Functional analysis of TRAIL receptors using monoclonal antibodies.

mAbs were generated against the extracellular domain of the four known TNF-related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel of human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4). Immobilized anti-TRAIL-R1 or -R2 mAbs were cytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to recombinant TRAIL were also resistant to these mAbs and only became sensitive when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced death was characterized by the activation of intracellular caspases, which could be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk). When used in solution, one of the anti-TRAIL-R2 mAbs was capable of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cells and prevented TRAIL-induced death of these cells, whereas two of the anti-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R1:Fc. Furthermore, use of the blocking anti-TRAIL-R2 mAb allowed us to demonstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 were necessary and sufficient to mediate cell death. In contrast, the expression of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in determining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.

Human neutrophils express the interleukin-15 receptor alpha chain (IL-15Ralpha) but not the IL-9Ralpha component.

The interleukin-15 receptor (IL-15R) is composed of at least three chains, namely gammac, IL-2Rbeta, and the recently identified IL-15Ralpha, while the IL-9R complex consists of gammac and a subunit designated IL-9Ralpha. Our previous work and that of others have shown that human neutrophils express gammac and IL-2Rbeta (two components shared with IL-2R) but not IL-2Ralpha and that IL-15 is a neutrophil agonist, whereas IL-2 is not. In this study, using flow cytometry with a specific anti-human IL-15Ralpha, we show for the first time that human neutrophils express surface IL-15Ralpha. Although we previously found that IL-15 is a neutrophil agonist, our present work shows that IL-15 does not trigger superoxide production nor cell spreading onto glass. In addition, we report that human neutrophils do not respond to IL-9 with respect to the functions/responses studied, namely, superoxide production, spreading onto glass, cell shape changes, phagocytosis, RNA synthesis, and apoptosis. Further, our results show that neutrophils do not express IL-9Ralpha as assessed by flow cytometry with a specific anti-human IL-9Ralpha antibody that stains the transfected cell line BW-h9R used as positive control. Finally, our results indicate that gammac expression was not modulated and remained stable for up to 24 h when neutrophils were stimulated with all currently known "gammac users," namely, IL-2, IL-4, IL-7, IL-9, and IL-15. We conclude that human neutrophils express all IL-15R components on their surface, including IL-15Ralpha, that IL-15 activates human neutrophils (as the IL-4 neutrophil agonist) by a mechanism which does not involve upregulation of gammac cell surface expression, and that IL-9 is not a neutrophil agonist as demonstrated by the inability to modulate the tested functions/responses that correlate with lack of the IL-9R component, namely, IL-9Ralpha.

TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL.

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL-R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL-R2, by ligand-based affinity purification and subsequent molecular cloning. TRAIL-R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL-R2 contains two extracellular cysteine-rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell-surface-expressed TRAIL-R2, and TRAIL-induced apoptosis is inhibited by a TRAIL-R2-Fc fusion protein. TRAIL-R2 mRNA is widely expressed and the gene encoding TRAIL-R2 is located on human chromosome 8p22-21. Like TRAIL-R1, TRAIL-R2 engages a caspase-dependent apoptotic pathway but, in contrast to TRAIL-R1, TRAIL-R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family.

A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells.

Mammalian cells proteolytically release (shed) the extracellular domains of many cell-surface proteins. Modification of the cell surface in this way can alter the cell's responsiveness to its environment and release potent soluble regulatory factors. The release of soluble tumour-necrosis factor-alpha (TNF-alpha) from its membrane-bound precursor is one of the most intensively studied shedding events because this inflammatory cytokine is so physiologically important. The inhibition of TNF-alpha release (and many other shedding phenomena) by hydroxamic acid-based inhibitors indicates that one or more metalloproteinases is involved. We have now purified and cloned a metalloproteinase that specifically cleaves precursor TNF-alpha. Inactivation of the gene in mouse cells caused a marked decrease in soluble TNF-alpha production. This enzyme (called the TNF-alpha-converting enzyme, or TACE) is a new member of the family of mammalian adamalysins (or ADAMs), for which no physiological catalytic function has previously been identified. Our results should facilitate the development of therapeutically useful inhibitors of TNF-alpha release, and they indicate that an important function of adamalysins may be to shed cell-surface proteins.

Dual oncostatin M (OSM) receptors. Cloning and characterization of an alternative signaling subunit conferring OSM-specific receptor activation.

Oncostatin M (OSM) is a cytokine whose structural and functional features are similar to other members of the interleukin (IL)-6 family of cytokines (IL-6, IL-11, leukemia inhibitory factor (LIF), granulocyte colonystimulating factor, ciliary neurotrophic factor, and cardiotrophin-1), many of which utilize gp130 as a common receptor subunit. A biologically active OSM receptor has been previously described that consists of a heterodimer of leukemia inhibitory factor receptor (LIFR) and gp130. This LIFR.gp130 complex is also a functional receptor for LIF. We have cloned and characterized an alternative subunit (OSMRbeta) for an OSM receptor complex (a heterodimer of gp130 and OSMRbeta) that is activated by OSM but not by LIF. The signaling capability of specific receptor subunit combinations was analyzed by independent assays measuring cell proliferation or induction of acute phase protein synthesis. Our results demonstrate that both LIF and OSM cause tyrosine phosphorylation and activation of the gp130.LIFR combination, but the gp130.OSMRbeta complex is activated by OSM only. OSM-induced cellular responses, initiated through low affinity binding to gp130, are mediated by two heterodimeric receptor complexes that utilize alternative signal transducing subunits that confer different cytokine specificities to the receptor complex.

CD30 ligand expression in nonmalignant and Hodgkin's disease-involved lymphoid tissues.

The CD30 ligand (CD30L) is a type II transmembrane glycoprotein of the tumor necrosis factor ligand superfamily. Recent cloning of CD30L has enabled studies to explore its function and tissue distribution. For instance, recombinant CD30L has been shown to co-stimulate T cells and to act as mitogen for Hodgkin's disease (HD)-derived cell lines. The counter-receptor for CD30L, ie, CD30, is a type I cytokine receptor that is highly expressed by activated T cells, Hodgkin and Reed-Sternberg (H-RS) cells, and anaplastic large cell lymphoma cells. In the present study, recombinant membrane-bound and soluble human CD30L were instrumental to raise a monoclonal antibody (M80) recognizing membrane-bound CD30L on transfected and native cells. With this reagent, a panel of cultured lymphoma-derived cell lines as well as primary normal, reactive, and HD-involved lymphoid tissues were examined for expression of CD30L by immunostaining and flow cytometry. In reactive lymphnodes and tonsils, CD30L was expressed by a small subset of lymphoid cells, histiocytes, and granulocytes. Higher levels of CD30L expression were noted in HD lesions among bystander cells; ie, T cells and granulocytes that surrounded H-RS cells. Native CD30L displayed at the cell surface was functionally active as shown by the ability of fixed granulocytes to interact with CD30+ cell lines. Moreover, CD30L was detectable, although to a lower staining intensity, in primary H-RS cells of all HD tissues investigated regardless of the histological subtype and the phenotype of H-RS cells (ie, CD30+/CD40+ versus CD30-/CD40+). Co-expression of CD30 and CD30L that was seen on H-RS cells of all, except the CD30- nodular lymphocyte predominant, subtypes of HD may point to the use of this pair of molecules in paracrine and/or autocrine mitogenic cell interactions. Monoclonal antibody M80 may thus represent a useful tool for studying CD30L expression on cultured cell lines and primary cells from normal, reactive, and malignant tissues.

Functional CD40 ligand expression on T lymphocytes in the absence of T cell receptor engagement: involvement in interleukin-2-induced interleukin-12 and interferon-gamma production.

Despite the fact that the great majority of T cells at the site of an inflammatory response are not antigen specific, the mechanisms leading to activation and recruitment of these bystander T cells are poorly understood. We previously reported that soluble (s)CD23 potentiated the interleukin (IL)-2-induced interferon (IFN)-gamma production by T cells co-cultured with autologous monocytes in the absence of T cell receptor (TCR) engagement. Our present data demonstrate that the IL-2-induced IFN-gamma secretion, in the presence but also in the absence of sCD23, is strictly IL-12 dependent, inasmuch as anti-IL-12 antibody abrogated both responses. Most interestingly, anti-CD40 ligand (CD40L) monoclonal antibody significantly inhibited IL-2-induced IL-12 as well as IFN-gamma production. These results suggest that CD40L was expressed on T cells in the absence of TCR engagement. Indeed, purified unstimulated T cells readily expressed CD40L. IL-2 and monocytes did not up-regulate CD40L on resting T cells. It is proposed that low levels of CD40L expression on non-antigen stimulated T cells are sufficient to signal through CD40 molecules on accessory cells and to induce IL-12 secretion, which in turn can synergize with IL-2 for the induction of IFN-gamma production, thus contributing to the inflammatory process.

Endogenous production of interleukin 15 by activated human monocytes is critical for optimal production of interferon-gamma by natural killer cells in vitro.

Natural killer (NK) cells are large granular lymphocytes that constitutively express functional IL-2 receptors. We have shown that recombinant human IL-15 uses the IL-2 receptor to activate human NK cells and can synergize with recombinant human IL-12 to stimulate NK cell production of IFN-gamma in vitro. IFN-gamma production by NK cells is critical in the prevention of overwhelming infection by obligate intracellular microbial pathogens in several experimental animal models. Herein, we demonstrate that human monocytes produce IL-15 protein within 5 h of activation with LPS. Using an IL-15-neutralizing antiserum in a coculture of LPS-activated monocytes and NK cells, we demonstrate that monocyte-derived IL-15 is critical for optimal NK cell production of IFN-gamma. Endogenous IL-15 activates NK cells through the IL-2 receptor, and with endogenous IL-12, regulates NK cell IFN-gamma after monocyte activation by LPS. These in vitro studies are the first to characterize a function for endogenous IL-15, and as such, suggest an important role for IL-15 during the innate immune response. IL-15 may be an important ligand for the NK cell IL-2 receptor in vivo.

Activation of HIV expression by CD30 triggering in CD4+ T cells from HIV-infected individuals.

CD30 is a member of the tumor necrosis factor receptor superfamily, preferentially expressed by T cells producing type 2 helper (Th2) cytokines, whose ligand (CD30L) has been identified on B cells, activated macrophages, and a subset of activated T cells. We show here that cross-linking CD30 with an agonistic CD30-specific monoclonal antibody, as well as with CD30L+ CD8+ T cell clones or CD30L+ B cells, enhanced HIV replication in CD4+ T cells from HIV-infected individuals, and such a potentiating effect was inhibited by anti-CD30L antibody. The anti-CD30L antibody also exerted a suppressive effect on spontaneous HIV replication occurring in lymph node cells from an HIV-sero-positive patient, showing CD30L expression by both B and CD8+ T lymphocytes. Thus, CD30 triggering by CD30L-expressing cells may plan an important role in the activation of HIV expression from latently infected CD4+ T cells.

Pleiotropic effects of the CD30 ligand on CD30-expressing cells and lymphoma cell lines.

CD30 is a member of the tumor necrosis factor receptor superfamily. CD30 was originally described as a cell surface antigen on primary and cultured Hodgkin's and Reed-Sternberg cells. In this study, recombinant human CD30 ligand was expressed on the surface of CV-1/EBNA cells and tested for biologic activities on a variety of different CD30+ human lymphoma cell lines. CD30 ligand enhanced Ig secretion of Epstein-Barr virus (EBV)-immortalized, CD30+ lymphoblastoid B-cell lines, but not Burkitt lymphoma lines. Recombinant CD30 ligand enhanced proliferation of "T-cell-like" Hodgkin's disease-derived cell lines and an adult T-cell leukemia cell line, but not "B-cell-like" Hodgkin's disease-derived cell lines, CD30+, EBV-immortalized lymphoblastoid B-cell lines, or CD30+ and EBV+ tumor B-cell non-Hodgkin's lymphoma cell lines. In addition, CD30 ligand mediated reduction of proliferation and viability, by induction of cytolytic cell death, of CD30+, large-cell anaplastic lymphoma cell lines. Two new antibodies, M44 and M67, against the CD30 antigen demonstrated similar biologic activities to the CD30 ligand. Taken together, these data demonstrate pleiotropic biologic activities of the CD30 ligand on different CD30+ lymphoma cell lines and indicate that the CD30-CD30 ligand interaction might have a pathophysiologic role in Hodgkin's and some non-Hodgkin's lymphomas.

Anti-cytokine antibodies as carrier proteins. Prolongation of in vivo effects of exogenous cytokines by injection of cytokine-anti-cytokine antibody complexes.

Anti-cytokine antibodies that block interactions between cytokines and cytokine receptors have been used to inhibit endogenous cytokine function. However, injection of mice with mixtures of IL-4 and either of two neutralizing anti-IL-4 mAb, at a cytokine/anti-cytokine mAb molar ratio of approximately 2:1, enhances and prolongs in vivo IL-4 activity, as measured by induction of increased spleen cell Ia expression. Although splenocyte Ia expression returns to baseline two days after mice are injected with free IL-4, soluble IL-4-anti-IL-4 mAb complexes still induce several-fold increases in Ia expression 3 days after injection. Complexes that contain as little as 400 ng of IL-4 have considerable in vivo stimulatory activity, and a maximal effect on splenocyte Ia expression is induced by injection of 2 micrograms of complexed IL-4. The stimulatory effect of IL-4-containing complexes on splenocyte Ia expression can be blocked by increasing the ratio of anti-IL-4 mAb to IL-4, by injection of anti-IL-4R mAb, and by in vivo aggregation of the complexes. Complexes of IL-4 with a non-neutralizing anti-IL-4 mAb do not have increased IL-4 agonist activity in vivo. These observations are most consistent with the possibility that neutralizing anti-IL-4 mAb act as carrier proteins that increase the in vivo half-life of IL-4 by preventing its excretion, and possibly, by preventing modification of its active site. The enhanced agonist effect of IL-4-anti-IL-4 mAb complexes is not unique; complexes of IL-3 with a neutralizing anti-IL-3 mAb have a greatly increased ability, compared with free IL-3, to stimulate mucosal mastocytosis, and complexes of IL-7 with a neutralizing anti-IL-7 mAb have a greatly increased ability, compared with free IL-7 or IL-7 complexed with a non-neutralizing anti-IL-7 mAb, to stimulate an increase in pre-B cell number. These observations suggest that complexes of cytokines and neutralizing anti-cytokine mAb may provide a generally useful way to increase the magnitude and duration of cytokine effects in vivo.

Inhibition of murine B and T lymphopoiesis in vivo by an anti-interleukin 7 monoclonal antibody.

The effects of interleukin 7 (IL-7) on the growth and differentiation of murine B cell progenitors has been well characterized using in vitro culture methods. We have investigated the role of IL-7 in vivo using a monoclonal antibody that neutralizes IL-7. We find that treatment of mice with this antibody completely inhibits the development of B cell progenitors from the pro-B cell stage forward. We also provide evidence that all peripheral B cells, including those of the B-1 and conventional lineages, are derived from IL-7-dependent precursors. The results are consistent with the rapid turnover of B cell progenitors in the marrow, but a slow turnover of mature B cells in the periphery. In addition to effects on B cell development, anti-IL-7 treatment substantially reduced thymus cellularity, affecting all major thymic subpopulations.

Interleukin-1 beta regulation of islet and thyroid autoimmunity in the BB rat.

Daily injections of high dose human recombinant interleukin-1 beta (IL-1 beta) accelerated the onset of both insulin-dependent diabetes mellitus and lymphocytic thyroiditis in genetically prone BB rats. In diabetes-resistant BB rats, high dose IL-1 beta failed to induce diabetes. Additionally, the presence of neutralizing IL-1 beta antibodies in these rats strongly correlated with inhibition of lymphocytic thyroiditis. Since low dose IL-1 beta protects diabetes-prone rats from IDDM, we conclude that IL-1 beta is a potent modulator of autoimmune diabetes and thyroid disease in genetically susceptible rats.