Interferon-gamma production by Listeria monocytogenes-specific T cells active in cellular antibacterial immunity

Cultures of peritoneal exudate T lymphocyte-enriched cells (PETLEC) from Listeria monocytogenes-immune mice, antigen-presenting cells (APC) and heat-killed L. monocytogenes produced high amounts of interferon-gamma (IFN-gamma). High IFN titers were also observed after stimulation of L. monocytogenes-immune cell cultures with the T cell mitogens concanavalin A and phytohemagglutinin. L. monocytogenes-immune PETLEC produced several fold higher IFN titers than normal cell cultures in response to mitogen and antigen. Under both circumstances, APC were required for optimum responses. L. monocytogenes-immune PETLEC participating in IFN production were Lyt 1+23-. IFN-gamma was also produced in cultures of cloned L. monocytogenes-specific T cells. Since the same T cell clone showed antigen-specific proliferative responses and interleukin production in vitro, and could adoptively mediate delayed-type hypersensitivity and anti-listerial protection in vivo, it is suggested that IFN production is a function of specific T cells active in cellular antibacterial immunity.     

Gamma interferon induction in human thymocytes activated by lectins and B cell lines.

Human thymocytes in culture synthesized small quantities of interferon (IFN) when stimulated by the lectins concanavalin A or phytohemagglutinin. IFN production by lectin-activated thymocytes was enhanced in the presence of live B lymphoblastoid cells, irradiated B lymphoblastoid cells, or the conditioned medium from B lymphoblastoid cell cultures. The IFN synthesized in mixed cultures had characteristics of IFN-gamma, whereas the IFN synthesized by B lymphoblastoid cells alone could be identified as IFN-alpha on the basis of its neutralization with specific antisera and stability at pH 2. These findings indicate that human thymocytes in culture synthesize IFN-gamma and that B lymphoblastoid cells and their products considerably stimulate IFN-gamma synthesis by lectin-activated human thymocytes in culture. This stimulation was not diminished in the presence of antibodies to IFN-alpha, indicating that IFN-alpha production by B lymphoblastoid cells was not responsible for the stimulatory effect. Removal of adherent cells from thymocyte suspensions did not abrogate IFN-gamma production.     

Lymphokines produced by herpesvirus-transformed marmoset monkey lymphoid cell lines. I. Characterization of a constitutively produced interferon

Conditioned media from cultures of marmoset monkey T-lymphoid cell lines transformed by Herpesvirus saimiri or Herpesvirus ateles were found to contain interferon (IFN) activity. Titers between individual cell lines varied by a factor of 100; large amounts (up to 10(5) units/ml, assayed on human cells) were produced in one of the cell lines. IFN production was enhanced by the diterpene tumor promoters, TPA and mezerein, but not by classical T-cell mitogens. The IFN resembles human IFN-gamma by the following criteria: lability at pH 2, stability against 2-mercaptoethanol, cross-species activity, shape of dose-response curves, and molecular weight determined by size-exclusion chromatography (50,000-55,000). Its activity was not inhibited, however, by antiserum against human IFN-gamma or antisera against human IFN-alpha or IFN-beta.     

Interleukin 2 enhances natural killer cell activity through induction of gamma interferon

Highly purified interleukin 2 (IL 2), free of interferon activity, enhanced natural killer (NK) cell activity against tumor cells in mouse spleen cell cultures and in human peripheral lymphocyte cultures in a manner similar to that of interferon (IFN). We determined that IL 2 enhanced NK activity indirectly in a cascade manner by the induction of gamma IFN (IFN-gamma) in the cultures, which actually mediated the enhanced killing. Accordingly, lymphocyte cultures treated with IL 2 alone produced 10 to 100 U of IFN per ml in 6 to 24 h of culture. The IFN was typed as IFN-gamma by specific antibodies. Specific antibodies either to natural IFN-gamma or to a synthetic peptide corresponding to the human IFN-gamma N-terminal amino acids, when added to cultures treated with IL 2, completely blocked IL 2 enhancement of NK cell activity for both the mouse and human systems. IL 2-induced proliferation was not affected by the antibodies. Thus, the enhancement of NK cell activity by IL 2 is completely mediated by IL 2-induced IFN-gamma. The findings clearly indicate a cascade effect whereby one lymphokine (IL 2) induces the production of another. The latter lymphokine (IFN-gamma) then mediates an important biological effect (natural killing).     

Production of human interferon-gamma in serum-free medium.

Interferon (IFN)-gamma was produced with a high yield in cultures of human peripheral mononuclear cells by combined stimulation with OK-432 and staphylococcal enterotoxin B. Human mononuclear cells cultured in serum-free medium produced several times as much IFN as those in RPMI-1640 medium containing 10% fetal bovine serum. A synergistic effect of OK-432 and staphylococcal enterotoxin B on the production of IFN-gamma was demonstrated. Ultrogel AcA54 column chromatography of crude IFN showed a single peak with an apparent molecular weight of 43,000. Our production system for human IFN-gamma offers a feasible approach to preparation of large quantities of purified IFN-gamma for structure studies, antibody production, and clinical applications.     

Defective gamma-interferon production in peripheral blood leukocytes of patients with acute tuberculosis

Production of interferon (IFN)-gamma by peripheral blood leukocytes (PBL) was examined in cultures of unseparated fresh whole blood exposed to phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). The yield of IFN-gamma was measured by a newly developed immunoradiometric assay. Nine of 14 patients with acute pulmonary tuberculosis (TB) showed a depressed IFN-gamma response to Con A and/or PWM. Only four of these TB patients also showed a depressed IFN-gamma response to PHA. Stimulation of the patients' PBL cultures with PHA in the presence of exogenous interleukin 2 (IL 2) produced normal IFN-gamma yields in all but the most severely depressed patients. PBL cultures of TB patients with defective IFN-gamma production in response to mitogenic lectins also produced less IFN-gamma after stimulation with tuberculin PPD. Although some patients showed a moderate degree of lymphopenia, their OKT4/T8 lymphocyte ratios were mostly normal or close to normal, with the notable exception of one TB patient who has been diagnosed to have the acquired immune deficiency syndrome (AIDS).     

Kinetics and characterization of interferon production by murine spleen cells stimulated with Legionella pneumophila antigens.

Formalin-killed Legionella pneumophila bacterial cells, as well as a purified cell wall preparation (designated F-1 antigen) containing lipopolysaccharide (LPS), stimulated production of interferons (IFNs) in mouse spleen cell cultures. L. pneumophila whole-cell vaccine induced an IFN that was pH 2 labile and neutralized by anti-IFN-gamma indicating that IFN-gamma was the dominant form present. F-1 antigen induced a mixture of IFNs, depending upon the age of the culture and cell types present. In freshly prepared whole-spleen cultures and in 2-h adherent cultures, F-1 induced predominantly IFN-alpha/beta. In whole-spleen cultures that were allowed to age for 24 to 48 h before stimulation, F-1 was seen to induce mostly IFN-gamma, with low levels of IFN-alpha/beta present. Since only IFN-alpha/beta was produced in T-cell-depleted populations (at 2 h or at 48 h), it is suggested that T cells are responsible for IFN-gamma production in aged cultures. Additionally, heat-treated F-1, Escherichia coli LPS, and heat-treated E. coli LPS all induced similar levels of IFN-gamma in whole-splenocyte or nonadherent cell cultures which were incubated 48 h before stimulation. This suggests that LPS present in F-1 is responsible for IFN-gamma production and that an activated cell population is required. These results show that L. pneumophila antigens can induce the production of various types of IFN in mouse spleen cell cultures through several mechanisms.     

Identification of Th0 cells responding to measles virus

Mechanisms involved in the induction of immunity to measles virus (MV) are not well understood. In the present study, we assessed proliferation, interferon (IFN)-gamma, and interleukin (IL)-4 production of MV-specific T cells after secondary in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from human donors. Such secondary stimulation resulted in responses substantially higher than after primary in vitro exposure. Most study participants produced both IFN-gamma and IL-4 after secondary in vitro stimulation. Patterns of secondary in vitro responses that use genetically disparate antigen-presenting cells were consistent with T-cell recognition restricted to human leukocyte antigen class II molecules. Limiting dilution analyses indicated that precursor frequencies of cytokine secreting and proliferating cells ranged from about 0.001% to 0.1% among fresh PBMCs. Split-well analyses of limiting dilution cultures suggested that virtually all putative T-cell clones produced either IFN-gamma alone or both IFN-gamma and IL-4. Intracytoplasmic flow cytometric analysis of polyclonal MV-specific secondary in vitro responding T cells revealed a similar pattern of cytokine expression. These results suggest that memory T cells responding in vitro to MV generate cells that produce either IFN-gamma alone (and resemble Th1-like cells) or secreted both IFN-gamma and IL-4 (resembling Th0-like cells) in vitro with few cells expressing a Th2-like pattern.     

Gamma interferon enhances mitogenic responses induced by pokeweed mitogen

Interferon (IFN)-induced suppression of the lectin-stimulated lymphoproliferative response was studied comparatively with human IFN-alpha, IFN-beta and IFN-gamma, using an equal unit of their antiviral activity ranging from 31.25 to 1000 IU/ml. Both IFN-alpha and IFN-beta inhibited phytohemagglutinin (PHA) and pokeweed mitogen (PWM)-stimulated lymphoblastogenic response similarly in a dose-related fashion, but the IFN-gamma effect was far less. Indeed, the PWM-stimulated lymphocyte blastogenesis in the cultures incubated for 7 days was enhanced in the presence of IFN-gamma at a concentration of 62.5 IU/ml. The enhancing effect was found to be highest at the lowest concentration of IFN-gamma examined. The IFN-gamma induced enhancement of lectin-stimulated blastogenesis was found mainly in the PWM cultures incubated for 7 days but less in cultures incubated for 5 days or in PHA cultures incubated for 3 days, suggesting that the observed effect might be caused by the activation of interleukin production.     

Effects of natural human interferon-alpha, -beta and -gamma on interleukin 2 production in human peripheral lymphocytes

Effects of interferon (IFN) on PHA-induced interleukin 2 (IL-2) production by human peripheral mononuclear cells were studied comparatively with natural human IFN-alpha, IFN-beta and IFN-gamma, using an equivalent unit of their antiviral activity ranging from 10 to 1000 IU/ml. IL-2 activity was assessed in cultures with or without IFN by a standard bioassay using murine CTLL-2 cells. PHA-induced production of IL-2 in cultures of peripheral mononuclear cells was unaltered or slightly suppressed by the simultaneous presence of IFN-alpha and IFN-beta. The effect was the same, whether or not indomethacin was present in the cultures. In contrast, the addition of IFN-gamma to the PHA-stimulated cultures markedly enhanced IL-2 production, while IFN-gamma per se had no effect on IL-2 production in the absence of PHA. The enhancement of IL-2 production due to IFN-gamma was more marked in cultures which did not include indomethacin than in cultures which contained indomethacin (1 x 10(-6) M).     

Spontaneous production of gamma interferon and induced production of beta interferon by human T-lymphoblastoid cell lines.

Two human T-lymphoblastoid cell lines, CCRF/CEM and Molt 4, produced beta interferon (IFN-beta) upon infection with Sendai virus. Molt 4, but not CCRF/CEM, spontaneously produced up to 300 U of IFN-gamma per ml, apparently not contaminated with IFN-alpha or -beta. Phytohemagglutinin, a T-cell mitogen, did not stimulate IFN production in these lines. A third T-lymphoblastoid line, CCRF/HSB2, produced no IFN either spontaneously or after infection with Sendai virus or treatment with phytohemagglutinin. The Molt 4 cells contained an mRNA which could be translated by oocytes to give IFN-gamma. Molt 4 cells therefore provide a convenient source of human IFN-gamma and its mRNA for experimental purposes.     

Selection of alpha/beta interferon- and gamma interferon-resistant rickettsiae by passage of Rickettsia prowazekii in L929 cells.

The ability of endogenously produced alpha/beta interferon (IFN-alpha/beta) to inhibit rickettsial growth in infected L929 cell cultures was evaluated by comparing the growth of Rickettsia prowazekii Madrid E in untreated cultures and cultures treated with anti-mouse IFN (alpha + beta) serum. The endogenously produced IFN was neutralized, and rickettsial growth was enhanced in the antiserum-treated cultures. This inhibitory effect of endogenously produced IFN-alpha/beta was used to select rickettsiae resistant to IFN-alpha/beta. Rickettsiae were screened for resistance to IFN-alpha/beta after being cultured in untreated L929 cells for several weeks to several months. Two isolates derived from R. prowazekii Madrid E and two isolates derived from plaque-purified R. prowazekii Madrid E were plaque-purified twice, grown in embryonated hen eggs, and evaluated for resistance to IFN-alpha/beta and IFN-gamma. Compared with the parental rickettsial strain, all four isolates were significantly resistant to IFN-alpha/beta and IFN-gamma. In addition, they were as resistant or more resistant to IFN-gamma when compared with two previously described IFN-gamma resistant isolates that were selected in IFN-gamma-treated L929 cells. One of the two isolates from IFN-gamma-treated L929 cells was also resistant to IFN-alpha/beta; the other isolate was similar to the parental Madrid E strain in sensitivity to IFN-alpha/beta.     

Stimulation of protein phosphorylation in mixed glial cell primary cultures and subcultures by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate

Glial cell primary cultures consisting of protoplasmic and fibrous astrocytes, oligodendrocytes and progenitor glial cells incubated in medium containing 0.5% foetal calf serum and treated with 25 nM 12-o-tetradecanoylphorbol-13-acetate (TPA) for periods between 15 and 60 min showed a stimulation of protein phosphorylation which was most prominent in a polypeptide with a molecular weight of about 80,000 Da. Glial subcultures consisting mainly of Type 2 astrocytes, oligodendrocytes and progenitor glia showed a similar TPA stimulation of 80,000 Da protein phosphorylation detectable within 1 min of phorbol ester addition. TPA treatment of primary glial cultures led to an enhancement of phospholipid turnover but exposure of primary glial cultures to concentrations of TPA up to 250 nM caused no morphological change in protoplasmic astrocytes. 4-Phorbol (4-PH) or dimethylsulfoxide (DMSO) was without effect on protein phosphorylation or lipid turnover in glial cultures.     

Gamma interferon production by combinations of human peripheral blood lymphocytes, monocytes, and cultured macrophages.

Mitogen-induced interferon (IFN) production was studied using human peripheral blood mononuclear cells and subpopulations of lymphocytes, monocytes, and cultured macrophages. Cell populations were prepared in suspension to permit quantitative analysis of the interactions among different cell types. After stimulation by staphylococcal enterotoxin A, nylon column-purified lymphocytes produced only 5% as much IFN as the peripheral blood mononuclear cells from which they were prepared. When lymphocytes were supplemented with as little as 2% monocytes, IFN production increased two- to eightfold; with the addition of up to 20% monocytes, IFN production increased further, to levels approximating those of peripheral blood mononuclear cells. Monocytes alone produced no or very little IFN. Macrophages were derived from monocytes by culturing in vitro for 7 days. The addition of 2 to 5% autologous macrophages augmented IFN production to the same extent as 2 to 5% monocytes. However, more macrophages consistently resulted in less, rather than more, IFN, so that lymphocytes with 20% monocytes produced three- to eightfold more IFN than did lymphocytes with 20% macrophages. Thus, whereas the addition of monocytes over a broad dose-response range (2 to 20%) progressively augmented IFN production, macrophages showed an optimal effect at 2 to 5%, with higher percentages being inhibitory. The IFN induced by stimulation with staphylococcal enterotoxin A was characterized as IFN-gamma by its resistance to neutralization by antibody to IFN- alpha and its inability to induce antiviral protection in embryonic bovine trachea cells.     

The preparation of interferon gamma-producing T-cell hybridomas from jacalin-stimulated T lymphocytes and the SH9 T-cell line.

Jacalin, a lectin(s) extracted from the seeds of Artocarpus integrifolia (Jackfruit), was shown to induce the production of gamma interferon (IFN gamma) in human peripheral blood mononuclear cells (PBMC) and human T-lymphocyte cultures. The amount of IFN gamma produced was enhanced in the presence of mezerein, a phorbol ester derivative. Fusion of jacalin stimulated T lymphocytes with the SH9 T-cell line resulted in the formation of T-cell hybridomas which spontaneously secreted IFN gamma. The spontaneous production of IFN gamma by T-cell hybridomas was not influenced by the presence of jacalin, although significant enhancement of production was observed when the cells were cultured in the presence of mezerein.     

In vitro regulation by muramyl dipeptide of interferon production from peripheral blood mononuclear cells of healthy volunteers

We investigated the effect of N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) on interferon (IFN)-alpha and -gamma production by peripheral blood mononuclear cells from healthy subjects. MDP, on its own, was found to lack the ability to induce IFN production. However, this synthetic adjuvant was able to modulate IFN production induced by other stimuli. In cultures from a considerable number of tested donors, MDP enhanced IFN-gamma levels induced by phytohaemagglutinin. This effect was further potentiated after depleting the PBMNC cultures of their adherent cells. In contrast, MDP significantly suppressed the Sendai virus-induced IFN-alpha and this effect was reversed following adherent cell depletion. Identical regulatory effects on IFN production were exerted by the adjuvant active analogue of MDP, namely murabutide. The adjuvant inactive stereoisomer, MDP (DD) exhibited a similar enhancing effect on IFN-gamma but had a significantly lower inhibitory activity on IFN-alpha production. The potential value of this generation of immunomodulators in the treatment of viral infections and in models for studying the regulation of IFN at the molecular level is discussed.     

Cytokine sensitivity and methylation of lysine in Rickettsia prowazekii EVir and interferon-resistant R. prowazekii strains.

Modified Rickettsia prowazekii strains have been derived from the avirulent Madrid E strain by passage in the lungs of white mice (strain EVir) or by selection for resistance to gamma interferon (IFN-gamma) (strains 427-19 and 87-17) or alpha/beta interferon (IFN-alpha/beta) (strains 83-2P, 60P, 103-2P, and 110-1P). Compared with the Madrid E strain, strain EVir has increased virulence (N. M. Balayeva and V. N. Nikolskaya, J. Hyg. Epidemiol. Microbiol. Immunol. 17:11-20, 1973) and a different lysine methylation profile in its surface protein antigen (A. V. Rodionov, M. E. Eremeeva, and N. M. Balayeva, Acta Virol. 35:557-565, 1991). The other six strains differ from the Madrid E strain in their resistance to IFN and their ability to grow well in untreated macrophagelike RAW264.7 cells. In the present study, to determine which properties are shared by these strains, we examined R. prowazekii EVir for the following: (i) the sensitivity of its growth in L929 cells to the cytokines IFN-alpha/beta, IFN-gamma, tumor necrosis factor alpha (TNF-alpha), and IFN-gamma plus TNF-alpha; (ii) the ability to grow in untreated RAW264.7 cells; and (iii) the ability to induce interferon in L929 cell cultures; we also evaluated strains 83-2P and 87-17 for lysine methylation. Multiplication of strain EVir in growing L929 cells was not markedly inhibited by either IFN-alpha/beta or IFN-gamma. In X-irradiated L929 cells, growth of strain EVir was slightly inhibited (11%) by TNF-alpha alone, somewhat inhibited (38%) by IFN-gamma alone, and markedly inhibited (87%) by IFN-gamma plus TNF-alpha. Nitrite production was induced in X-irradiated, strain EVir-infected L929 cell cultures treated with TNF-alpha alone or IFN-gamma alone; however, more nitrite was produced in infected cultures treated with IFN-gamma plus TNF-alpha. Nitrite production, the dramatic inhibitory effect of IFN-gamma plus TNF-alpha, and the modest inhibitory effect of IFN-gamma on the growth of strain EVir in X-irradiated L929 cells were all alleviated by the addition of the nitric oxide synthase inhibitor NG-methyl-L-arginine. Strain EVir grew very well in untreated macrophagelike RAW264.7 cells and appeared defective in the ability to induce IFN in L929 cell cultures. All strains grown in L929 cells in the presence of radiolabeled lysine had similar percentages of their radioactivity as methylated lysines.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytokine induction of neopterin production.

The pteridine neopterin is a marker of immunological activation and has been shown to be a useful marker of graft-versus-host disease (GVHD) in bone marrow transplant patients. High levels of both neopterin and interferon-gamma (IFN-gamma) were produced in vitro during mixed lymphocyte responses, which may be considered to be a model of the primary events leading to GVHD. Neopterin was shown to be produced by monocytes in response to stimulation with IFN-gamma, but not other cytokines. However, the interleukins IL-1 alpha, IL-1 beta, IL-2, and tumour necrosis factor (TNF) alpha and beta, but not IL-6, stimulated neopterin production by unfractionated peripheral blood mononuclear cells (PBMC), and culture supernatants from PBMC stimulated with IL-1 alpha, IL-1 beta, IL-2 and IL-6, but not TNF-alpha or TNF-beta induced neopterin production following transfer to fresh monocyte cultures. It therefore appears that cytokines may generate neopterin by induction of IFN-gamma, by synergy with low levels of induced IFN-gamma, or by non-IFN-gamma-dependent mechanisms.     

Simultaneous induction of interferon gamma and tumor necrosis factor alpha by different seleno-organic compounds in human peripheral blood leukocytes.

Ebselen is known as anti-inflammatory and anti-oxidant selenium containing drug. We have synthetized 13 seleno-organic compounds, analogs of ebselen. Seven of them were found to be inducers of interferon gamma (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) in human peripheral blood leukocytes (PBL) cultures. The most active cytokine inducers were: 2-phenyl-1,2-benzisoselenazol-3(2H)-one (1, ebselen), bis [2-(N-phenylcarbamoyl)]phenyl diselenide (7) and bis (2-[N-(2-pyridyl)carbamoyl])phenyl diselenide (8). The amounts of IFN and TNF produced by PBL cultures in response to the seleno-organic compounds were found to be similar to that induced by phytohemagglutinin (PHA). The activities of the seleno-organic compounds were dose-dependent and related to the chemical structure of the drugs suggesting involvement of the specific cytokine-inducer receptor. The simultaneous inductions of IFN-gamma and TNF-alpha were highly correlated, but independent on each other.     

Cytokines produced in lymph follicles

The events occurring inside lymph follicles during a germinal center reaction are poorly understood. Using B and T lymphoid cell populations prepared from human tonsillar lymph follicles, and enriched or not in macrophages or in follicular dendritic cells, we examined the production of cytokines by these cells in vitro. Interleukin 6 (IL-6) and tumor necrosis factor (TNF) were found in the supernatants of cultures stimulated with phytohemagglutinin or pokeweed mitogen. IL-1 beta was occasionally detected; its secretion apparently depends on the origin of the tonsils, the stimulation, and the cell populations. IFN-gamma and IL-2 were not produced in significant amounts by these lymph follicle cells. IL-4 was only found in very low concentrations in the supernatant of the different cell cultures. The cell populations containing follicular dendritic cells produced more IL-6 and TNF than the others, especially than those composed of only B and T cells.