Donna M. Paulnock

Regulation of innate and acquired immunity in African trypanosomiasis.

African trypanosomes are well known for their ability to avoid immune elimination by switching the immunodominant variant surface glycoprotein (VSG) coat during infection. However, antigenic variation is only one of several means by which trypanosomes manipulate the immune system of their hosts. In this article, the role of parasite factors such as GPI anchor residues of the shed VSG molecule and the release of CpG DNA, in addition to host factors such as IFN‐γ, in regulating key aspects of innate and acquired immunity during infection is examined. The biological relevance of these immunoregulatory events is discussed in the context of host and parasite survival.

CD40 Ligation Activates Murine Macrophages via an IFN-γ-Dependent Mechanism Resulting in Tumor Cell Destruction In Vitro

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-γ because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-γ mAb, as well as in IFN-γ-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-γ for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-γ. We confirmed this hypothesis by detecting intracytoplasmic IFN-γ in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-γ production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-γ and mediate tumor cell destruction.

Signaling pathways initiated in macrophages after engagement of type A scavenger receptors

Scavenger receptors are macrophage cell surface molecules associated with endocytic uptake of lipoproteins and binding of microbial ligands. Macrophage class A scavenger receptors (SR‐As) interact with ligands to induce cellular signaling leading to gene transcription and cytokine release. We used inhibitors of early and late signaling to block SR‐A‐mediated polyinosinic‐polycytidilic acid (poly I:C) and lipoteichoic acid (LTA) activation of RAW 264.7 macrophages. Effects of multiple inhibitors on tumor necrosis factor (TNF)‐α release were monitored to determine requirements for inflammatory cytokine production. Cycloheximide, monodansylcadaverine, and cytochalasin B all blocked TNF‐α release from macrophages stimulated with LTA or poly I:C, whereas monensin only nominally reduced TNF‐α production. Selected inhibitors of downstream signaling events reduced SR‐A‐dependent TNF‐α release by >95% after stimulation with either ligand, whereas others were ineffective. The PKC inhibitor H7 reduced LTA‐dependent secretion of TNF‐α by 94% but inhibited poly I:C‐dependent TNF‐α production only by 50%. Priming of RAW 264.7 cells with interferon‐γ potentiated the response to poly I:C but did not alter inhibitor effects. These results demonstrated that for both ligands tested here, early events of receptor internalization are requisite for cellular activation. The response pattern suggests that tyrosine phosphorylation and activation of the MAP kinase pathway are key components of SR‐A‐mediated signal transduction cascades.

Macrophage activation by T cells

In the past year, the application of biochemical and molecular approaches to the analysis of macrophage activation by T cells has provided new information concerning the regulation of gene expression during the activation process, and advanced our understanding of the multiple mechanisms that influence the development of macrophage function during an immune response.

Analysis of macrophage activation in African trypanosomiasis

African trypanosomes cause a fatal disease of man and animals that ischaracterized by extensive functional, histological, and pathologicalchanges in the lymphoid tissues of infected hosts, including anincrease in the numbers and activation state of macrophages. Macrophageactivation during infection is the result of exposure of these cells toparasite components and host‐derived IFN‐γ, produced in response toparasite antigens. The balance of these different activation signalsmay determine the outcome of infection. In the experiments describedhere, we assessed the ability of the variant surface glycoprotein (VSG)of the organism Trypanosoma brucei rhodesiense (T.b.rhodesiense) to activate macrophages directly. Our resultsdemonstrate that macrophages bind and are activated by the VSGmolecule. The resulting profile of activation differs from thatstimulated by IFN‐γ. These results suggest that the interaction ofhost macrophages with VSG released during parasite infection may be akey component of trypanosomiasis.

Glycosylinositolphosphate Soluble Variant Surface Glycoprotein Inhibits IFN-γ-Induced Nitric Oxide Production Via Reduction in STAT1 Phosphorylation in African Trypanosomiasis

Macrophages are centrally involved in the host immune response to infection with Trypanosoma brucei rhodesiense, a protozoan parasite responsible for human sleeping sickness in Africa. During trypanosome infections, the host is exposed to parasite-derived molecules that mediate macrophage activation, specifically GPI anchor substituents associated with the shed variant surface glycoprotein (VSG), plus the host-activating agent IFN-γ, which is derived from activated T cells and is essential for resistance to trypanosomes. In this study, we demonstrate that the level and timing of exposure of macrophages to IFN-γ vs GPI ultimately determine the macrophage response at the level of induced gene expression. Treatment of macrophages with IFN-γ followed by GIP-sVSG (the soluble form of VSG containing the glycosylinositolphosphate substituent that is released by parasites) stimulated the induction of gene expression, including transcription of TNF-α, IL-6, GM-CSF, and IL-12p40. In contrast, treatment of macrophages with GIP-sVSG before IFN-γ stimulation resulted in a marked reduction of IFN-γ-induced responses, including transcription of inducible NO synthase and secretion of NO. Additional experiments revealed that the inhibitory activity of GIP-sVSG was associated with reduction in the level of STAT1 phosphorylation, an event required for IFN-γ-induced macrophage activation. These results suggest that modulation of specific aspects of the IFN-γ response may be one mechanism by which trypanosomes overcome host resistance during African trypanosomiasis.

Analysis of interferon-gamma-dependent and -independent pathways of macrophage activation.

Macrophages are a cellular cornerstone of the innate immune response. The outcome of macrophage activity during development of an immune response to microbes results from macrophage activation by both organism‐derived and host‐derived factors. In order to more fully understand the spectrum of responses expressed by macrophages when encountering these distinct stimuli, we investigated the similarities and differences between interferon‐γ receptor (IFN‐γR)dependent macrophage activation and stimulation of macrophages through the Type A1 scavenger receptor (SR). We observed distinct patterns of macrophage activation depending on the nature of the ligand. IFN‐γ and the SR ligand lipotechoic acid (LTA) induced largely non‐overlapping sets of genes. The use of two additional SR ligands, maleylated bovine serum albumin and the polydeoxynucleo‐tide poly dI:dC, revealed differences within SR activation‐induced gene expression. We also observed that priming with IFN‐γ resulted in an enhanced response to subsequent SR‐mediated activation. These results suggest that full potentiation of macrophage activity during development of an antimicrobial immune response is achieved by activation of these cells through multiple receptors. J. Leukoc. Biol. 67: 677–682; 2000.

In vivo CD40 ligation can induce T cell‐independent antitumor effects that involve macrophages

We have previously demonstrated T cell‐independent antitumor and antimetastatic effects of CD40 ligation that involved natural killer (NK) cells. As CD40 molecules are expressed on the surface of macrophages (Mφ), we hypothesized that Mφ may also serve as antitumor effector cells when activated by CD40 ligation. Progression of subcutaneous NXS2 murine neuroblastomas was delayed significantly by agonistic CD40 monoclonal antibody (anti‐CD40 mAb) therapy in immunocompetent A/J mice, as well as in T and B cell‐deficient severe combined immunodeficiency (SCID) mice. Although NK cells can be activated by anti‐CD40 mAb, anti‐CD40 mAb treatment also induced a significant antitumor effect in SCID/beige mice in the absence of T and NK effector cells, even when noncytolytic NK cells and polymorphonuclear cells (PMN) were depleted. Furthermore, in vivo treatment with anti‐CD40 mAb resulted in enhanced expression of cytokines and cell surface activation markers, as well as Mφ‐mediated tumor inhibition in A/J mice, C57BL/6 mice, and SCID/beige mice, as measured in vitro. A role for Mφ was shown by reduction in the antitumor effect of anti‐CD40 mAb when Mφ functions were inhibited in vivo by silica. In addition, activation of peritoneal Mφ by anti‐CD40 mAb resulted in survival benefits in mice bearing intraperitoneal tumors. Taken together, our results show that anti‐CD40 mAb immunotherapy of mice can inhibit tumor growth in the absence of T cells, NK cells, and PMN through the involvement of activated Mφ.

Signal Transduction, Gene Transcription, and Cytokine Production Triggered in Macrophages by Exposure to Trypanosome DNA

ABSTRACT Activation of a type I cytokine response is important for early resistance to infection with Trypanosoma brucei rhodesiense, the extracellular protozoan parasite that causes African sleeping sickness. The work presented here demonstrates that trypanosome DNA activates macrophages to produce factors that may contribute to this response. Initial results demonstrated that T. brucei rhodesiense DNA was present in the plasma of C57BL/6 and C57BL/6-scid mice following infection. Subsequently, the effect of trypanosome DNA on macrophages was investigated; parasite DNA was found to be less stimulatory than Escherichia coli DNA but more stimulatory than murine DNA, as predicted by the CG dinucleotide content. Trypanosome DNA stimulated the induction of a signal transduction cascade associated with Toll-like receptor signaling in RAW 264.7 macrophage cells. The signaling cascade led to expression of mRNAs, including interleukin-12 (IL-12) p40, IL-6, IL-10, cyclooxygenase-2, and beta interferon. The treatment of RAW 264.7 cells and bone marrow-derived macrophages with trypanosome DNA induced the production of NO, prostaglandin E2, and the cytokines IL-6, IL-10, IL-12, and tumor necrosis factor alpha. In all cases, DNase I treatment of T. brucei rhodesisense DNA abolished the activation. These results suggest that T. brucei rhodesiense DNA serves as a ligand for innate immune cells and may play an important contributory role in early stimulation of the host immune response during trypanosomiasis.

The Soluble Variant Surface Glycoprotein of African Trypanosomes Is Recognized by a Macrophage Scavenger Receptor and Induces IκBα Degradation Independently of TRAF6-Mediated TLR Signaling

The GPI residues of soluble variant surface glycoprotein (sVSG) molecules released from the membrane of African trypanosomes during infection induce macrophage activation events. In this study, we demonstrate that the trypanosome sVSG molecule binds to the membrane of murine RAW 264.7 macrophages and activates the NF-κB cascade independently of a TLR-mediated interaction. The binding of fluorochrome-labeled sVSG molecules to macrophage membranes was saturable, was inhibited by the scavenger receptor-specific ligand maleylated BSA, and was followed by rapid intracellular uptake of the molecules and subsequent internalization to lysosomal compartments. Inhibition of cellular phagocytic and endocytic uptake processes by cytochalasin B and monodansylcadaverine, respectively, revealed that sVSG internalization was necessary for IκBα degradation and occurred by an actin-dependent, clathrin-independent process. Activation of RAW 264.7 cells by sVSG following treatment of the cells with the TRAF6 inhibitory peptide DIVK resulted in enhanced NF-κB signaling, suggesting both that TRAF6-dependent TLR activation of the pathway alone is not required for signaling and that TLR pathway components may negatively regulate expression of sVSG-induced signaling. These results demonstrate that stimulation of macrophages by sVSG involves a complex process of receptor-mediated binding and uptake steps, leading to both positive and negative signaling events that ultimately regulate cellular activation.