation by ConA does not prepare rabbit PBLs to respond to LPS.