Human Leukocyte Cultures Research Articles

Cytogenetic effects of exposure to zirconium oxychloride in human leucocyte cultures

The addition of different concentrations of aqueous zirconium oxychloride solution to human peripheral blood leucocyte cultures, obtained from healthy donors, increased the frequency of chromosomal aberrations in the cells compared with untreated controls. The degree of enhancement in the occurrence of aberrations and sister chromatid exchanges was directly related to the concentrations used. The most common aberrations were chromatid breaks. The clastogenic effects of this metal in human leucocyte cultures have been reported for the first time.

Dunaimycins, a new complex of spiroketal 24-membered macrolides with immunosuppressive activity. III. Immunosuppressive activities of dunaimycins.

The immunosuppressive effects of the dunaimycins, a new complex of spiroketal 24-membered macrolides, were compared to cyclosporin A, ascomycin, and rapamycin. Each dunaimycin was a potent inhibitor of the mitogenic response observed in mixed murine splenocyte or human leukocyte cultures, and like immunosuppressive drugs these compounds were relatively less potent inhibitors of the constitutive proliferation of murine EL4 thymoma cells. Dunaimycin D4S showed no selectivity in inhibiting the mitogenic response of spleen cells to concanavalin A, pokeweed mitogen, lipopolysaccharide, or phytohemagglutinin. Cyclosporin A and ascomycin did not inhibit interleukin 2 dependent proliferation, whereas the dunaimycins and rapamycin blocked the uptake of [3H]thymidine in mixed cultures supplemented with exogenous interleukin 2. In addition, dunaimycin D4S had no apparent affinity for cyclosporin A or FK-506 immunophilins. Although the dunaimycins inhibited the activity of Na+, K(+)-ATPase, inhibition of this enzyme appeared insufficient to explain the biological activity of these new macrolides. Over a narrow concentration range, dunaimycin D4S showed in vivo immunosuppressive activity in the murine popliteal lymph node hyperplasia model.

Immunophenotype and sister chromatid differentiation: A combined methodology for analyzing cell proliferation in unfractionated lymphocyte cultures

Combination of the MAC (morphology, antibody, chromosomes) and harlequin staining procedures offers a method for direct analysis of cell kinetics in cultures of unfractionated hematopoietic cells. In the present study unfractionated human mononuclear leukocyte cultures were stimulated with PHA or PWM mitogens and exposed to bromodeoxyuridine for various periods. For MAC, cytospin preparations were made and cells were classified with monoclonal B and T antibodies by the immunoperoxidase technique. After differentiation of the different lymphocyte subsets, the cells were stained by a fluorescence-plus-Giemsa method to distinguish sister chromatids and to determine the proportions of first, second, third, or subsequent mitoses among the previously identified subsets. The results showed (1) that the relative proportions of mitotic T and B cells are the same regardless of the mitogen used; (2) that T and B lymphocytes proliferate faster in cultures stimulated by PWM than in those stimulated by PHA; and (3) that T cells enter mitosis earlier than B cells when PHA or PWM are used as mitogens.

Mutagenic effects of TPA-induced clastogenic factor in Chinese hamster cells

Previous work in our laboratory has shown that the clastogenic and SCE-inducing effects of 12- O-tetradecanoylphorbol 12-acetate (TPA) are mediated by secondary products formed by the cell in response to the tumor promoter. A low-molecular-weight clastogenic factor (CF) was isolated from the medium of TPA-treated human leukocytes and caused chromosome aberrations and sister-chromatid exchanges (SCE) in fresh cultures not exposed to TPA itself. In the present study, we show that Chinese hamster fibroblasts (V79 cells) also produce CF when exposed to TPA. CF from V79 cells induced SCE not only in hamster cells, but also in human lymphocytes. Vice versa, Cf from human leukocyte cultures induced SCE in hamster cells. It also increased the frequency of 6-thioguanine-resistant mutants in this cell system. All cyto- and geno-toxic effects of TPA-induced CF were prevented if the cells were treated with superoxide dismutase before exposure. The lipophilic CF seems to be derived from arachidonic acid of cell membranes released as a consequence of oxidative damage and subsequently degraded to genotoxic adehydes in an autoxidative process. CF is formed only under culture conditions with low antioxidant content in culture media and sera. This may explain the discordant results obtained by different laboratories with regard to the genotoxic effects of TPA.

Interleukin 1 production during accessory cell-dependent mitogenesis of T lymphocytes.

We have studied the control and significance of IL-1 production in human leukocyte cultures during accessory cell-dependent, T lymphocyte mitogenesis using sensitive bioassays and immunolabeling techniques. In primary antigen-dependent systems like the MLR, IL-1 production was not detected in accessory cells (monocytes, dendritic cells) or T cells, suggesting that it is not an early product in these responses. However, monocytes could be induced to make IL-1 after interacting with sensitized antigen-specific T cells. Both alloreactive T cell clones or freshly prepared lymphoblasts induced IL-1 provided the monocytes carried the HLA-DR antigens to which the T cells were initially sensitized. Even in these circumstances, dendritic cells and B cells failed to make IL-1. The mechanism whereby activated T cells induce IL-1 in monocytes was explored. Supernatants from cocultures of monocytes and T cells or several recombinant cytokines induced little or no IL-1. A more potent antigen independent pathway of IL-1 induction was identified. IL-1 could be induced in third-party HLA-DR nonspecific monocytes in cocultures of alloreactive T cell clones or blasts and HLA-DR-specific dendritic cells. The induction was factor independent since dendritic cells and T blasts placed in a chamber separate from third-party monocytes by a semipermeable membrane did not induce monocyte IL-1. These results suggest that a cell contact mechanism rather than an IL-1-inducing factor leads to IL-1 production. The role of IL-1 in T cell proliferation was tested with a polyclonal anti-IL-1 antibody. The antibody failed to block the proliferation of primary T cells, or alloreactive T cell clones and blasts stimulated with HLA-specific monocytes or dendritic cells, even though IL-1 in the medium was neutralized.

Induction of interferon-a in human leukocytes by polioviruses: Wild-type strains are better inducers than attenuated strains

Cultures of human peripheral blood leukocytes were exposed to different poliovirus strains at a multiplicity of infection of 1 TCID 50/cell. Induction of interferon was a regular phenomenon in the cultures but the maximal yields varied between experiments. Resistance to moderate temperature (56°) and pH 2, as well as antigenic properties, indicated that most of the activity was due to interferon-α. Low levels of interferon-γ were also measured in the cultures using a radioimmunoassay. Using kinetic uv-inactivation we found that infectivity of the virus preparations was affected more rapidly than the capability to induce interferon-α. Dependence of interferon-a yields on the inducing virus strain was examined in 46 independent cultures by comparing the three serotypes of the attenuated poliovirus vaccine strains and two wild-type strains of serotype 3 (Saukett and P3/FIN/23127/84). The wild-type strains were significantly better inducers of interferon than the attenuated strains ( P < 0.001). Four other wild-type strains representing all serotypes tested in 12 independent cultures were also better interferon inducers than the corresponding Sabin strains ( P < 0.02). Induction of interferon may have a role in the poliovirus-induced inhibition of lymphocyte mitogenesis previously reported by others.

Human microvascular endothelial cells: coordinate induction of morphologic differentiation and twofold extension of life span.

Human microvascular endothelial cells (HMVEC) from adult adipose tissue were cultured in MCDB 131 medium supplemented with 10% fetal bovine serum. Under these conditions, HMVEC from seven different donors had finite proliferative life spans ranging from 14.5 to 23.5 population doublings (PD), with a mean life span of 19 PD. Addition of 10% conditioned medium from activated human leukocyte cultures (BM Condimed) extended the life span of HMVEC to 31 to 41 PD, with a mean life span of 37 PD. At the end of lifespan, HMVEC cultures both with and without BM Condimed had very low labeling indices (0 to 5% [3H]thymidine labeled nuclei) and consisted of enlarged cells. However, the morphologies of the two types of HMVEC cultures were very different. Untreated HMVEC were polygonal endothelial cells that formed cobblestonelike monolayers with no cell overlapping. In contrast, BM Condimed-treated HMVEC were more elongated, less regularly shaped cells that were not strictly inhibited from overlapping. When old, these cells accumulated numerous vacuoles. The BM Condimed-treated HMVEC expressed Factor VIII antigen, which confirms their identity as endothelial cells. These cells reverted rapidly to the polygonal morphology of untreated HMVEC when they were removed from BM Condimed. Likewise, their proliferative capacity was not extended further once BM Condimed was removed. These results suggest that HMVEC can exist in two distinct morphologic states in which the cells have different finite proliferative life spans.

Induction of interferon-γ in human leukocyte cultures stimulated by Zn 2+

Our studies have confirmed previous reports that Zn 2+ has a mitogenic effect on human lymphocytes. Furthermore, we have tested the ability of Zn 2+ to induce interferon production in human leukocyte cultures. Peripheral blood mononuclear cells were stimulated with ZnCl 2 at different concentrations. Interferon was detected in the supernatants of cultures stimulated with concentrations ranging from 5 × 10 −4 to 5 × 10 −5 M. Interferon was produced only in the presence of accessory cells. The type of interferon induced by Zn 2+ was interferon-γ, as proven by the use of specific antisera.

Products of activated lymphocytes and macrophages inhibit mouse embryo development in vitro.

The effects of activated leukocyte products on embryonic development were assessed by adding mouse and human leukocyte culture supernatants and purified murine and human lymphokines and monokines to mouse embryos in tissue culture. Supernatants from mitogen-stimulated and mixed lymphocyte cultures arrested embryonic development at the two-cell to morula stage. Of a panel of six individual lymphokines and monokines tested for effects in this system, both murine and human forms of the lymphokines colony-stimulating factor, interferon-gamma, and human B cell growth factor significantly arrested embryonic development over a wide concentration range. The monokines, interleukin 1 and tumor necrosis factor, also had significant effects but only at high doses. These results indicate that products of activated lymphocytes and macrophages can have detrimental effects on preimplantation embryos. Early abortion could result from local (intrauterine) production of such embryotoxic factors by activated lymphocytes and macrophages in response to stimulation by microorganisms or reproductive tissue antigens.

Induction of interleukin 1 by Legionella pneumophila antigens in mouse macrophage and human mononuclear leukocyte cultures

Exposure to Legionella pneumophila antigens has been reported to result in both an adjuvant effect and pathophysiological changes such as fever, headache, myalgia and arthralgias. Immunoenhancement and inflammatory changes have been associated with the production of interleukin 1, and we, therefore, sought an involvement of interleukin production in the alteration of biological responsiveness following exposure to Legionella pneumophila antigens. Killed Legionella pneumophila cells, incubated with mouse splenocytes, induced the formation of a soluble substance which enhanced splenocyte antibody production to heterologous antigen. The immunoenhancing substance was also produced by mouse peritoneal macrophages and supernatants from these cultures were demonstrated to also contain thymocyte co-mitogenic activity. Following gel filtration, this co-mitogenic activity eluted in the 15,000 molecular weight range suggesting an involvement of interleukin 1. Experiments with Legionella pneumophila cells, and cell extracts containing endotoxin, and purified endotoxin suggested that the interleukin 1 activity was induced by both endotoxin and non-endotoxin antigens. The Legionella pneumophila antigens were also found to be potent inducers of interleukin 1 activity in human peripheral blood mononuclear cell cultures. These results suggest that Legionella pneumophila antigens are potent inducers of interleukin 1 in both mouse and human cells. The induction of this monokine may partially acount for both the immunoenhancing property of this bacterial species and the associated pathophysiological changes following infection with this microorganism.

Inhibition of human lymphocyte proliferation and interleukin 2 production by platelet activating factor (PAF-acether]: Reversal by a specific antagonist, BN 52021

When added to a 72 h culture of human peripheral blood mononuclear leukocytes stimulated with phytohemagglutinin, PAF-acether caused a significant inhibition (40–65%) of proliferation at concentrations of 10 −8 to 10 −6M. This inhibition was reversed by the specific PAF antagonist, BN 52021. It was also reversed by indomethacin, suggesting that PAF-acether mediated this suppression via cyclooxygenase metabolites of arachidonic acid. IL-2 production, measured at 24 h of lymphocyte proliferation, was similarly impaired [50–66%] by 10 −8–10 −6M PAF-acether. IL-2 production was brought up to 90% of control values when both PAF-acether and BN 52021 (10 −4M) were added together to the lymphocyte cultures. These studies suggest a significant immunoregulatory role for PAF-acether and a potential use of BN 52021 as a biological response modifier.

Stimulation of lymphocytes with zinc ions

Publisher Summary This chapter describes methods for the in vitro stimulation of lymphocytes with zinc ions. The chapter describes the mitogenic effect of Zn 2+ . Second, it discusses the role of zinc ions in lymphocyte activation by mitogens in general. It also discusses the potential role of zinc in the immune response in vivo. Zn 2+ is an essential trace element, most important as a cofactor for many enzymes. Zn 2+ is of high nutritional and physiologic significance. Human lymphocyte stimulation by Zn 2+ has been confirmed by a number of workers. Low degrees of nonspecific lymphocyte stimulation by nickel compounds are also described in the chapter. This chapter discusses human leukocyte cultures. The optimal conditions for stimulation of lymphocytes by Zn 2+ are that the human peripheral blood lymphocytes, separated on Ficoll-Hypaque gradients and resuspended in RPMI 1640 medium, containing 5% heat-inactivated fetal calf serum are distributed in the wells of microfiltration plates at the density of 2 × 10 5 cells/well.

Lymphocyte activation and induction of interferon gamma in human leucocyte cultures by the mitogen in Mycoplasma arthritidis supernatant (MAS).

This report succinctly summarizes a number of experiments in which human leucocytes were activated by the mitogen in the supernatant of cultured Mycoplasma arthritidis (MAS). Lymphocytes from a large number of donors were found to be reactive when the two parameters of lymphoproliferation and of production of gamma interferon were analysed. Lymphocytes of cord blood origin were also reactive in lymphoproliferation. Their interferon production was low, but this has been established previously for mitogens in general. During the course of the studies, different batches of MAS were tested, including most recently a purified preparation. All of these were active. Removal of accessory cells adherent to nylon wool column abolished MAS reactivity, whereas it has little effect on lymphoproliferation induced by phytohaemagglutinin (PHA). MAS reactivity was also strongly reduced when monoclonal anti-HLA-DR antibodies were added to the leucocyte cultures.

Modulation of lymphocyte proliferation and immunoglobulin synthesis by interferon-γ and “type I” interferons

Interferon (IFN)-α and IFN-β (“type I” IFNs), but not IFN-γ reduced phytohemagglutininor pokeweed mitogen (PWM)-induced proliferation in cultures of human mononuclear leukocytes. Proliferation induced by specific antigens (tuberculin PPD or tetanus toxoid) or by exogenous interleukin 2 (IL-2) was strongly inhibited by type I IFNs and, to a lesser extent, by IFN-γ as well. Inhibition of proliferation in mitogen-stimulated cultures was not due to a reduced production of IL-2 or to an inhibition of IL-2 receptor expression. Type I IFNs inhibited immunoglobulin (Ig) production in PWM-stimulated unseparated mononuclear cells, whereas IFN-γ enhanced Ig production in such cultures. In cultures of purified B cells type I IFNs caused a stimulation of Ig production and this B-cell differentiation factor (BCDF) hike activity of IFNs was synergistically enhanced in the presence of IL-2. IFN-γ produced less BCDF-like activity than type I IFNs. These results show that in some instances type I IFNs can be more potent in affecting functions of cells of the immune system than IFN-γ.

Supernatants of human leukocytes contain mediator, different from interferon gamma, which induces expression of MHC class II antigens.

In this report, data are presented on the regulation of MHC class II antigen expression by a mediator present in supernatants of human mixed leukocyte cultures (MLC-SN), and which is different from IFN-gamma. The capacity of supernatants to induce antigen expression did not correspond to titers of IFN-gamma. Removal of IFN-gamma using either dialysis against pH 2 or neutralizing mAb against human IFN-gamma did not abrogate the MHC class II antigen expression-inducing capacity of MLC-SN when tested on adenocarcinoma cell lines, kidney epithelial cells, and fibroblasts in vitro in an indirect immunofluorescence assay. Therefore, supernatants of human leukocytes contain a mediator, different from IFN-gamma, which induces expression of MHC class II antigens. Dose-response studies revealed that the mediator is produced after allogeneic and lectin stimulation of human leukocytes, and by unstimulated leukocytes. Activation of leukocytes resulted in increased titers of the mediator. The mediator markedly enhances expression of both HLA-DR and HLA-DQ antigens, whereas IFN-gamma had a similar effect on HLA-DR antigens, and only a minor effect on HLA-DQ antigens. Interaction of the mediator and IFN-gamma resulted in a potentiating effect of these two factors on MHC class II antigen expression. Biochemical analysis revealed a mediator, distinguishable by FPLC from IL-1, IL-2, and human IFN-gamma, and which has a molecular mass of 32 kD.

Synergy between recombinant interleukin 2 (rIL 2) and IL 2-depleted lymphokine-containing supernatants in facilitating allogeneic human cytolytic T lymphocyte responses in vitro.

Supernatants from human mixed leukocyte cultures or lectin-depleted supernatants from cultures of PHA-activated human peripheral blood leukocytes were depleted of IL 2 by passage over an anti-human rIL2 immunoadsorbent column. The column eluates were concentrated, dialyzed, and tested for their ability to synergize with human rIL 2 in facilitating human cytolytic T lymphocyte (CTL) responses to allogeneic, uv-irradiated HT144 melanoma cells in vitro. CTL were generated in the presence of 1 X 10(-4) M hydrocortisone sodium succinate in order to minimize the generation of nonspecific lymphokine-activated killer (LAK) cells. IL 2-depleted lymphokine-containing supernatant (LKS), alone or in the presence of less than or equal to U/ml rIL 2 did not stimulate significant CTL responses. Recombinant IL 2 at greater than 2 U/ml stimulated weak CTL responses in the absence of LKS. However, strong synergistic CTL responses were observed when both IL 2-depleted LKS and greater than 2 U/ml rIL 2 were added to the cultures. CTL generated in these cultures could be distinguished from nonspecific LAK cells on the basis of their i) specificity, ii) T3 phenotype, and iii) kinetics of generation. Nevertheless, rIL 2 and IL 2-depleted LKS were sometimes observed to synergize in facilitating the generation of nonspecific LAK cells as well as the generation of specific CTL. When the times at which rIL 2 and IL 2-depleted LKS were added to the cultures were varied, IL 2 was found to be required early in CTL responses, whereas the synergistic factor(s) in LKS seemed to act later. Recombinant human interferon-gamma was unable to replace LKS in synergizing with rIL 2 to elicit CTL responses. In summary, these experiments suggest that LKS contains a late-acting factor(s), antigenically distinct from IL 2, which synergizes with IL 2 in facilitating human CTL responses.

Variation in sister chromatid exchange frequencies between human and pig whole blood, plasma leukocyte, and mononuclear leukocyte cultures.

Sister chromatid exchange (SCE) induction by ultraviolet (UV) light was studied in both human and pig whole blood cultures (WBC) and plasma leukocyte cultures (PLC). No variation in SCE frequency was observed between pig WBC and PLC in control as well as in treated cells. Conversely, SCE frequencies of human PLC were consistently higher than those of WBC in control and UV-exposed cells. Thus, red blood cells (RBCs) do not influence the sensitivity of lymphocytes to UV light exposure, and there must be some different culture condition(s) in the induction of SCEs between human WBC and PLC but not in swine lymphocyte cultures. Since the BrdUrd/lymphocyte ratio of WBC was halved in PLC, the effect of BrdUrd concentration in inducing the SCE baseline frequency of PLC may be ruled out. Also, Ficoll-Hypaque-separated human mononuclear leukocytes in culture (MLC), but not pig MLC, showed a two-fold increase in SCE frequency over WBC values. Thus, neither the cell separation technique nor polymorphonuclear leukocytes had a significant role in the elevated SCE frequency of human PLC or MLC. Experiments where human RBCs were titrated into human PLC showed that the induction of an elevated SCE frequency of PLC was suppressed in a dose-dependent manner by the presence of RBCs in the culture medium. Since the incorporation of pig or human RBCs into human PLC as well as into MLC reduced the SCE frequency to that of WBC, a common component and/or function existing in these cells is suggested. Analysis of different RBC components showed that RBCs, specifically RBC ghosts, release a diffusible but not dialyzable "corrective" factor into culture medium that is able to reduce the SCE frequencies of PLC. Antioxidant enzymes such as catalase and horseradish peroxidase were unable to reduce the SCE frequency of human PLC to WBC values.

Characterization of human tumor necrosis factor produced by peripheral blood monocytes and its separation from lymphotoxin.

Cultures of human peripheral blood leukocytes (PBL) induced with phytohemagglutinin (PHA) and the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) produced two types of cytotoxic proteins, indistinguishable in the in vitro assay employing murine L 929 cells as targets. One of these proteins had the antigenic and physicochemical properties of lymphotoxin (LT). We have identified the other cytotoxin as tumor necrosis factor (TNF), mainly on the basis of antigenic cross-reactivity demonstrated with antiserum to TNF, and also by its characteristic physicochemical properties and cell source. Unlike LT, PBL-derived TNF did not bind to Concanavalin A-Sepharose or to several other agglutinin-Sepharose columns specific for carbohydrate moieties common in glycoproteins. The molecular weight of native TNF determined by gel filtration was approximately 40,000 while SDS-PAGE revealed a single sharp peak of 16,500 +/- 500. When cultures of monocytes and lymphocytes separated by elutriation were stimulated with PHA and/or TPA, monocytes were the major source of TNF. In contrast, only lymphocytes produced LT. A mixture of antisera to TNF and LF neutralized all cytotoxicity of crude human lymphokine preparations for L 929 cells, suggesting that TNF and LT are either the only, or the major, cytotoxic proteins present in such crude lymphokine preparations demonstrable in this assay.

Influence of prostaglandin E2 and indomethacin on interferon-gamma production by cultured peripheral blood leukocytes of multiple sclerosis patients and healthy donors.

The addition of indomethacin to concanavalin A (Con A)-induced cultures of human peripheral blood leukocytes (PBL) caused an increase in interferon response, regardless of whether the PBLs were derived from multiple sclerosis (MS) patients or from control donors. Specifically the response rates increased from 71 to 100% in controls and from 24 to 53% in MS patient-derived cultures. The amounts of interferon produced also increased in both groups by 0.8 log U/ml. However, interferon yields of nonresponsive cultures becoming interferon-producing only after indomethacin treatment remained relatively low. In control cultures, maximal increases of interferon production were obtained with doses of 0.05 to 0.1 microgram/ml indomethacin; for MS patients higher doses were needed--0.1 to 0.5 microgram/ml. Conversely, a relatively low dose (0.05 microgram/ml) of exogenous prostaglandin E2 (PGE2) was able to inhibit interferon production completely in MS patient-derived cultures, whereas in control cultures higher doses were needed (0.1 to 1.0 microgram/ml). Analysis of endogenous PGE2 levels in the PBL cultures revealed that PGE2 production was similar in nonresponder MS cultures and responder control cultures but that MS leukocytes were more sensitive to the inhibitory effect of PGE2 on interferon production. We conclude that in a minor percentage of MS patient-derived PBL cultures, the deficiency in interferon-gamma (IFN-gamma) production can be (partially) overcome by treatment of the cells with indomethacin. However, in the major part of nonresponder MS cultures, indomethacin has no effect, indicating that the PG system is not the major cause for the defective interferon response in MS.

SLO4, A New Interferon Inducer Isolated from Klebsiella Pneumoniae and Escherichia Coli

A product isolated from Klebsiella pneumoniae and Escherichia coli, coded SLO4, has been shown to be effective in endogenous interferon induction in vivo in mouse when administered IP or IV, and in vitro with human leukocyte cultures. In these two systems induced interferon was defined. The inducer has not yet been characterized but seems not to belong to any components known to be interferon inducers such as viral particles, nucleic acids or endotoxins. An analytical study will be carried out to specify the constitution of this interferon inducer.