Effect of synthesized constituents in the L-tryptophan product on the differentiation of eosinophils and the induction of IL-6: a possible cause of eosinophilia-myalgia syndrome.

While the pathogenesis of eosinophilia-myalgia syndrome (EMS) remains obscure, the ingestion of L-trypophan (LT) and possibly certain constituents in the LT product might be associated. We investigated the effect of chemically synthesized substances, 1,1'-ethylidene bis[tryptophan] (EBT) and its decomposition product, 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA) recently identified in the implicated LT, on the eosinophil differentiation and the induction of IL-1 and IL-6. EBT and MTCA alone did not support colony formation. However, EBT or MTCA in conjunction with IL-2 induced colony-forming activity containing a small number of eosinophils. In addition, these LT constituents induced a significant level of IL-6 but not IL-1 beta in the mononuclear cells from normal volunteers and a patient with hypereosinophilic syndrome. These results suggest that certain constituents of LT product are associated with the pathogenesis of EMS through the induction of colony-stimulating factors and IL-6, hence giving rise to eosinophilia and inflammation.

IL-1-induced production of IL-2 and IFN-gamma in subclones of human T-cell derived leukaemia HSB.2 cells: regulation by phytohaemagglutinin-mediated (poly)phosphoinositide breakdown and cyclic AMP.

A human T-leukaemic cell line, HSB.2-C5B2, which produces high levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) when stimulated with phytohaemagglutinin (PHA) plus IL-1, was recloned to obtain spontaneous variants in IL-2 production in response to the stimuli. In these subclones, the ability of one clone to produce IL-2 correlated well with that to produce IFN-gamma. Three C5B2 subclones: clone no. 28, a high IL-2 producer, clone no. 61, an intermediate IL-2 producer, and clone no. 40, a non-producer, were selected and examined for differences in signal transduction mechanisms. Since the three subclones were shown to express about the same number of IL-1 binding sites with similar affinities, the loss of ability to produce IL-2 was not due to decreased cell-surface receptor or changes in receptor property. In support of this, IL-1 induced expression of the IL-2 receptor (Tac/p55 antigen) to the same extent on the three subclones. The levels of conventional intracellular second messengers were compared and it was revealed that loss of responsiveness was closely related to the subclones' degree of (poly)phosphoinositide (PI) turnover, protein kinase C (PKC) activation and cyclic AMP formation in response to PHA. Moreover, resting intracellular cyclic AMP concentrations were found to be increased in subclones with attenuated IL-2 production. These results indicate that the variation of IL-1-induced production of IL-2 and IFN-gamma in this T-cell line is attributed to the difference in the PHA-mediated signal transduction pathway and, presumably, to the different regulation of intracellular cyclic AMP.

Interleukin 1 regulation of interleukin 2 and interferon-gamma gene activation in a human leukemic HSB.2 subclone.

The role of interleukin 1 (IL1) in causing IL2 and interferon-gamma (IFN-gamma) production and their associated gene activation has been studied in a human leukemic HSB.2 subclone. One of the subclones, HSB.2-C5B2 clone #28, which produced only low levels of IL2 and IFN-gamma when stimulated with phytohemagglutinin (PHA) or with a combination of phorbol-myristate-acetate (PMA) and Ca2+ ionophore, ionomycin, showed marked IL2 and IFN-gamma production in the presence of IL1. The augmentation by IL1 was observed in both dot and Northern blot analysis, indicating that IL1 regulates lymphokine genes at the pretranslational level. The kinetics of IL2 and IFN-gamma production were essentially similar for both lymphokines except that there was a faster accumulation of IFN-gamma mRNA than IL2 mRNA. In contrast to the IL2 and IFN-gamma gene activation, IL2 receptor (Tac/p55 antigen) expression was induced directly by IL1 itself as with PMA in this subclone. In these studies, IL1 action was not replaced by the drugs raising intracellular cyclic AMP (cAMP). Taken collectively, these experiments support the notion that IL1 does not trigger IL2 gene activation per se, but amplifies the preactivated lymphokine genes initiated by PMA and ionomycin, whereas IL1 can activate the IL2 receptor gene without any other known signal requirements.

Signal requirement for interleukin-1-dependent interleukin 2 production by a human leukemia-derived HSB.2 subclone.

We have previously established subclones from human leukemia-derived HSB.2 cell line that produced high levels of interleukin (IL) 2 when stimulated with phytohemagglutinin (PHA) and IL-1. Herein, we investigated the signal requirement for IL-2 production, particularly concerning the role of IL-1 in this system. PHA but not IL-1 rendered marked protein kinase C (PKC) activation and IL-2 production induced by PHA plus IL-1 was totally abrogated by a potent PKC inhibitor, H-7. Concomitantly, PHA alone caused marked Ca2+ influx, whereas IL-1 neither induced Ca2+ influx nor augmented PHA-induced Ca2+ influx. As expected, a signal delivered by PHA could be substituted by phorbol 12-myristate 13-acetate (PMA) and ionomycin while IL-1 was still indispensable, indicating that at least three signals, i.e., those delivered by IL-1 as well as PKC activation and Ca2+ influx were required for optimal IL-2 production. Kinetic study indicated that while PMA and ionomycin should be added at the initiation of culture, delayed addition of IL-1 up to 4 hr later induced even higher levels of IL-2 production, demonstrating the requirement for IL-1 after PKC activation and Ca2+ influx. In this system, it was revealed that IL-1 was not involved in PKC activation, Ca2+ influx, and breakdown of phosphatidylinositols. Whereas PMA, ionomycin, and IL-1 stimulated high levels of IL-2 production, those combinations of signals did not induce breakdown of phosphatidylinositols. It should be noted that IL-2 production induced by these three signals seemed to bypass hydrolysis of phosphatidylinositols in contrast to PHA plus IL-1 stimulation that was accompanied with a marked breakdown of phosphatidylinositols.

Effects of anti-Tac antibody on the response of large granular lymphocytes to interleukin-2.

Large granular lymphocytes (LGL), which consist of a unique population comprising almost all of the natural killer (NK) cell activity, were separated from peripheral blood mononuclear cells by sequential depletion of monocytes and conventional discontinuous Percoll density gradient sedimentation. The LGL populations thus obtained exhibited significant levels of interferon-gamma (IFN-gamma) production and proliferation, as well as augmentation of NK cell activity in response to interleukin-2 (IL-2). Among these IL-2-driven phenomena, only IFN-gamma production was markedly inhibited by the monoclonal anti-Tac antibody, which presumably recognized the IL-2 receptor, whereas the proliferative response and the augmentation of NK cell activity were only minimally affected by the same antibody, even at the higher concentration. The dissociation of the effects of anti-Tac antibody on these IL-2-driven events gives us some insight on the role of IL-2 receptors involved in these immunologically interesting functions of IL-2.

Immunoenzymatic detection of three kinds of 43,000-molecular-weight antigens by monoclonal antibodies in the insoluble fraction of Toxoplasma gondii.

Monoclonal antibodies (TpM 3, TpM 6, and TpM 19) against Toxoplasma gondii insoluble antigens were produced by the hybridization of NS-1, a mouse myeloma cell line, with spleen cells from mice immunized with T. gondii insoluble antigens. TpM 3, TpM 6, and TpM 19 were characterized by the dye test, the latex agglutination test, two types of enzyme-linked immunosorbent assays, using either T. gondii supernatant antigens or T. gondii insoluble antigens, and immunoperoxidase staining. TpM 3, TpM 6, and TpM 19 antigens were analyzed by the immunoblotting method, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer of the antigens to nitrocellulose sheets. TpM 3, TpM 6, and TpM 19 were all negative by the dye test, the latex agglutination test, and the enzyme-linked immunosorbent assay, using T. gondii supernatant antigens but positive by the enzyme-linked immunosorbent assay, using T. gondii insoluble antigens. Each antibody gave unique spot patterns in the cytoplasm of tachyzoites, and each detected antigens of ca. 43,000 molecular weight with different electrophoretic patterns. Purified TpM 3 antigen bound only to TpM 3 but not to TpM 6 or TpM 19. Comparable results were obtained with TpM 6 and TpM 19 antigens. Rabbit anti-T. gondii sera reacted with both TpM 3 and TpM 19 antigens. However, human anti-T. gondii sera reacted only with TpM 3 antigen. These results indicate that T. gondii insoluble antigens contained at least three types of 43,000-molecular-weight antigens that have been revealed for the first time in this paper.

Interleukin 2-dependent T cell line acquires responsiveness to phorbol myristate acetate and lipopolysaccharide in the course of long-term culture.

We have observed that CT6 cell line, a murine interleukin 2 (IL 2)-dependent T cell line, was highly responsive to phorbol myristate acetate (PMA) and to a lesser extent but significantly responsive to lipopolysaccharide (LPS) in the short-term proliferation assay. In contrast, two other murine IL 2-dependent cell lines, CTLL-2 and NK clone 7, were totally unresponsive to these stimulants. Even when CT6 was recloned by a limiting dilution, no unresponsive clone to PMA was obtained, whereas several clones unresponsive to LPS were obtained. PMA, unlike to IL 2, could not support a long-term culture of CT6. There are no differences between CT6 and CTLL-2 except that the former possessed asialo GM1 while the latter lacked it. Though it is unknown whether this difference is involved in the mechanism of the response of CT6 to PMA or LPS, these data give caution to us against the use of CT6 for the determination of IL 2 activity contaminated with PMA and LPS.

Detection of toxoplasma membrane antigens transferred from SDS-polyacrylamide gel to nitrocellulose with monoclonal antibody and avidin-biotin, peroxidase anti-peroxidase and immunoperoxidase methods.

Toxoplasma (Tp) membrane antigens separated by discontinuous SDS-polyacrylamide gel electrophoresis were electrophoretically transferred to nitrocellulose and detected with avidin-biotin (AB), peroxidase anti-peroxidase complex (PAP) or indirect immunoperoxidase (IIP) methods. In the AB method, the nitrocellulose was treated with biotinylated monoclonal antibodies and avidin-labeled peroxidase. In the PAP method, it was treated with monoclonal antibody, rabbit anti-mouse IgG antibody, goat anti-rabbit IgG antibody and PAP. Of the two, the AB method was the more sensitive and specific for Tp membrane antigen. The PAP method was less sensitive, but did not require chemical manipulation of the antibodies and was convenient and useful for analyzing Tp membrane antigens. The IIP method was more convenient, but had a lower sensitivity than the other methods.

Distinction of two subtypes of human leukocyte interferon (IFN-alpha) on B cell activation. B cell proliferation by two subtypes of IFN-alpha.

We examined the effect of interferon (IFN), with particular emphasis on the effects of the two subtypes of IFN-alpha (IFN-alpha A and IFN-alpha B) on the B cell proliferation induced by Staphylococcus aureus Cowan I bacterium (SpA Col). An increase of SpA Col-induced proliferation was observed in the presence of 100 to 1000 U/ml of IFN-alpha, but a decrease of SpA Col-induced proliferation was observed in the presence of 1000 to 10,000 U/ml of IFN-beta. The two subtypes of IFN-alpha had different effects on cell proliferation; a significant enhancement was shown in the presence of 1000 to 10,000 U/ml of IFN-alpha A, but inhibition was shown in the presence of 1000 to 10,000 U/ml of IFN-alpha B. In the reconstitution test of the two subtypes of IFN-alpha, the boundary between enhancement and inhibition of SpA Col-induced proliferation was revealed when the proportion of IFN-alpha A and IFN-alpha B (IFN-alpha A:IFN-alpha B) ranged between 8:2 and 9:1. Toward the SpA Col-induced responses, the above IFN were all found to act on B cells directly, independent of the presence of T cells. Proliferative responses by IFN-alpha and IFN-alpha A, however, were shown to be slightly dependent on the presence of monocytes. The lymphocyte proliferation induced by other mitogens (phytohemagglutinin, concanavalin A, pokeweed mitogen, and protein A of S. aureus) were all inhibited by the above IFN.

Isolation of Ia positive human leukocytes by a direct rosette assay.

A direct rosette assay (DRA) using ox erythrocytes coated with monoclonal antibody (AF-10) to human Ia-like antigen (OE-anti-Ia) is a rapid and convenient method for detection and isolation of Ia-like antigen-bearing cells. Various cell lines and peripheral blood lymphocytes were tested by DRA. Cell lines expressing Ia-like antigen formed rosettes, while cell lines lacking Ia-like antigen did not. In a highly purified T cell fraction, a small number of T cells formed rosettes; but in a Con A-stimulated T cell fraction, about 40% of the cells formed rosettes. The majority of purified B cells and monocytes formed rosettes. Rosette-forming and non-rosette-forming mononuclear cells could be separated by centrifugation on Ficoll-Urografin. Rosette-forming cells reacted positively by indirect immunofluorescence (IIF), while non-rosette-forming cells reacted negatively. The results obtained by DRA were consistent with those obtained by IIF with OKIa*1 and AF-10. DRA is thus suitable not only for detection of Ia-like antigen-bearing cells, but also for separation of Ia-like antigen-positive (Ia+) and -negative (Ia-) cells.

Effect of Staphylococcus aureus Cowan i bacteria on the mitogenic response of human B-cell subsets.

We have made a detailed investigation to determine which of the B-cell subsets could be stimulated by Staphylococcus aureus Cowan I bacterium (SpA CoI). B-cell subsets were separated from peripheral blood and tonsil lymphocytes by means of rosette formation with E, EAIgG, anti-immunoglobulin (Ig) conjugated OE (OE-Pro A) or by separation on a bovine serum albumin (BSA) discontinuous density gradient. The cells responding to SpA CoI included E receptor negative (E-), C3 receptor positive (C3R+), and surface Ig positive (SIg+) B-cell subsets. Among these B-cell subsets, FcR-n cells were more responsive than FcR+ cells. These B-cell subsets responded alone to SpA CoI and significantly proliferated, although, they failed to respond alone to pokeweed mitogen (PWM) and Protein A of S. aureus (Protein A). Among the SIg+ B-cell subsets stimulated with SpA CoI, IgM+ and IgG+ B cells showed much less response. Both Protein A receptor positive (Pro A . R+) and negative (Pro A . R-) cells responded well to SpA CoI. Fractionation of B cells on a BSA gradient revealed that comparatively small sized and denser B-cell subsets responded well to SpA CoI. From these criteria, it is suggested that B cells responding to SpA CoI are capable of stimulating not only mature B cells, but can also stimulate immature B cells.

Potentiation of natural killer cell activity of human lymphocytes in vitro: the participation of interferon in stimulation with Staphylococcus aureus Cowan I bacteria but not with protein A.

In the previous paper we reported that human natural killer (NK) cell activity was augmented greatly by preincubation with Staphylococcus aureus Cowan I bacteria (SpA CoI) or its Protein A. We examined here whether the augmentation with these stimulants is ascribable to the direct activation of NK cells or mediated by some soluble factors produced by the stimulants. It was found that a significant amount of interferon (IFN) was produced by the SpA CoI-stimulation but not by the Protein A-stimulation, although the latter usually induced augmentation of NK-cell activity not less than SpA CoI-stimulation. IFN produced by SpA CoI was considered to belong to alpha-type IFN, because it was stable at pH 2.0 and could be neutralized effectively by anti-IFN alpha antibody. Kinetics of NK-cell activation by SpA CoI (but not by Protein A) were very similar to those by IFN alpha. Furthermore, augmentation of NK-cell activity with SpA CoI-stimulated supernatant was inhibited almost completely by diluted anti-IFN alpha antibody, whereas augmentation with Protein A-stimulated supernatant could not be abolished by the same treatment. It was, therefore, suggested that augmentation of NK-cell activity with SpA CoI might be ascribable in most part to the IFN induced, whereas Protein A can stimulated NK or T cells directly or soluble factors other than IFN might work as well.

Potentiation of natural killer activity of human lymphocytes by Staphylococcus aureus bacteria and its protein A.

Cytotoxic activity of human lymphocytes against the myeloid cell line K-562 was augmented greatly by 24-h incubation with Staphylococcus aureus Cowan I bacteria (SpA CoI) and its protein A. This effect was not observed when these stimulants were added after preincubation, suggesting that this activity was different from so-called lectin-induced cellular cytotoxicity. Potentiation required at least 12 to 18 h incubation of lymphocytes with these stimulants. Macrophage depletion did not affect the potentiation by protein A or SpA coI, although the potentiation by poly I:C or OK-432, an immunopotentiator of Streptococcus pyogenes was completely reduced. Further cell separation procedures revealed that neither T cells nor FcR- cells, which showed little natural killer (NK) activity, were enhanced by protein A or SpA coI. On the other hand, (a) null cells which were obtained from nylon column (NC)-passed fraction by depleting T cells and surface membrane Ig-positive cells, and (b) FcR+ E- cells which were obtained from NC-passed fraction by depleting FcR- cells and T cells, showed marked NK activity by themselves and were further augmented by these stimulants. FcR+ E+ cells failed to show NK activity even if they were stimulated by these stimulants. Thus, it was found that protein A and SpA CoI, as well as human interferon, could stimulate NC-non-adherent, FcR+, E- NK cells and potentiate markedly their NK activity.

A simple and rapid latex fixation test for measuring immunoglobulins produced in cell cultures.

A rapid and simple latex fixation test (LFT), which quantifies immunoglobulin (Ig) released into culture supernatants is described. Latex particles are coated with rabbit anti-human IgG, IgA or IgM antibodies. With this LFT technique the concentration of Ig is determined within a few minutes. The LFT is as sensitive and quantitative as double-antibody radioimmunoassay and is capable of detecting 35, 68 and 225 ng/ml of IgG, IgA and IgM, respectively.

Stimulation of Immunoglobulin production from human B lymphocytes by Staphylococcus aureus: effects of monocytes and con A-induced suppressor cells.

Significant immunoglobulin (Ig) production by human peripheral blood lymphocytes was induced in vitro by stimulating the cells with pokeweed mitogen (PWM) and Staphylococcus aureus Cowan I (SpA CoI). IgG, IgM, and IgA were determined by a combination of the latex fixation test and radioimmunoassay. High levels (1,000 to 5,000 microgram/ml of IgG and IgM and a lesser amount of IgA were constantly produced during 7 to 8 days of incubation with both stimulants. Ig production induced by SpA CoI stimulation was independent of the presence of T cells, while Ig production induced by PWM required T cells exclusively. Depletion of monocytes in the culture caused but a slight decrease in Ig production (particularly in the case of IgG). While the addition of a small number of monocytes enhanced IgG induction by both stimulants, coculture with an excess number of monocytes inhibited Ig induction (particularly IgG) by PWM stimulation but not by SpA CoI stimulation. Marked suppression of Ig production (IgG, IgM, and IgA) was observed in cocultures with Con A-activated T cells. The phenomena of suppression were observed in both the SpA CoI-stimulated and PWM-stimulated lymphocytes. These data indicated that Ig production from B cells and relatively of independent of monocytes, but could be subjected to the regulation of the Con A-induced suppressor T cells.

Low responsiveness of human neonatal lymphocytes to a B-cell mitogen, Staphylococcus aureus Cowan I (SpA CoI).

Responses of neonatal and adult lymphocytes to various mitogens were studied. Lymphocytes from umbilical cord blood (UCB) responded well to both phytohemagglutinin and concanavalin A, and also to pokeweed mitogen and Staphylococcus aureus Protein A. The responses of UCB lymphocytes to these mitogens were not significantly lower than those of adult peripheral blood lymphocytes (PBL). In contrast, UCB lymphocytes showed only a minimal response to killed Staphylococcus aureus Cowan I (SpA CoI), a potent B-cell mitogen for human PBL, although the proportion of B cells in UCB was not less than that in PBL. The low level of response of lymphocytes from UCB to SpA CoI was not ascribed to differences in dose response or kinetics. Purified B cells from UCB were not stimulated by SpA CoI either, suggesting tht the low responsiveness was not due to the suppressive effect of T cells or macrophages, but to some intrinsic defect in B cells in UCB. These results suggest that the B cells in neonates may be more immature than the T cells.