Interleukin 4 inhibits the interleukin 2-induced production of its functional antagonist, interferon gamma

Interferon gamma (IFN gamma) inhibits many effects of interleukin (IL)-4. Its production largely parallels cell proliferation but is regulated independently. As IL-4 inhibits several IL-2-induced effects including proliferation of human peripheral blood mononuclear cells, we investigated its influence on the IL-2-induced IFN gamma production. We found that both absolute IFN gamma production and IFN gamma production per proliferation unit (1000 cpm) in response to IL-2 were inhibited by IL-4. IL-2-induced interferon production was inhibited by IL-4 in both sheep red blood cell rosetting and non-rosetting cells. In bidirectional mixed lymphocyte culture, IL-4 alone enhanced proliferation but suppressed IFN gamma production. As 48% of IFN gamma-producing cells are known to show T cell phenotype, the nearly total inhibition of IFN gamma production is evidence for suppression of T cell response to IL-2. Additionally, we found that the inhibition of IL-2-induced proliferation by IL-4 was significantly more pronounced at low cell densities. Basal proliferation was only inhibited in serum-containing media. These data stress the importance of other lymphokines or accessory cells in the regulation of early lymphocyte activation.     

A macrophage-derived factor that inhibits the production and action of interleukin 2

The human monocytic leukemia cell line THP-1 produces an immunosuppressive factor that inhibits interleukin 1 (IL-1)-dependent proliferation of mouse thymocytes as well as the mitogenic effects of concanavalin A (Con A) and phytohemagglutinin (PHA) on human peripheral blood mononuclear cells. The mechanism of action of this factor includes interference with both the production of interleukin 2 (IL-2) and its effects on target cells. Thus, the suppressor abrogates the proliferation of an IL-2-dependent cytotoxic T cell line (CTLL), but not of IL-2 independent cells like the L929 fibroblasts or the EL4 T lymphoma and U937 histiocytic lymphoma lines. It also suppresses IL-2 production by human peripheral blood enriched T cells and mouse splenocytes. The mediator has a molecular weight of 60,000-70,000 dalton, as determined by gel filtration chromatography, is heat labile, and is sensitive to trypsin, chymotrypsin, and protease.     

Interferon-γ-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-γ production

The novel cytokine interferon-γ-inducing factor (IGIF) augments natural killer (NK) cell activity in cultures of human peripheral blood mononuclear cells (PBMC), similarly to the structurally unrelated cytokine interleukin (IL)-12. IGIF has been found to enhance the production of interferon-γ (IFN-γ) and granulocyte/macrophage colony-stimulating factor (GM-CSF) while inhibiting the production of IL-10 in concanavalin A (Con A)-stimulated PBMC. In this study, when anti-CD3 monoclonal antibody (mAb)-stimulated human enriched T cells were exposed to IGIF, the cytokine dose-dependently enhanced the proliferation of the cells and this could be completely inhibited by a neutralizing antibody against IL-2 at lower concentrations of IGIF. Neutralizing antibody against IFN-γ had only insignificant inhibitory effects on T cell proliferation at higher concentrations of IGIF. Enzyme-linked immunosorbent assays (ELISA) revealed that, like PBMC, T cells exposed to IGIF produced large amounts of IFN-γ; however, changes in the production of IL-4 and IL-10 were minimal. IGIF, but not IL-12, significantly enhanced IL-2 and GM-CSF production in T cell cultures, as determined by CTLL-2 bioassay and ELISA, respectively; however, both IGIF and IL-12 enhanced IFN-γ production by the T cells. When T cells were exposed to a combination of IGIF and IL-12, a synergistic effect was observed on the production of IFN-γ, but not on production of IL-2 and GM-CSF. In conclusion, IGIF enhances T cell proliferation apparently through an IL-2-dependent pathway and enhances Th1 cytokine production in vitro and exhibits synergism when combined with IL-12 in terms of enhanced IFN-γ production but not IL-2 and GM-CSF production. Based on structural and functional differences from any known cytokines, it was recently proposed that this cytokine be designated interleukin-18.     

Bacterial lipopolysaccharide induces proliferation of IL-6-dependent plasmacytoma cells by MAPK pathway activation

Mouse plasmacytomas are appropriate models to study the biology of human multiple myeloma (MM). Growth of murine interleukin-6 (IL-6)-dependent hybridoma/plasmacytoma lines can be stimulated by bacterial lipopolysaccharides (LPS). However, the molecular mechanisms of this phenomenon are still not elucidated. In this study the in vitro action of bacterial LPS on the mouse IL-6-dependent B9 hybridoma/plasmacytoma cell line and two IL-6-dependent hybridomas was investigated. The involvement of different signal transduction pathways was established using specific kinase inhibitors in proliferation assays and immunoblotting analysis of the kinase activity. Selective mitogen-activated protein kinase (MAPK) kinase inhibitor PD989059 inhibited both IL-6- and LPS-induced B9 cell proliferation. In contrast, in H187 and H188 cells, PD98059 inhibited only LPS-, but not IL-6-stimulated cell growth. The kinetics of MAPK activation in all cell lines showed that phosphorylation of p42 MAPK (encoded as ERK2) but not of p44 MAPK (ERK1), was considerably increased after treatment with LPS. We found that in H187 and H188 hybridomas IL-6 induced proliferation by a different STAT3-dependent mechanism. This study demonstrates the key role of the MAPK pathway in LPS-stimulated growth of mouse IL-6-dependent plasmacytoma cells. These findings suggest the presence of signaling mechanism in MM cells inducible by bacterial mitogens and possibly mediated by Toll-like receptors (TLR) – evolutionarily conserved molecules playing a central role in the microbial recognition and initiation of the cellular innate immune response.     

A rapid limiting dilution assay for measuring frequencies of alloreactive, interleukin-2-producing T cells in humans.

A limiting dilution analysis (LDA) has been established which measures the total numbers of alloreactive interleukin-2 (IL-2)-secreting T cells in human peripheral blood mononuclear cells (PBMC). A significant advantage over most previous LDA is that the assay may be completed in approximately 48 h since an IL-2-dependent 'indicator' cell line is used to reduce assay time. Results are reproducible and correlate with the degree of HLA class II antigenic disparity between responder and stimulator cells. Use of both PBMC and Epstein-Barr virus-transformed B lymphoblastoid cell lines (B-LCL) as stimulator cells permits estimation of the frequency of Epstein-Barr virus-specific T cells in different responder individuals. A modification of the assay may also be used to measure the frequencies of 'primed' alloreactive cells, i.e., those alloreactive cells which have previously encountered their specific stimulating alloantigen. Use of the assay in the clinical context of bone marrow and renal transplantation is discussed.     

Suppression of interleukin-2 production and activity by factor(s) released by peripheral blood mononuclear cells during papillomavirus infections.

Supernatant fluids from cultured peripheral blood mononuclear cells (PBMC) obtained from patients with extensive papillomavirus infections such as condyloma acuminatum (CA) and epidermodysplasia verruciformis (EV) depressed the proliferative responses of T cells to phytohemagglutinin-P (PHA-P) and the production of interleukin-2 (IL-2) from those preparations. Fluids from the same cultures also inhibited the mitogenic activity of IL-2 on CTLL-2 cells as IL-2-dependent target cells. These soluble suppressor factors (SSF) from PBMC were present in significantly higher concentrations in fluids from cultured PBMC from patients in comparison to healthy controls. A soluble suppressor factor was characterized also from cultured rabbit PBMC after the rabbits had been infected with Shope papillomaviruses. This suppressor factor likewise inhibited IL-2 production and IL-2 activity.     

Proliferative response of human CD4+ T lymphocytes stimulated by the lectin jacalin

The Galβ(1–3)GalNAc-binding lectin jacalin is known to specifically induce the proliferation of human CD4⁺ T lymphocytes in the presence of autologous monocytes and to interact with the CD4 molecule and block HIV-1 infection of CD4⁺ cells. We further show that jacalin-induced proliferation is characterized by an unusual pattern of T cell activation and cytokine production by human peripheral blood mononuclear cells (PBMC). A cognate interaction between T cells and monocytes was critical for jacalin-induced proliferation, and human recombinant interleukin (IL)-1 and IL-6 did not replace the co-stimulatory activity of monocytes. Blocking studies using monoclonal antibodies (mAb) point out the possible importance of two molecular pathways of interaction, the CD2/LFA-3 and LFA-1/ICAM-1 pathways. One out of two anti-CD4 mAb abolished jacalin responsiveness. Jacalin induced interferon-γ and high IL-6 secretion, mostly by monocytes, and no detectable IL-2 synthesis or secretion by PBMC. In contrast, jacalin-stimulated Jurkat T cells secreted IL-2. CD3^? Jurkat cell variants failed to secrete IL-2, suggesting the involvement of the T cell receptor/CD3 complex pathway in jacalin signaling. IL-2 secretion by CD4^? Jurkat variant cells was delayed and lowered. In addition to CD4, jacalin interacts with the CD5 molecule. Jacalin-CD4 interaction and the proliferation of PBMC, as well as IL-2 secretion by Jurkat cells were inhibited by specific jacalin-competitive sugars.     

A recombinant extracellular domain of the human interleukin 4 receptor inhibits the biological effects of interleukin 4 on T and B lymphocytes

Human interleukin 4 (IL4) acts on various hematopoietic cell types through interaction with a specific cell surface receptor (IL4R), whose cDNA has been cloned. We have produced a cDNA encoding a soluble form of the extracellular domain of the human IL 4R (sIL4R) and describe here the capacity of sIL4R to antagonize the in vitro activities of IL4 on normal B and T lymphocytes. sIL4R inhibited IL4-induced proliferation of both phytohemagglutinin-preactivated peripheral blood mononuclear cells (PBMC) and anti-IgM co-stimulated tonsil B cells with similar efficiency. This inhibitory activity was specific since sIL4R did not affect IL2-dependent proliferation of these cells. sIL4R also blocked IL4-dependent induction of the low-affinity receptor for IgE on B cells and inhibited IgE production by IL4-activated PBMC. Thus, in contrast to the IL6R extracellular domain which stimulates IL6 biological activity, the IL4R extracellular domain is a powerful antagonist of its specific ligand.     

Transforming growth factor beta from multiple myeloma cells inhibits proliferation and IL-2 responsiveness in T lymphocytes

Multiple myeloma (MM) is a cancer of plasma cells, characterized by profound suppression of host immune responses. Here we show that MM cell lines significantly suppress the proliferation, blasting, response to interleukin-2 (IL-2), and expression of CD25 by concanavalin A (Con A)-activated or allostimulated peripheral blood T lymphocytes. T cells arrest in the G1 stage of the cell cycle, and do not enter the IL-2 autocrine growth pathway. T cell inhibition was mediated by a soluble factor. MM cell lines did not produce IL-10 but did produce large amounts of transforming growth factor beta1 (TGF-beta1). T cells were assessed for their ability to respond to IL-2 when co-cultured with MM cells in the presence or absence of the TGF-beta inhibitor, TGF-beta latency-associated peptide (LAP). MM cells suppressed IL-2 responses but this inhibition was completely reversed by TGF-beta LAP. A CD25-, IL-2-dependent blast cell line was not inhibited by MM cells or rhTGF-beta, confirming the specificity of the inhibition mechanism for the IL-2 autocrine growth pathway. We conclude that MM cells suppress T cells in their entry into the autocrine IL-2/CD25 pathway and in response to IL-2, and that TGF-beta has a significant role to play.     

Mitogenic response of human thymocytes: identification of functional Ca2+-dependent and independent signals.

In contrast to peripheral blood mononuclear cells (PBMC), human thymocytes do not exhibit a proliferative response to the T cell mitogens phytohaemagglutinin (PHA), concanavalin A (Con A), or Staphylococcal protein A (SPA). In thymocytes and PBMC, Con A and PHA induce increases in free cytosolic calcium concentrations [( Ca2+]i). Since both Con A and PHA induce similar increases in [Ca2+]i in thymocytes and PBMC, the absence of thymocyte proliferation was not due to an inability to induce an increase in [Ca2+]i. The lack of proliferative response was secondary to the failure of the mitogens to induce interleukin 2 (IL-2) production. Incubation of mitogen-treated thymocytes with phorbol esters reconstituted IL-2 production and the proliferative response indicating that the cells were indeed activated by the mitogens. Similarly, addition of exogenous recombinant IL-2 also induced mitogen-treated thymocytes to proliferate. This IL-2-dependent proliferation established that SPA, Con A, and PHA triggered the expression of biologically active IL-2 receptors. Since an increase in [Ca2+]i is a prerequisite, and possibly a trigger, for IL-2 production, the failure of PHA, Con A, or SPA to result in thymocyte proliferation may be due to an inability of thymocytes to respond to increases in [Ca2+]i with subsequent IL-2 production.     

Activation induced changes in expression and structure of the IL-7 receptor on human T cells.

Activation of human peripheral blood T cells renders them capable of proliferating to IL-2, -4, and -7, and upregulates the receptors for IL-2 and -4. In this study the effect of activation on the receptor for IL-7 has been investigated. Scatchard analysis showed dual affinity binding of IL-7 to peripheral blood mononuclear cells (PBMC). Furthermore, activation of PBMC with anti-CD3 antibodies resulted in a 4-fold downregulation of both the high and low affinity IL-7 receptors. SDS-PAGE analysis of [125I]IL-7 cross-linked resting PBMC revealed a major complex of 104/107 kDa (reduced/non-reduced) and a minor complex of 184/178 kDa (reduced/non-reduced). In contrast, cross-linking of activated PBMC revealed a third prominent complex of 93 kDa (non-reduced) not seen on unstimulated cells. This 93 kDa complex was observed on purified activated peripheral blood T cells and T cell blasts. Moreover, on a panel of IL-7 responsive T cell clones the 93 kDa complex was the only major cross-linked product observed. These results demonstrate that T cell activation causes changes in both the level of expression of the IL-7 receptor and the nature of the proteins associated with the receptor. It is postulated that these changes in receptor structure may be related to the acquisition of responsiveness to the IL-7 growth signal.     

Impaired T and NK cell response of bone marrow and peripheral blood stem cell products to interleukin (IL)-2.

The function of steady-state and interleukin (IL)-2-co-cultured mononuclear cells differs significantly between bone marrow (BM) products, growth factor-mobilized peripheral blood stem cell (PSC) products and normal peripheral blood mononuclear cells (PBMC). The natural killer (NK) cell activity and T cell proliferative response of PSC products from non-Hodgkin's lymphoma (NHL) patients are significantly higher than that of BM products and similar to normal PBMC. However, following a five-day co-culture with IL-2 (100 IU/ml), the NK activity of PSC, PBMC, and BM products (lytic units) was increased 176-, 40-, and 14-fold, respectively, compared to that observed prior to IL-2 culture. In contrast, lymphokine activated killer (LAK) cytotoxicity prior to IL-2 culture was low in PSC and BM products and normal PBMC, but was significantly increased in PSC products and PBMC following IL-2 co-culture. The proliferative response of PSC and BM products to the T cell mitogen phytohemagglutinin (PHA) was significantly lower than that observed with normal PBMC; however, PSC had a significantly higher response than cells from BM products. Similar patterns of T cell PHA mitogenic response were observed after IL-2 co-culture. In addition, the IL-2 mitogenic responses of IL-2-co-cultured PSC and BM products were also significantly lower than that observed with PBMC co-cultured with IL-2. The IL-2 mitogenic response of PBMC was also significantly increased compared to prior to IL-2 co-culture; whereas, the IL-2 mitogenic responses from PSC and BM cells were not. In summary, co-culture with IL-2 can increase the NK and LAK cell cytotoxicity of PSC and BM products from NHL patients, but IL-2 co-culture does not improve T cell function within either BM or PSC products.     

Genistein, a selective protein tyrosine kinase inhibitor, inhibits interleukin-2 and leukotriene B4 production from human mononuclear cells.

In this study, genistein, a selective protein tyrosine kinase (PTK) inhibitor, inhibited peripheral blood mononuclear cell (PBMC) proliferation and interleukin-2 production from cultures that were stimulated with phytohemagglutinin (PHA), phorbol 12-myristate 13-acetate (PMA) plus A23187, or PHA plus PMA, and genistein effectively blocked the PHA plus IL-2-induced PBMC proliferation. Further, we also found that genistein inhibited LTB4 production from A23187-stimulated cultures whereas H-7, a PKC inhibitor, had no effect on LTB4 production. Our results suggest that PTK may be necessary for the synthesis of LTB4.     

Interleukin 7 is a growth factor for mature human T cells

We have studied the capacity of interleukin (IL) 7 to support the growth and expansion of human T cell clones of different phenotype and function. All the clones studied (CD4+CD8+, CD4-CD8- Tcell receptor alpha/beta or gamma/delta) responded to IL 7. The proliferative response of all the T cell clones induced by IL 7 was routinely less than to IL2, but comparable to the IL4 response. IL 7 also induced the proliferation of resting, freshly prepared peripheral blood mononuclear cells (PBMC) or Tcell-enriched (E+) cells. The pattern of proliferation observed in the presence of IL 7 was similar, but lower in magnitude, to that induced by IL 2. In both these cells populations the response to lymphokines alone was always less than the response to lymphokines plus insolubilized anti-CD3 monoclonal antibody. In contrast IL4 produced a different pattern of responsiveness, as significant proliferation was observed only on PBMC costimulated with anti-CD3. The possibility that IL 7 mediates its growth stimulation by the IL2 pathway was excluded by the incapacity of anti-IL2 or anti-Tac monoclonal antibody, in concentrations which blocked IL2-dependent proliferation, to inhibit IL 7-dependent growth. We conclude that IL 7 is a major growth factor for human mature T cells, and its activity is not limited to lymphocyte progenitors.     

Inhibition of T cell-dependent human B cell proliferation and B cell differentiation by polyspecific monomeric IgG.

A commercially available polyspecific, monomeric IgG preparation suitable for intravenous administration (IgSRK; Sandoglobulin) can inhibit pokeweed mitogen (PWM)-induced proliferation of peripheral blood mononuclear cells (PBMC) by a small, but statistically significant, amount compared to control cultures. Such inhibition could not be demonstrated when PBMC were stimulated with the T cell mitogen phytohaemagglutinin. Surface phenotype analysis of the PWM-stimulated cells indicated that in IgSRK-containing cultures, the proportion of B cells was decreased and the proportion of T cells was increased compared to control cultures. This alteration in T:B ratio was not due to antigenic modulation of B or T cell markers from their surfaces. In addition, IgSRK inhibited the proliferation of T cell-depleted PBMC cultures stimulated by B cell proliferation factors (BCPF) but not by fixed protein A-bearing Staphylococcus aureus strain Cowan I. The capacity to inhibit B cell proliferation was independent of and distinct from its capacity to inhibit B cell differentiation, since IgSRK inhibited the differentiation of a B cell differentiation factor (BCDF)-sensitive line by BCDF (which contains no BCPF activity). IgSRK inhibited PWM-induced generation of cytoplasmic Ig+ cells but had no effect on Ig secretion from mature Ig-secreting cells. Taken together, these findings suggest that IgSRK (which contains the IgG fraction from pooled plasma from 2,000 healthy donors) can inhibit T cell-dependent or T cell factor-dependent B cell proliferation and B cell differentiation.     

An OKT4+ T-Cell Population in Séezary Syndrome: Attempts to Elucidate Its Lack of Proliferative Capacity and Its Suppressive Effect

We have previously reported a case of Sézary Syndrome (SS), in which an OKT4+ T-cell population exhibited a defective response to non-specific mitogens, and an ability to suppress lectin-induced T-cell proliferation and pokeweed mitogen (PWM)-induced B-cell differentiation of normal donor peripheral blood mononuclear cells (PBMC). We report now that resting Sézary cells (SC) were essentially negative for activation antigens (Ag) detected by monoclonal antibodies (MoAb) B1.49.9, anti-Tac, OKT9, OKT10, and OKIa1. After phytohaemagglutin (PHA) stimulation, all these Ag were expressed with the notable exception of OKT10. Further investigations of SC functions indicated that no interleukin 2 (IL-2) biological activity was detected in culture supernatants of SC constimulated with PHA and phorbol myristate acetate (PMA). Interestingly, such stimulated SC exhibited a marked capacity to absorb exogenous IL-2 while remaining unable to proliferate. These data suggest that patient's unresponsiveness to PHA may be unrelated to IL-2 as an extracellular growth signal, but may instead be due to a failure in a later cellular activation event, subsequent to the binding of IL-2 to its receptors. Lack of T10 Ag expression may be involved as a cause or a consequence. Kinetic study of suppression of PHA-induced T-cell proliferation of normal PBMC revealed that inhibition occurred during the first 24 h; moreover we showed that it was not due to limitation of available IL-2 since it persisted in excess of IL-2; remarkably the growth of an IL-2-dependent murine cell line was unaffected by the presence of SC. Further, inhibition was also observed on IL-2-independent calcium ionophore A 23187-induced T-cell proliferation of normal PBMC. Taken together, the data suggest that the target of suppressor activity is probably an important obligatory intracellular event controlling DNA replication, which is common to both IL-2-dependent and IL-2-independent T-cell activation processes. Human T-cell leukaemia/lymphoma virus I (HTLV-I) related p.19 and p.24 Ag were absent on fresh and 30-day cultured SC, suggesting the absence of HTLV-I infection, although not ruling out a proviral integration in the SC DNA.     

Antilymphoproliferative activity of ethanolic extract of Boerhaavia diffusa roots

Extracts of plants have been widely evaluated for possible antiproliferative and anticarcinogenic properties. The antiproliferative activity of ethanolic extract of Boerhaavia diffusa, a plant used in traditional medicine, was evaluated in several cells. It inhibited T cell mitogen phytohemagglutinin and concanavalin A-stimulated proliferation of human peripheral blood mononuclear cells (PBMC). It also inhibited purified protein derivative antigen-stimulated PBMC proliferation and human mixed lymphocyte culture. In addition, B. diffusa extract inhibited the growth of several cell lines of mouse and human origin, such as mouse macrophage cells (RAW 264.7), human macrophage cells (U937), human monocytic cells (THP-1), mouse fibroblast cells (L929), human embryonic kidney cells (HEK293), mouse liver cells (BNLCL.2), African green monkey kidney cells (COS-1), mouse lymphoma cells (EL-4), human erythroleukemic cells (K562), and human T cells (Jurkat). The present study has demonstrated the antiproliferative potential of ethanolic extract of B. diffusa in vitro.     

Influence of growth hormone-releasing hormone (GHRH) on phytohemagglutinin-induced lymphocyte activation: comparison of two synthetic forms. GHRH and PHA-induced lymphocyte activation.

The in vitro effect of synthetic human growth hormone-releasing hormone (GHRH) on mitogen-induced lymphocyte proliferation and lymphokine secretion was investigated. Peripheral blood mononuclear cells (PBMC) of healthy adults were incubated in the presence and absence of increasing concentrations (from 0.006 to 50 micrograms/ml) of two forms of GHRH differing in amino-acid sequence (GHRH 1-44 and GHRH 1-29) or of increasing concentrations (from 0.0012 to 20 U/ml) of recombinant human insulin (rh-insulin). Low concentrations of GHRH 1-29 increased phytoemoagglutinin (PHA)-induced lymphoproliferation, while high concentrations inhibited lymphocyte response, interleukin-2 (IL-2) secretion and IL-2 receptor expression on activated cells. A toxic effect was excluded since no differences in cell viability were observed between cells cultured with and without hormone. GHRH 1-44 did not affect PHA-induced lymphoproliferation, IL-2 production and IL-2 receptor expression. Low concentrations of rh-insulin increased PHA-elicited lymphoproliferation, while high concentrations did not decrease lymphocyte response. The present study suggests that GHRH modulates in vitro human T lymphocyte functions.