CD47 ligation induces caspase-independent cell death in chronic lymphocytic leukemia.

Thrombospondin forms a 'molecular bridge' between phagocytic and apoptotic cells through interaction with alphavbeta3/CD36. We report here that engagement of CD47, a newly described thrombospondin receptor, by immobilized monoclonal antibody against CD47 or by thrombospondin induced in all B-cell chronic lymphocytic leukemia clones the cytoplasmic features of apoptosis (cell shrinkage, decrease in mitochondrial transmembrane potential and phosphatidylserine externalization) without the nuclear features (chromatin condensation, appearance of single-stranded DNA, DNA fragmentation and cleavage of poly ADP-ribose polymerase). These cytoplasmic events of apoptosis were not prevented by the addition of caspase inhibitor z-VAD-fmk, or by the presence of survival factors (such as interleukin-4 and gamma interferon) or cell activation. Morphological studies confirmed the integrity of the nucleus and showed swelling of the mitochondria. This caspase-independent death pathway may be relevant to the development of alternate therapeutic strategies in chronic lymphocytic leukemia, which remains an incurable disease.

Regulation of cytokine production by soluble CD23: costimulation of interferon gamma secretion and triggering of tumor necrosis factor alpha release.

Soluble CD23 (sCD23) has multiple IgE-independent biological activities. In the present study, we examined the regulatory effect of sCD23 on cytokine production by human peripheral blood mononuclear cells (PBMC). We show that sCD23 enhances by about 80-fold the interleukin 2 (IL-2)-induced interferon gamma (IFN-gamma) production and by about 10-fold the response to IL-12. This potentiating activity is time and dose dependent and is not associated with a significant effect on DNA synthesis. The sCD23 costimulatory activity for IFN-gamma synthesis is drastically reduced in monocyte-depleted PBMC, suggesting that monocytes may be the target for sCD23. This hypothesis was supported by the following observations. First, sCD23 alone is a potent inducer of tumor necrosis factor alpha (TNF-alpha) production by PBMC and this effect disappears after monocyte depletion. The triggering of TNF-alpha release is specifically inhibited by neutralizing anti-CD23 monoclonal antibody (mAb). In addition, IL-2 and IL-12 synergize with sCD23 to induce TNF-alpha production. Second, sCD23 triggers the release of other inflammatory mediators such as IL-1 alpha, IL-1 beta, and IL-6. Finally, TNF-alpha production in response to IL-2 and sCD23 precedes IFN-gamma and IFN-gamma secretion is significantly inhibited by anti-TNF-alpha mAb, indicating that the sCD23 costimulatory signal for IFN-gamma production may be partially mediated by TNF-alpha release. It is proposed that sCD23 is a proinflammatory cytokine that, in addition, may play an important role in the control of the immune response via the enhancement of IFN-gamma production.

CD47 ligation selectively downregulates human interleukin 12 production.

Interleukin (IL)-12 plays a key role not only in protective innate and adaptive T helper cell type 1 (Th1) responses but also in chronic inflammatory diseases. We report here that engagement of CD47 by either monoclonal antibody, its natural ligand thrombospondin (TSP), or 4N1K (a peptide of the COOH-terminal domain of TSP selectively binding CD47) inhibits IL-12 release by monocytes. The suppression occurred after T cell-dependent or -independent stimulation of monocytes and was selective for IL-12 inasmuch as the production of tumor necrosis factor (TNF)-alpha, IL-1, IL-6, and granulocyte/macrophage colony-stimulating factor was not inhibited. CD47 ligation did not alter transforming growth factor (TGF)-beta and IL-10 production, and the suppressive effect on IL-12 was not due to autocrine secretion of TGF-beta or IL-10. The IL-12 inhibition was not mediated by Fcgamma receptor ligation, did not require extracellular Ca(2+) influx, but was reversed by two phosphoinositide 3-kinase inhibitors (wortmannin and Ly294002). Thus, engagement of CD47 on monocytes by TSP, which transiently accumulates at the inflammatory site, is a novel and unexplored pathway to selectively downregulate IL-12 response. The pathway may be relevant in limiting the duration and intensity of the inflammatory response, and in developing novel therapeutic strategies for Th1-mediated diseases.

The vitronectin receptor and its associated CD47 molecule mediates proinflammatory cytokine synthesis in human monocytes by interaction with soluble CD23.

The vitronectin receptor, alphavbeta3 integrin, plays an important role in tumor cell invasion, angiogenesis, and phagocytosis of apoptotic cells. CD47, a member of the multispan transmembrane receptor family, physically and functionally associates with vitronectin receptor (VnR). Although vitronectin (Vn) is not a ligand of CD47, anti-CD47 and beta3 mAbs suppress Vn, but not fibronectin (Fn) binding and function. Here, we show that anti-CD47, anti-beta3 mAb and Vn, but not Fn, inhibit sCD23-mediated proinflammatory function (TNF-alpha, IL-12, and IFN-gamma release). Surprisingly, anti-CD47 and beta3 mAbs do not block sCD23 binding to alphav+beta3+ T cell lines, whereas Vn and an alphav mAb (clone AMF7) do inhibit sCD23 binding, suggesting the VnR complex may be a functional receptor for sCD23. sCD23 directly binds alphav+beta3+/CD47(-) cell lines, but coexpression of CD47 increases binding. Moreover, sCD23 binds purified alphav protein and a single human alphav chain CHO transfectant. We conclude that the VnR and its associated CD47 molecule may function as a novel receptor for sCD23 to mediate its proinflammatory activity and, as such, may be involved in the inflammatory process of the immune response.

IL-2 and IL-7 but not IL-12 protect natural killer cells from death by apoptosis and up-regulate bcl-2 expression.

Human natural killer cells (NK) respond to interleukin-2 (IL-2) with augmented cytolytic activity, cytokine secretion and cell proliferation. Here we show that IL-2 protects NK cells from death by apoptosis (programmed cell death; PCD). Highly purified NK cells (CD3- CD56+) were isolated from peripheral blood lymphocytes (PBL) of either control donors or of an asymptomatic donor with 60% NK cells. Glucocorticosteroids (GCS) induced PCD in NK cells, as shown by nuclear condensation and DNA fragmentation. IL-2 completely prevented GCS-induced PCD in a dose-dependent manner without overcoming GCS-induced inhibition of NK cell proliferation. The IL-2 protective effect was mediated through the p75 beta chain of the IL-2R, as neutralizing monoclonal antibody (mAb) to the p75 beta chain but not to the p55 alpha chain completely abolished the IL-2 anti-apoptotic activity. In addition to IL-2, the cytokines IL-7 and IL-12 have been reported to regulate NK cell functions. Our present data showed that IL-7 but not IL-12 rescued NK cells from apoptosis, but to a lesser extent than IL-2. Although IL-4 had a marginal protective effect, IL-1, IL-3, IL-6, IL-8, interferon-gamma (IFN-gamma) and IFN-alpha, tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and granulocyte-macrophage colony-stimulating factor (GM-CSF) displayed no significant activity. Finally, we report that IL-2 and IL-7 enhanced bcl-2 expression in NK cells, suggesting the existence of a bcl-2-dependent survival pathway. In addition to regulating various functions, it is concluded that IL-2 and IL-7 have the ability to prevent PCD in NK cells.

Soluble CD23 directly activates monocytes to contribute to the antigen-independent stimulation of resting T cells.

The majority of T cells at the site of an inflammatory lesion do not appear to be Ag-specific, but they still contribute to the inflammatory response. Herein, we report that sCD23 activates monocytes to participate in the stimulation of resting T cells in the absence of TCR engagement. First, sCD23 selectively triggers monokine release by purified monocytes in the absence of costimulus. It induces TNF-alpha, IL-1 beta, IL-8, granulocyte-macrophage-CSF, and prostaglandin E2 but no IL-10, IL-12, TGF-beta, or leukotriene B4. The sCD23-induced TNF-alpha production is significantly inhibited by IL-4 and IL-10 but not by TGF-beta. Also, monocytes activated by sCD23 express decreased levels of HLA-DR and increased levels of CD14, CD54, CD40, and B7 Ag. Next, we show that, in the presence of monocytes, sCD23 is a potent costimulator of IL-2 or IL-12-induced IFN-gamma production by resting T cells in the absence of exogenous Ag and that this effect is partially reduced by anti-TNF-alpha mAb. B cells cannot substitute for monocytes, and CD4+ and CD8+ T cells are equal responders. The data further indicate that monocyte-T cell contact, more particularly CD40-CD40L interactions, is required for IFN-gamma production in response to IL-2 plus sCD23, and the response to IL-12 plus sCD23 is CD40- and B7-independent but is still partially contact-dependent. It is proposed that sCD23, when produced locally at a site of immune response, may trigger an inflammatory process via monokine release and may further amplify it via the stimulation of bystander non-Ag-specific T cells.

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.

IL-15 promotes IL-12 production by human monocytes via T cell-dependent contact and may contribute to IL-12-mediated IFN-gamma secretion by CD4+ T cells in the absence of TCR ligation.

At inflammatory sites, the number of activated bystander T cells exceeds that of Ag-activated T cells. We investigated whether IL-15, a monocyte-derived cytokine that shares several biologic activities with IL-2, may contribute to bystander T cell activation in the absence of IL-2 and triggering Ag. The addition of IL-15 to cocultures of monocytes and T cells stimulates CD4+ but not CD8+ T cells to produce IFN-gamma. IFN-gamma production requires endogenous IL-12, the production of which in turn is dependent upon CD40/CD154 interactions between CD4+ T cells and monocytes. Indeed, non-TCR-activated CD4+ but not CD8+ T cells express significant levels of CD154. IL-15 may enhance IFN-gamma in this system by up-regulating CD40 expression on monocytes and IL-12Rbeta1 expression on CD4+ T cells. Conversely, using neutralizing anti-IL-15 mAb, we show that the ability of IL-12 to augment IFN-gamma secretion is partly mediated by endogenous IL-15. Finally, in the absence of monocytes, a synergistic effect between exogenous IL-12 and IL-15 is necessary to induce IFN-gamma production by purified CD4+ T cells, while IL-15 alone induces T cell proliferation. It is proposed that this codependence between IL-12 and IL-15 for the activation of inflammatory T cells may be involved in chronic inflammatory disorders that are dominated by a Th1 response. In such a response, a self-perpetuating cycle of inflammation is set forth, because IL-15-stimulated CD4+ T cells may activate monocytes to release IL-12 that synergizes with IL-15 to induce IL-12 response and IFN-gamma production.

Staphylococcal enterotoxin B induces an early and transient state of immunosuppression characterized by V beta-unrestricted T cell unresponsiveness and defective antigen-presenting cell functions.

Staphylococcal enterotoxin B (SEB) is a bacterial enterotoxin able to simultaneously bind to class II molecules on APCs and to selected V beta regions (including V beta 8) of the TCR complex. Administration of SEB to adult BALB/c mice results in clonal activation of T cells bearing V beta 8 receptors, leading to an excessive release of proinflammatory cytokines. This initial immune response is followed by a long-lasting state of V beta 8-specific unresponsiveness, thought to benefit both the host (as it contributes to the down-regulation of the inflammatory response) and the bacterium (through ligand-specific T cell anergy). However, it is not clear how this type of restricted unresponsiveness can effectively impair the generation of an antibacterial response. To gain insight into the mechanism by which Gram-positive bacteria subvert the host immune response, we have investigated the immune competence of SEB-treated mice 48 h following SEB administration. We demonstrate in this report that in vivo, SEB induces a transient but profound state of unresponsiveness affecting both T and Ag-presenting cell functions. Although in vivo activation by SEB appears to be V beta-restricted under our experimental conditions, SEB-treated mice displayed an early (lasting 48 to 72 h postinjection) and V beta-unrestricted unresponsive state characterized by the inability to produce IL-2 in response to polyclonal TCR mitogens including third party bacterial superantigens (staphylococcal enterotoxin A and toxic shock syndrome toxin 1, SEA and TSST-1, respectively), Abs to non-SEB reactive V beta regions (V beta 6), anti-CD3 epsilon Abs, and a lectin (Con A). Spleen cell populations from SEB-treated mice also displayed defective APC functions, possibly related to a selective decrease in splenic dendritic cells numbers. Taken together, these observations indicate that SEB induces an early and transient state of immunodeficiency in vivo, representing a potential mechanism for escaping host immune surveillance.

Potent suppression of IL-12 production from monocytes and dendritic cells during endotoxin tolerance.

Endotoxin tolerance, the down-regulation of a subset of endotoxin-driven responses after an initial exposure to endotoxin, may provide protection from the uncontrolled immunological activation of acute endotoxic shock. Recent data suggest, however, that the inhibition of monocyte/macrophage function associated with endotoxin tolerance can lead to an inability to respond appropriately to secondary infections in survivors of endotoxic shock. IL-12 production by antigen-presenting cells is central to the orchestration of both innate and acquired cell-mediated immune responses to many pathogens. IL-12 has also been shown to play an important role in pathological responses to endotoxin. We therefore examined the regulation of IL-12 during endotoxin tolerance. Priming doses of lipopolysaccharide ablate the IL-12 productive capacity of primary human monocytes. This suppression of IL-12 production is primarily transcriptional. Unlike the down-regulation of TNF-alpha under such conditions, the mechanism of IL-12 suppression during endotoxin tolerance is not dependent upon IL-10 or transforming growth factor-beta, nor is IL-12 production rescued by IFN-gamma or granulocyte-macrophage colony-stimulating factor. Of note, human dendritic cells also undergo endotoxin tolerance, with potent down-regulation of IL-12 production. Endotoxin tolerance-related suppression of IL-12 production provides a likely mechanism for the anergy seen during the immunological paralysis which follows septic shock.