Interleukin-10 induces macrophage apoptosis and expression of CD16 (FcgammaRIII) whose engagement blocks the cell death programme and facilitates differentiation.

The development of monocytes into macrophages is regulated by helper T cells (Th) cells and, vice versa, the differentiation of Th cells into Th1 and Th2 is regulated by macrophages. Herein we examined the role of the Th2 cytokine, interleukin-10 (IL-10), on the development of macrophages. IL-10 is known to block the expression of antigen-presenting major histocompatibility complex (MHC) II and of costimulatory B7 molecules but it induces the expression of FcRs, especially the FcgammaRIII (CD16). The expression of CD16 enables the macrophage to carry out antibody-dependent cell-mediated cytotoxicity (ADCC) functions. However, this differentiation step is largely undercut by the capacity of IL-10 to induce macrophage apoptosis before the process of differentiation ensues. We found that the negative effect of IL-10 on the survival of macrophages is reversed in an environment that contains immunoglobulin G (IgG). IgG, especially when immune complexed with antigen, stimulates CD16 to transmit survival signals in macrophages which enable them to complete the differentiation process into CD16+ cells. Thus, IL-10 suppresses macrophage accumulation in healthy tissues where IgG is absent, and facilitates macrophage accumulation and differentiation in tissues that contain IgG, for example inflamed tissues or tissues that present autoreactive antibodies.

Singular anti-RNA virus-directed proteins.

AIMS: To additionally purify and characterise the anti-RNA virus-directed protein termed p14. MATERIALS AND METHODS: Antiviral assays of p14 against RNA and DNA viruses were carried out and its antigenic similarities with chicken interferon (CIFN) were studied. HPLC-Reverse Phase of p14 was performed to further purify p14. RESULTS: p14 showed antiviral activity against RNA viruses only and not against DNA viruses. It was antigenically distinct from CIFN. Purification of p14 yielded three proteins with antiviral activity, which had different physico-chemical properties than those described for interferons. CONCLUSIONS: The data presented on the antiviral, immunological and physico-chemical properties, establish the unique nature of p14 vis-á-vis those of interferons.

Polyspecific self-reactive antibodies in individuals infected with human immunodeficiency virus facilitate T cell deletion and inhibit costimulatory accessory cell function.

Self-reactive polyspecific IgG antibodies (PSAs) arise in human immunodeficiency virus (HIV)-seropositive subjects before they develop AIDS. Self-reactive PSA levels correlate with the destruction of CD8 T cells in HIV-infected individuals and mediate the antibody-dependent cellular toxicity-based destruction of human T cells in tissue culture. PSAs react across the species barrier and bind to T cell antigens in mice. Such reactivity with mouse lymphocytes was not detected in normal human serum. Injection of human PSA IgG causes massive T cell depletion in the spleen, lymph nodes, and thymus in mice: evidence that PSA IgG facilitates T cell destruction in vivo. In addition to facilitating macrophage cytotoxicity, self-reactive PSA IgG inhibits the macrophage-mediated activation of T cells with antigen receptor-specific monoclonal antibody or with antigen. Exogenous costimulatory stimuli or interleukin (IL)-12 can reverse the inhibition. In contrast, exogenous IL-10 mimics this inhibition. These data implicate PSA IgG as a pathogenic factor in the development of HIV disease.

Regulation of IL-6 signaling by p53: STAT3- and STAT5-masking in p53-Val135-containing human hepatoma Hep3B cell lines.

The influence of p53 on cytokine-triggered Janus kinase-STAT signaling was investigated in human hepatoma Hep3B cell lines engineered to constitutively express the temperature-sensitive Val135 mutant of p53. In comparison to the parental p53-free Hep3B cells, these p53-Val135-containing Hep3B cell lines displayed a reduced response to IL-6 at the wild-type-like p53 temperature (32.5 degrees C). In these cells, IL-6 induced a marked reduction in the immunologic accessibility of cytoplasmic and nuclear STAT3 and STAT5 within 20 to 30 min that lasted 2 to 4 h (STAT-masking) provided that the cells had been previously cultured at 32.5 degrees C for at least 18 to 20 h. The onset of IL-6-induced STAT-masking required protein tyrosine kinase, protein tyrosine phosphatase, proteasomal, phospholipase C, and mitogen-activated protein kinase kinase 1 activities. The maintenance of IL-6-induced STAT-masking was dependent on continued signaling through the phosphatidylinositol-dependent phospholipase C pathway. Despite a reduction in IL-6-induced STAT3 DNA binding activity in the nuclear compartment during STAT-masking, there was increased and prolonged accumulation of tyrosine-phosphorylated STAT3 in both the cytoplasmic and nuclear compartments, indicating that the capacity of tyrosine-phosphorylated STAT3 to bind DNA was reduced during STAT-masking. Thus, IL-6-induced STAT-masking, as dramatically evident on immunomicroscopy, is a visible consequence of a novel cellular process by which a p53-Val135-induced gene product(s) regulates the association of masking protein(s) with and the DNA-binding capacity of STAT3.

Distinct classes of chaperoned IL-6 in human blood: differential immunological and biological availability.

Transport of IL-6 in blood is fundamental to the biology of this cytokine. In the present study, IL-6 transport, immunological reactivity, and biological availability were investigated in blood from melanoma patients subjected to different active specific immunization regimens (an anti-idiotypic mAb immunization protocol (mAb-keyhole limpet hemocyanin (KLH)-Calmette-Guerin bacillus (BCG), an autologous anti-cancer vaccine protocol (AAAP), or both). Sera were subjected to Sephadex G-200 gel filtration chromatography, and the structure and biological activity of IL-6 complexes in the eluate fractions were probed using five IL-6 ELISAs and two bioassays. Sera from patients administered mAb-KLH+BCG followed by AAAP contained three distinct classes of IL-6 eluting at 30, 200, and 450 kDa, each with its characteristic ELISA reactivity and bioactivity: the 30- and 450-kDa complexes were bioactive in the B9 and Hep3B assays, but the 200-kDa complex was not. The 30- and 450-kDa IL-6 complexes were preferentially reactive in the 7IL6/5IL6 ELISA, the 200-kDa IL-6 complexes were preferentially reactive in the 4IL6/5IL6 ELISA, while the three commercial ELISAs (R&D, Endogen, and Genzyme) detected essentially only the 30-kDa IL-6. In contrast, 1) sera from AAAP patients contained biologically active 30- and 450-kDa IL-6 complexes, while 2) sera from mAb-KLH+BCG patients contained 200-kDa IL-6 complexes inactive in ex vivo bioassays. Both the 450- and 200-kDa complexes included soluble IL-6R, with the 200-kDa complexes additionally containing ligand-occupied anti-IL-6 and anti-soluble IL-6R IgG. The data indicate the existence of specific mechanisms that regulate the transport and function of IL-6 in vivo.

Proteasome- and p53-dependent masking of signal transducer and activator of transcription (STAT) factors.

Hepatoma Hep3B cell lines stably expressing a temperature-sensitive p53 species (p53-Val-135) displayed a reduced response to interleukin-6 (IL-6) when cultured at the wild-type (wt) p53 temperature (Wang, L., Rayanade, R., Garcia, D., Patel, K., Pan, H., and Sehgal, P. B. (1995) J. Biol. Chem. 270, 23159-23165). We now report that in such cultures IL-6 caused a rapid (20-30 min) and marked loss of cellular immunostaining for STAT3 and STAT5, but not for STAT1. The loss of STAT3 and STAT5 immunostaining was transient (lasted 120 min) and tyrosine kinase-dependent, and even though the loss was blocked by the proteasome inhibitors MG132 and lactacystin it was not accompanied by changes in cellular levels of STAT3 and STAT5 proteins suggesting that IL-6 triggered a rapid masking but not degradation of these transcription factors. STAT3 and STAT5 masking was accompanied by a reduction in IL-6-induced nuclear DNA-binding activity. The data suggest that p53 may influence Jak-STAT signaling through a novel indirect mechanism involving a wt p53-dependent gene product which upon cytokine addition is activated into a "STAT-masking factor" in a proteasome-dependent step.

Modulation of interleukin-6-induced plasma protein secretion in hepatoma cells by p53 species.

The ability of p53 species (wild-type and mutant) to modulate the "differentiated" response of human hepatoma cell lines Hep3B and HepG2 to interleukin-6 (IL-6) was investigated. Transient transfection experiments were carried out in Hep3B and HepG2 cell cultures in which IL-6 was used to activate a beta-fibrinogen (beta Fib) enhancer/reporter construct containing two copies of the 36-base pair IL-6-response element (IL-6RE) (p beta FibCAT). Cotransfection with constitutive expression vectors for wild-type (wt) human or murine p53 inhibited the activation of the p beta FibCAT reporter by IL-6 in both Hep3B and HepG2 cells. Several mutant p53 species either did not inhibit the activation of p beta FibCAT or up-regulated the response. Hepatoma cell lines stably expressing the Val-135 temperature-sensitive mutant of murine p53 (wt-like at 32.5 degrees C and mutant-like at 37 degrees C) were derived from Hep3B cells and tested for the temperature-sensitive phenotype of their ability to synthesize and secrete fibrinogen and alpha 1-antichymotrypsin in response to IL-6. In an experimental protocol in which the parental Hep3B cells did not show a significant difference in plasma protein secretion at the two temperatures, hepatoma line 3 (p53Val-135+) had a greater response to IL-6 at 37 degrees C than parental Hep3B cells, while line 3 cells had a reduced response to IL-6 at 32.5 degrees C. Similarly, hepatoma lines 1 and 2 (both p53Val-135+) had reduced IL-6 responsiveness at 32.5 degrees C, whereas line 22 (transfected with pSVneo alone) and the parental Hep3B cells did not. These data indicate that mutations in p53 contained in tumor cells can modulate the "differentiated" response of these cells to cytokines.