Murine macrophages (M phi) are activated either by interferon-gamma (IFN-gamma) or interferon-alpha/beta (IFN-alpha/beta) in combination with bacterial lipopolysaccharide (LPS) to induce synthesis of tumor necrosis factor alpha (TNF-alpha) and nitric oxide synthase (iNOS) mRNA synthesis for generation of tumor cytotoxic nitric oxide (NO). In the present study, the effect of exogenous IFN-gamma on the induction of endogenous mRNA synthesis and secretion of IFN-alpha/beta by murine M phi was investigated. Neutralizing antibodies to IFN-alpha/beta reversed TNF-alpha and NOS mRNA synthesis, as well as nitric oxide (NO)-mediated tumor cytotoxicity. Quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed that treatment of M phi with IFN-gamma induced increases in both IFN-alpha and IFN-beta mRNA synthesis by approximately 2-fold and 10-fold, respectively, which corresponded to a 2-fold increase in secretion of IFN-alpha/beta by ELISA. These data indicate that exogenous IFN-gamma induces endogenous synthesis and secretion of IFN-alpha/beta by M phi, which appears to act in concert with endogenously synthesized TNF-alpha for the autocrine induction of NOS mRNA synthesis.
We investigated the effects of bacterial lipopolysaccharide (LPS), immune complexes (IC), and C3b opsonized zymosan (AZ) alone and in combination with interferon-gamma (IFN-gamma) priming on macrophage synthesis and secretion of C1q. Our results indicated that LPS, IC, and AZ alone stimulated C1q mRNA and secretion in the absence of IFN-gamma. The increase in mRNA accumulation was detectable after 3 h, peaked at 6 h and was maintained at constitutive levels for 24 h. There was a corresponding early burst of increased secretion of functional C1q after 3 to 6 h which declined rapidly after 9 to 24 h culture of LPS-stimulated macrophages. Priming of macrophages with IFN-gamma and simultaneous triggering with LPS, IC, or AZ produced additive rather than synergistic increases in C1q mRNA accumulation. These same agents inhibited constitutive secretion of C1q in the absence of IFN-gamma priming as determined by autoradiographic analysis of metabolically radiolabeled secretory C1q. Triggering of IFN-gamma primed macrophages with LPS, IC, or AZ also markedly suppressed the increased rate of C1q secretion induced by IFN-gamma in a dose-related fashion. A corresponding dose-dependent increased accumulation of endogenous C1q in cell lysates was detected by Western blot analysis of macrophages which had been stimulated by LPS, IC, or AZ alone or in combination with IFN-gamma. Our findings indicate that LPS as well as FcR and C3bR triggering agents stimulate early and sustained C1q synthesis accompanied by an early and short-lived burst of C1q secretion which rapidly diminished and results in an increased intracellular accumulation of C1q due to ongoing synthesis. IFN-gamma appeared to further amplify the same kinetics of increased C1q mRNA accumulation and decreased extracellular accumulation mediated by LPS, IC, and ZM. Our results suggest that LPS, IC, and AZ alone or in combination with IFN-gamma stimulate early C1q production to modulate macrophage effector functions followed by an inhibition of C1q secretion when the activation process has been culminated.
Human natural killer (NK) cells and T lymphocytes can bind to and inhibit the growth of the yeast-like organism Cryptococcus neoformans. Binding of target cells to NK or T cells also has the potential to modulate cytokine production by the effector cells. In this study, we assessed the ability of C. neoformans to modulate NK cell production, or in some cases T-cell production, of granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor alpha (TNF-␣). We found that freshly isolated human NK cells from most individuals make GM-CSF and TNF-␣ constitutively when cultured in vitro. The addition of C. neoformans to T-cell fractions which do not make GM-CSF constitutively did not affect GM-CSF production, but the addition of C. neoformans to NK cell fractions significantly reduced the amounts of GM-CSF produced in most NK cell samples. The reduction in the amount of GM-CSF in C. neoformans-NK cell cocultures could not be attributed to loss of lymphocyte viability or to C. neoformans adsorbing or degrading the cytokine and was dependent on direct contact between the NK cells and cryptococcal cells. GM-CSF was not the only cytokine to be downregulated. TNF-␣ production was also diminished when NK cells were incubated with C. neoformans. The regulation of both cytokines was at the transcriptional level because GM-CSF and TNF-␣ mRNA levels were lower in NK cell samples incubated with C. neoformans than in NK cell samples incubated without C. neoformans. Diminished production of constitutively produced cytokines resulting from the interaction of NK cells with cryptococcal cells has the potential to affect phagocytic cells in the immediate regional environment and to damp the immune response. MATERIALS AND METHODSReagents. The reagents used in these studies were purchased from the following vendors: RPMI 1640 medium and pooled human serum (type AB) from Gibco Laboratories, Grand Island, N.Y.; fetal bovine serum (FBS) from Hy-Clone Laboratories, Inc., Logan, Utah; penicillin, streptomycin, Ficoll-Hypaque, Percoll, and phorbol myristate acetate (PMA) from Pharmacia, Uppsala, Sweden; nylon wool from Robbins Scientific Corp., Sunnyvale, CA.; fluorescein isothiocyanate-conjugated mouse anti-human CD3 monoclonal antibody (immunoglobulin G1 IgG1), phycoerythrin (PE)-conjugated mouse anti-human CD16 monoclonal antibody (IgG1), PE-conjugated mouse anti-human CD14 monoclonal antibody (IgG2), and fluorescein isothiocyanate-conjugated and PE-conju-* Corresponding author. Mailing address:
Complement subcomponent C1q has been recently implicated in the modulation of autocrine binding of TNF‐α to murine macrophages for induction of nitric oxide synthase. In the present study, the putative role of C1q in increasing TNF‐α binding to L929 cells to mediate cytotoxicity was explored. TNF‐sensitive L929 cells (L929‐S) had higher total endogenous cellular and surface C1q levels and bound correspondingly more phycoerythrin‐labelled rTNF‐α (PE‐TNF) than did a TNF‐resistant L929 variant (L929‐R). Pretreatment of L929‐S with soluble C1q increased their sensitivity to TNF‐mediated cytotoxicity coincident with increased binding of PE‐TNF, but similar treatment of L929‐R had no effect. Pretreatment of L929‐S with an inhibitor of C1q secretion, 3,4 dehydro‐D,L‐proline (DHP), resulted in a decrease in their TNF‐mediated cytotoxicity, as well as reduced binding of PE‐TNF. Subsequent exposure of DHP‐treated L929‐S with exogenous soluble C1q restored their TNF‐mediated cytotoxicity and binding of PE‐TNF. These results provide evidence for the modulation of TNF‐α binding to TNF sensitive tumour targets L929 by either endogenously synthesized or exogenously added C1q to promote TNF‐mediated cytotoxicity by mechanisms which remain to be elucidated.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.