Simple, sensitive and specific bioassay of interleukin-1

This paper describes a convenient method for the culture of sub-lines of the murine T cell cloned line, D10.G4.1, and the use of these lines in a highly sensitive and specific bioassay for interleukin-1 (IL-1). The cells are cultured with IL-1, interleukin-2 (IL-2), and concanavalian A (ConA), in the absence of feeder cells or antigen. Assays are routinely carried out in the presence of saturating IL-2, which enhances sensitivity and ensures that further IL-2 will not give false positives. Addition of interleukin-4 (IL-4) has a similar effect and can be used together with IL-2 where there is a potential for interference from either cytokine. The assay is not affected by high concentrations of human interleukin-6 or tumour necrosis factor- (TNF-) and only minimally affected by high concentrations of murine TNF-.     

A simple microassay for interleukin-2 activity

A simple in vitro assay is described to quantitate human interleukin-2 (IL-2). It is based on the spontaneous' activation of human peripheral T cells in tissue culture and the subsequent expression of the IL-2 receptor. The results obtained with this assay agree with those of assays which utilise IL-2 dependent T cell clones. The method is highly sensitive, reproducible and easy to perform.     

Enzyme-linked immunosorbent assay of mouse interleukin-2 receptors

An enzyme-linked immunosorbent assay (ELISA) which measures soluble mouse interleukin-2 receptor (IL-2R) was developed, using two monoclonal anti-mouse IL-2R antibodies directed against two different epitopes of the IL-2R molecule. The ELISA-reactive material was identical with the IL-2R material which was capable of binding to affinity support beads coupled with recombinant interleukin-2 (IL-2). Quantitation of IL-2R in detergent-lysed cells was compared to that of the purified IL-2R, and the detection limit was estimated to be within the range of 2-10 ng/ml of a test sample. This sensitivity made it possible to determine soluble IL-2R levels in cell culture supernatants and mouse sera.     

Biological activity of recombinant murine interleukin-6 in interleukin-1 T cell assays

Interleukin-6 (also called B cell stimulatory factor 2, hepatocyte activating factor, interferon-β₂) has been shown to have effects on various lineages of hemopoietic cells. Some of its activities appear to overlap those of interleukin-1. In particular, recombinant murine IL-6 induced proliferation of phytohemagglutinin-activated thymocytes, an assay widely used to detect IL-1. In this report, we compared several features of IL-1 and IL-6 dependent thymocyte proliferation. The results indicate that IL-2 is the major second mediator of both IL-1 and IL-6 dependent proliferation. Finally, we tested whether IL-6 would also have activity in other T cell-based IL-1 assays using the T cell lymphoma LBRM33 1A5 and the T cell clone D10-G4.1. IL-6 had no activity in the latter two assays. These results indicate that IL-1 assays using LBRM33 1A5 and D10-G4.1 selectively detect Il-1, and are more specific assays for the detection of IL-1 in samples that may also contain IL-6.     

A modified assay for interleukin-1 (IL-1)

A simple and reliable biological assay for interleukin-1 (IL-1) was developed, based on the production of interleukin-2 (IL-2) from the EL-4 murine T-cell lymphoma cell line, in the presence of 2-5 X 10(-7) M calcium ionophore A23187. The assay was generally performed in 2 stages ((a) IL-1-dependent IL-2 production, and (b) IL-2 assay) and took 36-48 h to complete. This assay was found to be 10-25 times more sensitive than the mouse thymus cell assay, was not sensitive to the presence of bacterial endotoxin, and had the advantage of not requiring the use of animal tissue as a source of cells. The assay was used in our laboratory to detect human, mouse, rat, and rabbit IL-1 of all isoelectric-point types.     

The use of an HTLV-I-infected human T-cell line (ATH8) in an interleukin-2 bioassay

Biological interleukin-2 (IL-2) assays require cells that proliferate only in the presence of IL-2. The most often used human cells for this purpose have been phytohaemagglutinin (PHA)-stimulated buffy coat lymphocytes made dependent on IL-2. The results obtained by this T-blast method have not been easily reproducible and/or comparable due to differences in individual buffy coat batches. Murine cytotoxic T-cell lines (CTLL and CTLL-2) have been widely used in human IL-2 assays, but there remains a need for a human cell line. We have developed and IL-2 bioassay, based on the use of human HTLV-I-infected T_h-cell line, ATH8. These cells express IL-2 receptor and are totally dependent on extrinsic IL-2 for proliferation. The results of the ATH8 IL-2 assay were reproducible and comparable to those obtained with successful T-blast assays. ATH8 cells do not require a prolonged culture period before they are suitable for the IL-2 assay as do T-blasts.     

Human recombinant interleukin-2 is mitogenic to human lymphocytes

Reported herein are the results of studies demonstrating that purified recombinant human interleukin-2 (hrlL-2) is a potent mitogen for lymphocytes of healthy human donors. The specificity of the hrlL-2-induced response was defined in experiments in which mitogenicity of this T cell growth-promoting lymphokine was completely abrogated by blocking the T cell membrane receptor for IL-2 with the anti-Tac monoclonal antibody. Depletion of adherent mononuclear leukocytes markedly reduced lymphocyte reactivity to hrlL-2, but the response could be fully recovered by the addition of interleukin-1 (IL-1). Increased proliferative responses were observed using a combination of hrlL-2 and a monoclonal antibody OKT3 that defines a T cell membrane antigen. These studies demonstrate that hrlL-2, as with antigens and phytomitogens, may serve as the first signal of T cell proliferation.     

Establishment of a highly sensitive enzyme-linked immunosorbent assay for interleukin-1 alpha employing a fluorogenic substrate

We have previously established a non-competitive solid-phase enzyme-linked immunosorbent assay (ELISA) specific for interleukin-1 alpha (IL-1 alpha) using a combination of polyclonal antibody as the immobilized antibody, biotinylated monoclonal antibody as the second antibody and avidin-peroxidase. The level of detection of that ELISA was 200-500 pg/ml. In order to improve its sensitivity, we have used streptavidin-beta-D-galactosidase and the fluorogenic substance 4-methylumbelliferyl-D-galactopyranoside as enzyme substrate. With this system IL-1 alpha could be detected at concentrations as low as 10-50 pg/ml, which was about 10-20 times more sensitive than conventional mouse thymocyte co-stimulator assays. Furthermore, the assay system was specific for IL-1 alpha in that neither IL-1 beta nor interleukin-2 (IL-2) interfered.     

T cell sensitivity to HLA class I-mediated apoptosis is dependent on interleukin-2 and interleukin-4

Antibody interaction with a specific epitope of the HLA class I α1 domain triggers apoptosis of activated but not resting T and B cells by a pathway which involves neither Fas ligand nor tumor necrosis factor-α. We have investigated at which stage of activation and proliferation T cells become sensitive to HLA class I-mediated apoptosis, using two monoclonal antibodies (mAb) which recognize the same monomorphic epitope of the HLA class I α1 domain (mAb90, mouse IgG1, and YTH862, rat IgG2b) and can induce apoptosis of phytohemagglutinin (PHA)-activated peripheral blood lymphocytes. Sensitivity to apoptosis develops after the expression of G1 markers (CD69 expression) but it is accelerated by addition of recombinant interleukin-2 (rIL-2). Blocking the IL-2 pathway by cyclosporin A, FK506, rapamycin, anti-IL-2 or CD25 antibodies, prevented the development of sensitivity to apoptosis. Addition of IL-2 and, to a lesser extent, IL-4, reversed the inhibitory effect of cyclosporin A. Conversely, rIL-7 and recombinant interferon-γ restored proliferation of peripheral blood lymphocytes stimulated by PHA in the presence of cyclosporin A but did not restore sensitivity to class I-mediated apoptosis. Finally cells stimulated in the presence of the DNA polymerase inhibitor aphidicolin did not enter into S phase of the cell cycle but secreted IL-2 and underwent apoptosis when exposed to mAb90 or YTH862. Together, the data indicate that sensitivity of peripheral T cells to HLA class I-mediated apoptosis depends on both activation signals and IL-2 or IL-4, but does not require cell proliferation. These data suggest that YTH862 and mAb90 might be used for achieving clonal deletion of antigen-activated peripheral T cells in vivo, provided that the IL-2 pathway is not blocked by other immunosuppressive agents.     

Cellular bases of the production of and response to interleukin-2 in man: role of autologous rosette-forming T-cell subsets defined with monoclonal antibodies

In this paper we present experiments that indicate that, in man, most T-cell subpopulations produce interleukin-2 (IL-2), but that the main cell subpopulation which produces it, both upon activation with phytohaemagglutinin or in autologous mixed lymphocyte cultures, is that of autologous rosette-forming (Tar) T4+ T cells. Conversely, the main IL-2-responding T-cell subpopulation is that composed of T cells depleted of Tar (T-Tar) that are T8+. IL-2 was also found to be more effectively produced by Tar cells that do not bind peanut agglutinin (PNA) than by those that do. The PNA-T4+Tar cells were also found to respond best to interleukin-1 (IL-1).     

Specificity of alloactivated human T lymphocyte clones in secondary proliferation, cell-mediated lympholysis and interleukin-2 release

Alloreactive cytolytic T cell clones were generated from mixed leucocyte reactions (MLRs) between unrelated individuals. Two clones exhibited proliferative responses specific for class I HLA antigens B21 and B17. They were also found to be cytolytic toward cells bearing these HLA-B antigens as measured in cell-mediated lympholysis (CML) assays. Four clones exhibited primed lymphocyte test (PLT) and CML activity specific for various class II HLA antigens, namely DR1, DR5, DQw3 and DRw53. For each of these six clones, the CML specificity was identical to the PLT specificity. Both class I and class II specific clones released interleukin-2 (IL-2) upon restimulation with irradiated cells carrying the relevant HLA specificity. This stimulator-induced IL-2 release showed the same specificity pattern as that observed in the PLT assay. Monoclonal antibody (mAb) inhibition studies on alloreactive T cell clones showed similar inhibition profiles of PLT, CML and IL-2 release assays. These findings suggest that cytolytic activity, secondary proliferation and IL-2 release by alloactivated T cells may be induced by the same HLA encoded determinant.     

Inhibition of the antigen activated T cell response by methylprednisolone is caused by inhibition of interleukin-2 (IL-2) production

A mechanism by which the steroid methylprednisolone inhibits the T cell proliferation normally seen in antigen driven cultures was investigated. At methylprednisolone concentrations less than 10 micrograms ml-1 inhibition of cellular proliferation was almost exclusively via the inhibition of the production of interleukin-2 by activated T cells. The steroid seemed to inhibit IL-2 production directly and not via reduced sensitivity to interleukin-1 by IL-2 producer T cells, or reduced IL-1 production by the antigen presenting monocytes. At methylprednisolone concentrations of greater than 10 micrograms ml-1 direct inhibition of the T cell receptor to antigen complexes presented by cooperating monocytes seemed the most likely explanation. T cells in the presence of antigen and monocytes and MP concentrations of greater than 10 micrograms ml-1 did not produce IL-2 and were incapable of expressing IL-2 receptors. The inhibiting effects of methylprednisolone therefore are via several mechanisms depending on the concentration of the steroid employed.     

Soluble interleukin-2 receptor, interleukin-2 and interleukin-4 in sera and supernatants from patients with progressive systemic sclerosis.

We studied the sera of patients with progressive systemic sclerosis (PSS) for elevated levels of soluble interleukin-2 receptor (sIL-2R), interleukin-2 (IL-2) and interleukin-4 (IL-4). We also measured IL-2, IL-4 and B cell growth factor (BCGF) activity in supernatants of peripheral blood mononuclear cells from the same patients. The finding of elevated serum sIL-2R and IL-2, and the increased levels of IL-2, IL-4 and BCGF activity in culture supernatants indicates that T lymphocyte hyperactivity likely play a major role in PSS. The failure to detect under our experimental conditions a direct proliferative effect of recombinant IL-2 on enriched normal B cells might suggest that IL-4 is the cytokine mainly responsible of the BCGF activity recovered in PSS supernatants.     

Characterization of the immunoregulatory properties of thymosin alpha 1 on interleukin-2 production and interleukin-2 receptor expression in normal human lymphocytes

Thymosin alpha 1 (T alpha 1) and thymosin fraction 5 (TF5) have been shown to induce lymphocyte maturation and differentiation as well as to modulate mature immune responses to antigens and mitogens. The present study focused on the characterization of the mechanisms involved in T alpha 1 and TF5 enhancement of phytohemagglutinin (PHA)-induced interleukin-2 (IL-2) secretion and interleukin-2 receptor (IL-2R) expression in human mononuclear cells. We provide evidence that TF5 and T alpha 1 modulate an early event(s) during lymphocyte activation by mitogens. A short preincubation period (30 min) of non-adherent cells with thymosins, followed by extensive washing and subsequent exposure to PHA, was sufficient to enhance the production of IL-2 and the expression of IL-2R induced by the mitogen. Furthermore, the concomitant addition of PHA and thymosin during the preincubation period is not necessary for the enhancing effects to occur. We have also studied the role of macrophages on thymosin modulation of these responses. Results presented here indicate that macrophages are not essential for the interaction of thymosins with T-cells. However, macrophages are an absolute requirement during the exposure to the mitogen after preincubation with thymosins for the manifestation of TF5- and T alpha 1-mediated enhancing effects on IL-2 production and IL-2R expression. Human recombinant interleukin-1 beta (rIL-1 beta) was able to replace this macrophage requirement, indicating that production of IL-1 by these cells is a critical event in thymosin modulation of the IL-2 system. Two-color flow cytometric analysis and experiments involving the use of highly purified helper/inducer (Th, CD4+) and cytotoxic/suppressor (Tc, CD8+) T-cell populations indicated that both, Th and Tc cell populations are targets of thymosin activity. These studies provide additional evidence that thymosins play an important role in the modulation of the normal immune response and begin to define the mechanisms underlying T alpha 1 immunoregulatory properties.     

Costimulation by interleukin-1 of multiple activation responses in a developmentally restricted subset of immature thymocytes

An intriguing feature of thymocyte differentiation is that the competence to express both interleukin-(IL)2 and CD25 is acquired even prior to T cell recept or (TcR) expression. When T cell receptor-independent stimuli are used, immature cells can express IL-2 at levels comparable to mature cells, but unlike the mature cells, immature cells require IL-1 as a costimulus. Here we present evidence that IL-1 affects a variety of responses by members of the CD25⁺ subset of immature thymocytes. Cells in this population are IL-1 dependent not only for induction of IL-2 expression, but also for high-level maintenance of CD25 expression. CD²⁵⁺ expression is amplified by IL-1 through a mechanism highly sensitive to changes in Ca²⁺ ionophore concentration. The effects of IL-1 on CD25 maintenance are not mediated by IL-2, because of the divergent effects of cAMP on IL-2 and CD²⁵⁺ expression. IL-1 costimulation also increases RNA accumulation in the cell cycle, and this effect too seems to be separable from the effects on IL-2 and CD²⁵⁺ expression. All these effects of IL-1 are developmentally stage-specific, manifest in the CD25⁺ subset of immature thymocytes but not in later-stage thymocytes or splenic T cells. Multiparameter cell sorting experiments that dissect the transitional stages between immature and TcR⁺ thymocytes imply that all immature cells pass through an IL-1 responsive state. Responsiveness to IL-1 costimulation is then lost by these cells, apparently irreversibly, at a stage just prior to detectable cell-surface TcR expression. These results indicate that IL-1 responsiveness is a defining characteristic of the activation physiology of cells in a particularly important developmental stage.     

Detection of individual Peyer's patch T cells that produce interleukin-5 and interferon-gamma.

A method was developed to detect individual interferon-gamma (IFN-gamma)- and interleukin-5 (IL-5)-producing cells among freshly isolated T cell populations and long term lines of CD4+ Peyer's patch T cells using frozen semi-thin sections of paraformaldehyde fixed-T cells and immunofluorescence techniques. Using this method, individual CD4+ Peyer's patch T cells could be shown to produce IFN-gamma, characteristic of the T helper 1 (Th1) T cell type, IL-5, characteristic of the T helper 2 (Th2) T cell type, as well as both IFN-gamma and IL-5. These data support the notion that Th1 and Th2 cells derive from a common T cell precursor.     

Leukotriene B4 augments interleukin-2 receptor-beta (IL-2R beta) expression and IL-2R beta-mediated cytotoxic response in human peripheral blood lymphocytes.

Interleukin-2 can induce cytolytic activity in human peripheral blood lymphocytes and this activation is mediated by the beta chain of the interleukin-2 receptor-beta (IL-2R beta). Leukotriene B4 (LTB4) is a potent lipid inflammatory mediator which induces IL-2 and interferon-gamma (IFN-gamma) production from T cells. We examined the ability of LTB4 to modulate IL-2-induced cytolytic activity. Peripheral blood lymphocytes which had been preincubated for 24 hr in the presence of LTB4 responded to 100-fold lower concentrations of IL-2 with an augmentation of natural killer (NK) cell cytotoxic activity. Furthermore, incubation of lymphocytes with graded concentrations of LTB4 augmented the proportion of IL-2R beta+ cells. Peak activity was seen at 10 nM LTB4 and was comparable to that of PHA. By two-colour cytofluorometry, the increased expression of IL-2R beta was found predominantly on CD56+ cells and to a lesser extent on CD8+ cells, while CD4+ cells were unaffected. These observations were correlated at the messenger RNA (mRNA) level with increased IL-2R beta mRNA accumulation following stimulation of purified CD56+ and CD8+ cells with LTB4. CD56-, CD8- cells did not respond to LTB4 by increased IL-2R beta mRNA accumulation. Our data indicate, for the first time, that LTB4 can markedly increase the sensitivity of non-major histocompatibility complex (MHC)-restricted cytotoxic lymphocytes to IL-2, in terms of IL-2-dependent cytotoxic responses, and that this sensitivity is associated with augmented IL-2R beta gene message and cell surface expression.