David O. Lucas

Type I and type II cyclic AMP-dependent protein kinase as opposite effectors of lymphocyte mitogenesis

THE role of cyclic AMP in the regulation of cell growth and differentiation has been extensively investigated in many cell types following various growth stimuli1,2. In recent years, peripheral blood lymphocytes treated with antigens3, plant lectins4, lipopolysaccharides5, or periodate oxidation6 have served as a model system for assessing whether cyclic AMP is involved in the regulation of the mitogenic response promoted in these cells by the above classes of agents7,8. There are two conflicting hypotheses, as outlined by Friedman1, concerning the role of cyclic AMP in lymphocyte proliferation. The first hypothesis suggests that the increase in cyclic AMP levels following mitogen stimulation8 and the subsequent increase in 32P incorporated into F1 histones9 and certain cystosol proteins10 implicates cyclic AMP as a positive mediator of mitogenesis in lymphocytes. The other hypothesis reasons that cyclic AMP inhibits proliferation, and this hypothesis is supported primarily by observations from several laboratories that raising the intracellular cyclic AMP level through the use of phosphodiesterase inhibitors11, prostaglandins12, or cyclic AMP analogues11,13–15 effectively inhibits mitogen-stimulated RNA and DNA synthesis. We present here evidence that may help resolve this apparent paradox. Incubation of Ficoll–Hypaque purified human peripheral blood lymphocytes with concanavalin A (conA) leads to the activation of only type I cyclic AMP-dependent protein kinase in these cells even though there are significant amounts of both type I and type II kinase present. But, the addition of dibutyryl cyclic AMP (DBcAMP) to the conA-stimulated lymphocytes at a concentration sufficient to block the synthesis of RNA and DNA results in the activation of both type I and type II cyclic AMP-dependent protein kinase. It therefore seems likely that while the activation of type I protein kinase represents a positive component in the progression of events promoted in a lymphocyte by a mitogenic signal (as it does in other cell types in which a trophic response is evoked), the activation of type II protein kinase (or perhaps types I and II in concert) represents a mechanism by which a negative influence can be imposed on the proliferative process by the generation of abnormally high levels of intracellular cyclic AMP.

Immune function in chronic active Epstein-Barr virus infection

The spectrum of illness attributed to Epstein-Barr virus (EBV) includes patients with symptoms persisting for more than 1 year without any other obvious underlying disease. High titers of antibodies to EBV, either IgG anti-viral capsid antigen or anti-early antigen, can be demonstrated. In this study, 13 patients diagnosed as having chronic active EBV infection were examined to determine aspects of their immunologic status. Morphological examination and fluorescent antibody analysis revealed no abnormalities in the phenotypes of peripheral blood white cells present in these patients. Compared to those from healthy control individuals, mononuclear cells from the patients showed a markedly depressed ability to produce both interleukin-2 and interferon after stimulation with mitogen and a phorbol ester. Studies of natural killer (NK) cell activity revealed that unfractionated mononuclear cells from patients with chronic active EBV infection were significantly lower in killing activity compared to the control group. Fractionation procedures to enrich for large granular lymphocytes resulted in an increase in NK activity for all individuals, but killing activity still remained slightly lower in the patients than in the control group. The dysfunctions which were found in patients with chronic active EBV infection may reflect a primary defect in natural immune functions of the patients predisposing them to a chronic or intermittent clinical disease rather than a self-limiting illness. Alternatively, the abnormalities detected in these experiments may be a result of the viral infection itself.

Heavy-Metal Mitogenesis: Zn++ and Hg++ Induce Cellular Cytotoxicity and Interferon Production in Murine T Lymphocytes

Splenic and lymph node lymphocytes from Balb/c mice were activated in vitro by the heavy-metal cations, Zn++ and Hg++, as noted by the several-fold increases in 3H-thymidine incorporation at 144 h of culture. Optimal mitogenic concentrations of Zn++ and Hg++ were 200 microM and 10 microM, respectively. Data from experiments using T or B splenic lymphocytes enriched by cell passage over nylon wool columns, through use of athymic Nu/Nu mouse spleen cells, or by cell lysis with monoclonal anti-Thy-1 and antibody plus complement, indicated than Zn++ and Hg++ are mitogens for T cells. Removal of macrophages from spleen cells by treatment with carbonyl iron, followed by cell passage through nylon wool, eliminated the lymphocyte responses to Zn++ and to Hg++. Moreover, addition of macrophage-depleted lymphocytes to monolayers of resident peritoneal macrophages restored the lymphocyte responses to these mitogens. Both Zn++ and Hg++ activated splenic lymphocytes to display lectin-dependent cytotoxicity and to produce acid-labile interferon.

Interferon and the cellular immune response: separation of interferon-producing cells from DNA-synthetic cells.

Abstract Spleen cells from mice were separated by albumin discontinuous density gradient centrifugation into six fractions. Cells from each fraction were cultured with a variety of general mitogens and a specific antigen PPD. The cultures then were assayed for mitogenesis and interferon production. The fraction of cells producing interferon were found to belong to a different population of cells from those undergoing a DNA synthetic response. Treatment of the interferon-producing fraction of cells with anti-theta serum and complement eliminated the interferon-producing capabilities of the remaining cells after exposure to PHA, Con A, and PPD but not after exposure to PWM. The results suggest at least a two-cell requirement for the production of interferon in response to PHA, Con A or PPD stimulation. They also suggest that the interferon-producing cells do not belong to the thymus-dependent lymphocyte subpopulation.

Differential production of interferon and lymphotoxin by human tonsil lymphocytes

Abstract Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [ 3 H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [ 3 H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.

Lymphotoxin and interferon production by rosette-separated human peripheral blood leukocytes☆☆☆

Abstract Experiments were undertaken to investigate the abilities of human T and B lymphocytes to produce the lymphokines interferon (IF) and lymphotoxin (LT). Sheep erythrocyte rosette-forming cells (E-RFC) were separated from nonrosetting cells by sedimentation on Ficoll-Hypaque gradients. Unseparated peripheral blood mononuclear cells and T-cell-enriched and B-cell-enriched populations were assayed for lymphokine production and proliferative response after stimulation with the T-cell mitogen, phytohemagglutinin (PHA), or the B- and T-cell mitogen, staphylococcal phage lysate (SPL). PHA induced LT production and cell proliferation primarily in the E-RFC-enriched population. In contrast, SPL preferentially induced LT production in the E-RFC-depleted population. SPL stimulated cell proliferation and induced equivalent IF production in both populations. Thus, PHA and SPL have differential and contrasting effects in terms of the induction of LT production and cell proliferation in T-cell and B-cell populations. In the same experiments there was no population specificity in induction of IF production. Therefore, the potentials to produce LT and IF appear to be inherent in both cell populations with their in vitro expression being dependent upon the nature of the stimulus.

Heavy-metal mitogenesis: thymocyte activation by Zn++ requires 2-mercaptoethanol and lipopolysaccharide as cofactors.

Thymocytes from Balb/C mice were activated in vitro by Zn++ as noted by the several-fold increases in 3H-thymidine incorporation at 144 h of culture. Thymocyte activation by Zn++ required the presence of 2-mercaptoethanol (2-ME) and bacterial lipopolysaccharide (LPS) in concentrations as low as 1.0 ng/ml. Thymocytes were not stimulated by these agents in the absence of Zn++. Bovine serum products, thought to contain trace amounts of LPS, appeared to satisfy this LPS requirement. Interleukin 1 (IL 1) could not replace LPS as a cofactor. Thymocytes did not respond to Hg++ under the culture conditions used here. Thymocyte subpopulation studies showed that cell preparations enriched for peanut lectin receptor-negative, mature thymocytes were activated by Zn++ and required LPS for the response.

Remote simulation to evaluate real-time traffic control strategies

Simulation is a valuable tool for evaluating the effects of various changes in a transportation system. This is especially true in the case of real-time traffic-adaptive control systems, which must undergo extensive testing in a laboratory setting before being implemented in a field environment. Various types of simulation environments are available, from software-only to hardware-in-the-loop simulations, each of which has a role to play in the implementation of a traffic control system. The RHODES (real-time hierarchical optimized distributed effective system) real-time traffic-adaptive control system was followed as it progressed from a laboratory project toward actual field implementation. The traditional software-only simulation environment and extensions to a hardware-in-the-loop simulation are presented in describing the migration of RHODES onto the traffic controller hardware itself. In addition, a new enhancement to the standard software-only simulation that allows remote access is described. The enhancement removes the requirement that both the simulation and the traffic control scheme reside locally. This architecture is capable of supporting any traffic simulation package that satisfies specific input-output data requirements. This remote simulation environment was tested with several different types of networks and was found to perform in the same manner as its local counterpart. Remote simulation has all of the advantages of its local counterpart, such as control and flexibility, with the added benefit of distribution. This remote environment could be used in many different ways and by different groups or individuals, including state or local transportation agencies interested in performing their own evaluations of alternative traffic control systems.

Serum Interferon in Navajo Children with Severe Combined Immunodeficiency Disease Inhibits Lymphoblastogenesis

Two Navajo Indian children with severe combined immunodeficiency disease (SCID) lost reconstituted immune function after virus infections. A serum factor which inhibited normal lymphocyte response to mitogens was found in one of them and led to the examination of sera from five other Navajos with SCID. Mean inhibition by six Navajo sera was 67%; no inhibitor was found in sera from normal adults and children. The inhibitor activity was nondialyzable and heat stable, yet partially sensitive to pH 2.0, suggesting that interferon(s) was present. Interferon (IFN) activity in patient sera ranged from 10 to 300 U/ml. Normal children had peak serum IFN levels of 100 and 30 U/ml in the acute and convalescent periods, respectively, of virus infections. IFNα, IFNβ, and IFNγ were identified in SCID sera by specific antisera. Both inhibitor and IFN activities in three Navajo sera were 88–95 and 89–100%, respectively, removed with anti-IFN antisera. Similar patterns of inhibition of lymphoblastogenesis were seen with IFN standards. IFN levels in the SCID patients did not correlate with documented infections; elevated levels were present when no infections could be documented. The immunologic imbalances in some forms of SCID may be related to circulating inhibitors, possibly interferon.

Antiautologous lectin-dependent cell-mediated cytotoxicity: Evidence for prekiller cells as effector cells

Abstract Spleen cell killing of target cells can manifest through spleen cell-target cell interaction in the presence of mitogenic lectin, lectin-dependent cell-mediated cytotoxicity (LDCC). Spleen cells from C57B1/6 mice immunized with C3H mouse cells were found to be capable of cytotoxicity against autologous and other C57B1/6 spleen cells in the presence of Con A. Thus, alloimmune spleen cells are capable of an anti-self cytotoxic response in the presence of mitogenic lectin, antiautologous LDCC. Antiautologous LDCC is blocked by preincubation of cytotoxic cells with colchicine, an inhibitor of the cytotoxic effector mechanism. Analysis of alloimmune spleen cell subpopulations suggests that the antiautologous LDCC cell is an immature alloimmune cytotoxic cell (prekiller cell). Potent LDCC was found in alloimmune spleen cell preparations depleted of alloimmune cytotoxic T cells (killer-depleted) by three passes on allogeneic cell monolayers genetically identical with the immunizing cell. However, some LDCC effectors were also found to adhere to the adsorbing target, suggesting that there is some maturational diversity among LDCC effectors.