Sabine Ring

Presenilin-Dependent Transcriptional Control of the Aβ-Degrading Enzyme Neprilysin by Intracellular Domains of βAPP and APLP

Amyloid beta-peptide (Abeta), which plays a central role in Alzheimers disease, is generated by presenilin-dependent gamma-secretase cleavage of beta-amyloid precursor protein (betaAPP). We report that the presenilins (PS1 and PS2) also regulate Abeta degradation. Presenilin-deficient cells fail to degrade Abeta and have drastic reductions in the transcription, expression, and activity of neprilysin, a key Abeta-degrading enzyme. Neprilysin activity and expression are also lowered by gamma-secretase inhibitors and by PS1/PS2 deficiency in mouse brain. Neprilysin activity is restored by transient expression of PS1 or PS2 and by expression of the amyloid intracellular domain (AICD), which is cogenerated with Abeta, during gamma-secretase cleavage of betaAPP. Neprilysin gene promoters are transactivated by AICDs from APP-like proteins (APP, APLP1, and APLP2), but not by Abeta or by the gamma-secretase cleavage products of Notch, N- or E- cadherins. The presenilin-dependent regulation of neprilysin, mediated by AICDs, provides a physiological means to modulate Abeta levels with varying levels of gamma-secretase activity.

Immunosuppressive Therapy in Whipple's Disease Patients is Associated with the Appearance of Gastrointestinal Manifestations

OBJECTIVES:Whipples disease is a rare chronic disorder, which is caused by systemic infection with Tropheryma whipplei. The first symptom of Whipples disease usually is a nondestructive polyarthritis resembling in many aspects seronegative rheumatoid arthritis. This polyarticular inflammatory arthropathy preceding the diagnosis of Whipples disease for several years frequently is treated with nonsteroidal antiinflammatory drugs (NSAIDs) and with immunosuppressive therapy. There is evidence that altered immune functions play a role in the manifestation of the disease and that Whipples disease is associated with opportunistic infections. We therefore asked whether immunosuppressive treatment for arthropathy may alter the course of Whipples disease.PATIENTS AND METHODS:In a series of 27 patients with Whipples disease clinical data were documented and the patients were followed for 3–4 yr. The patients were classified into three groups according to their medication: (i) patients with immunosuppressive therapy preceding the diagnosis, (ii) patients with NSAIDs before diagnosis, and (iii) patients without such therapies.RESULTS:Arthropathies occurred in the mean 8 yr before diagnosis and were the first symptom in 63% of the patients. Gastrointestinal involvement usually became evident later on and frequently led to the diagnosis of Whipples disease. In patients with immunosuppressive treatment, diarrhea occurred in the median 4 months after the initiation of such therapy and diagnosis of Whipples disease was made after another 2 months. In contrast, other medical treatments were not closely followed by the onset of diarrhea.CONCLUSIONS:These results indicate an association between immunosuppressive therapy and the onset of diarrhea in Whipples disease and thus support the concept that immunologic factors play a role in disease pathogenesis. Further investigation on the interaction of the immune system and Tropheryma whipplei infection are required to understand the factors contributing to the clinical manifestation of this rare disorder and possibly to introduce preventive interventions.

CD4+CD25+ regulatory T cells suppress contact hypersensitivity reactions by blocking influx of effector T cells into inflamed tissue

CD4+CD25+ regulatory T cells (Treg) exert suppressive functions on effector T cells in vitro and in vivo. However, the exact cellular events that mediate this inhibitory action remain largely unclear. To elucidate these events, we used intravital microscopy in a model of contact hypersensitivity (CHS) and visualized the leukocyte‐endothelium interaction at the site of antigen challenge in awake C57BL/6 mice. Injection of Treg i.v. into sensitized mice at the time of local hapten challenge significantly inhibited rolling and adhesion of endogenous leukocytes to the endothelium. A similar inhibition of leukocyte recruitment could be recorded after injection of Treg‐derived tissue culture supernatant. Thus, these data indicate that soluble factors may account for the suppressive effects. Accordingly we found that IL‐10, but not TGF‐β, was produced by Treg upon stimulation and that addition of anti‐IL‐10 antibodies abrogated the suppressive effects of Treg and tissue culture supernatant in CHS reactions. Moreover, CD4+CD25+ T cells isolated from IL‐10–/– mice were not able to suppress the immune response induced by hapten treatment in C57BL/6 mice. In conclusion, our data suggest that cytokine‐dependent rather than cell‐cell contact‐dependent mechanisms play a pivotal role in the suppression of CHS reactions by Treg in vivo.

Engagement of αIIbβ3 (GPIIb/IIIa) with ανβ3 Integrin Mediates Interaction of Melanoma Cells with Platelets A CONNECTION TO HEMATOGENOUS METASTASIS

A mutual relationship exists between metastasizing tumor cells and components of the coagulation cascade. The exact mechanisms as to how platelets influence blood-borne metastasis, however, remain poorly understood. Here, we used murine B16 melanoma cells to observe functional aspects of how platelets contribute to the process of hematogenous metastasis. We found that platelets interfere with a distinct step of the metastasis cascade, as they promote adhesion of melanoma cells to the endothelium in vitro under shear conditions. Constitutively active platelet receptor GPIIb/IIIa (integrin αIIbβ3) expressed on Chinese hamster ovary cells promoted melanoma cell adhesion in the presence of fibrinogen, whereas blocking antibodies to aνβ3 integrin on melanoma cells or to GPIIb/IIIa significantly reduced melanoma cell adhesion to platelets. Furthermore, using intravital microscopy, we observed functional platelet-melanoma cell interactions, as platelet depletion resulted in significantly reduced melanoma cell adhesion to the injured vascular wall in vivo. Using a mouse model of hematogenous metastasis to the lung, we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a mAb blocking the aν subunit of aνβ3 integrin. This effect was significantly reduced when platelets were depleted in vivo. Thus, the engagement of GPIIb/IIIa with aνβ3 integrin interaction mediates tumor cell-platelet interactions and highlights how this interaction is involved in hematogenous tumor metastasis.

Regulatory T Cells Stimulate B7-H1 Expression in Myeloid-Derived Suppressor Cells in ret Melanomas

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells, and they promote an immunosuppressive environment in tumor-bearing hosts. To characterize MDSCs in melanoma, we examined the expression of inhibitory B7 molecules by CD11b(+)Gr1(+) cells isolated from mice with transplantable ret tumors. B7 molecules were expressed on CD11b(+)Gr1(+) cells, which also expressed CD124 and inducible nitric oxide synthase, thus verifying their relation to MDSCs. In developing melanomas, CD11b(+)Gr1(+) cells express only low levels of B7-H1. In contrast, B7-H1 is upregulated in large tumors, and functional analysis demonstrates that CD11b(+)Gr-1(+) cells suppress the proliferation of CD4(+) T cells through B7-H1. Depletion of regulatory T cells (Tregs) significantly downregulated the expression of B7-H1, B7-H3, and B7-H4 on MDSCs and reduced tumor growth, indicating a concerted immunosuppressive activity of Tregs and MDSCs. No differences in the suppressive function of MDSCs between CD25-depleted and non-depleted mice were recorded. Instead, tumor-derived MDSCs from Treg-depleted hosts produced less IL-10 and more IFN-γ as compared with Treg-harboring mice. These studies indicate that Tregs in tumors not only suppress effector T cells directly, but also modify the phenotype of tumor-infiltrating CD11b(+) cells to express inhibitory B7-H molecules and to produce IL-10.

Skin melanoma development in ret transgenic mice despite the depletion of CD25+Foxp3+ regulatory T cells in lymphoid organs.

CD4+CD25+Foxp3+ regulatory T cells (Treg) known to mediate self-tolerance were also shown to contribute to tumor progression. In mouse melanoma transplantation models, Treg depletion resulted in the stimulation of antitumor immune responses and tumor eradication. To study Treg in conditions close to the clinical situation, we used a ret transgenic mouse spontaneous melanoma model, which, in contrast to transplantation models, resembles human melanoma regarding clinical development. Significantly higher numbers of Treg were found in skin tumors and metastatic lymph nodes at early stages of melanoma progression compared with more advanced stages accompanied by the elevated CCR4 expression on Treg and higher production of its ligand CCL2 in tumor lesions. Numbers of tumor infiltrating Treg inversely correlated with Treg amounts in the bone marrow, suggesting their possible recruitment to melanoma lesions from this organ. The immunosuppressive function of Treg from transgenic tumor-bearing mice was similar to that from transgenic tumor-free mice or nontransgenic littermates. Although anti-CD25 mAb injections resulted in the efficient Treg depletion from lymphoid organs of transgenic mice, melanoma development was not significantly delayed. Furthermore, the treatment of mice with macroscopical tumors also failed to inhibit tumor progression, which correlated with the inability to deplete intratumoral Treg. We suggest that in the autochthonous melanoma genesis, other immunosuppressive cells could play an important role and replace immunosuppressive, tumor-promoting functions of Treg. Therefore, effective melanoma immunotherapy should include the inhibition of Treg migration into the tumor combined with neutralization of other immunosuppressive cells and factors in the tumor microenvironment.

Inhibition of Melanoma Growth by Targeting of Antigen to Dendritic Cells via an Anti-DEC-205 Single-Chain Fragment Variable Molecule

Purpose: Our goal was to target melanoma antigens to the dendritic cell-specific receptor DEC-205. DEC-205 is an antigen receptor expressed on dendritic cells and has been shown to guide antigens to MHC class I and II compartments for processing and presentation to T cells. Experimental Design: The melanoma tumor-associated antigen (TAA), gp100, was fused to the single-chain fragment variable (scFv) specific for DEC-205. The binding capacity of the scFv was tested on lymph node-isolated CD11c+ cells. Mixed lymphocyte reactions were carried out to show an increased proliferative capacity of gp100 antigen-specific CD4 and CD8 T cells. Furthermore the scFv-TAA was used in a therapeutic setting using two different melanoma mouse models. Results: C57Bl/6 mice were injected with scFv-DEC-205-gp100, monoclonal antibody anti-DEC-205, or PBS. Using fluorescence-activated cell sorting, we showed that lymph node CD11c+ dendritic cells stained positive for the binding of the scFv-mDEC-205-gp100 and the anti-DEC-205 monoclonal antibody, whereas the PBS-injected animals were negative. In mixed lymphocyte reactions, bone marrow-derived dendritic cells pulsed with scFv-mDEC-205-gp100 significantly increased proliferation of gp100-specific CD8+ and CD4+ T cells beyond gp100 peptide-pulsed or nonpulsed bone marrow-derived dendritic cells. Finally, in B16/F10 and RET models, a concentration-dependent suppression of tumor growth using scFv-mDEC-205-gp100 (66% reduction of tumor volume), in comparison with gp100 peptide vaccination, was observed. Conclusions: Our results indicate that the scFv-mDEC-205-gp100 targets TAA to dendritic cells in vivo for presentation on both MHC class I and II molecules. In vivo, this leads to an improved immune response and a decrease in tumor growth rate.

Antigen‐induced mucosal T cell activation is followed by Th1 T cell suppression in continuously fed ovalbumin TCR‐transgenic mice

We investigated kinetics and dose‐dependent features of mucosal and peripheral immune responses following oral antigen application in a TCR‐transgenic mouse model. Ovalbumin (OVA) TCR‐transgenic mice were fed OVA at different doses (5–250 mg) and various frequencies (one to seven times, or continuous feeding). Low‐ and medium‐dose (10, 100 mg) OVA feeding resulted in priming of immune responses, i.e. increased antigen‐specific proliferation as well as IL‐2, IL‐4 and IFN‐γ secretion upon in vitro restimulation in Peyers patches and spleen. Immune responses were suppressed with doses of one or three times 250 mg OVA feeding in the spleen. However, only the highest OVA feeding doses (7×250 mg OVA) or continuous feeding (5 mg daily in the drinking water over a 12‐week period) actively suppressed immune responses and were associated with production of TGF‐β and IL‐10 in the spleen and Peyers patches. Thus, the cell population generated by continuous antigen feeding was characterized by production of suppressive cytokines and seems to be based on a counter‐regulation with Th1 cytokines. These data further define the regulation of suppressive immune functions following antigen feeding in the periphery and the mucosal immune system.

Targeting of Autoantigens to DEC205+ Dendritic Cells In Vivo Suppresses Experimental Allergic Encephalomyelitis in Mice

The dendritic and epithelial cell receptor with a m.w. of 205 kDa (DEC205) is expressed by dendritic cells (DCs) and facilitates Ag presentation. After injection of Ags coupled to Abs specific for DEC205 into mice, Ag presentation occurs by nonactivated DCs, which leads to induction of regulatory T cells (Tregs). To test this system for tolerance induction in experimental allergic encephalomyelitis (EAE), we created single-chain fragment variables (scFv) specific for DEC205 and fused the scFv to the self-Ag myelin oligodendrocyte glycoprotein (MOG; scFv DEC:MOG). An anti–β-galactosidase scFv:MOG fusion protein (scFv GL117:MOG) served as isotype control. After staining of DCs in vitro with purified scFv DEC:MOG, binding to DCs and colocalization with MHC class II was apparent, whereas isotype controls did not bind. We next injected scFv DEC:MOG into mice and observed elevated numbers of highly activated, IL-10–producing CD4+CD25+Foxp3+ Tregs (17% of CD4) in spleens, as compared with isotype controls and uninjected mice (12% of CD4). Furthermore, DCs isolated from scFv DEC:MOG-injected animals produced significantly increased levels of TGF-β. Most importantly, when EAE was induced in scFv DEC:MOG-injected mice, 90% of the mice were protected from EAE, whereas all mice in the isotype controls (scFv GL117:MOG) experienced development of EAE. When applying scFv DEC:MOG to mice that had already experienced EAE symptoms, abrogation of the disease in 90% of the animals was apparent, whereas all animals in the control groups experienced development of severe EAE. Thus, these data indicate that targeting of MOG to “steady-state” DCs in vivo may provide a tool to prevent and to treat EAE by a DC/Treg-driven mechanism.

ERK/p38 MAP-kinases and PI3K are involved in the differential regulation of B7-H1 expression in DC subsets

Regulatory molecules of the B7‐H‐family expressed by DC are important for immune homeostasis, but their regulation is largely unknown. When investigating the pathways regulating B7‐H1 expression in monocyte‐derived DC (MoDC), freshly isolated myeloid DC (mDC) and plasmacytoid DC, respectively, we showed that in MoDC and mDC B7‐H1 expression was upregulated by a standard cytokine cocktail, poly I:C or LPS. MoDC utilize ERK and PI3K pathways to upregulate B7‐H1 in response to cytokines, whereas p38 kinase was predominantly utilized in response to poly I:C. In mDC, ERK and p38 pathways are involved in B7‐H1 regulation, and similar to MoDC, mainly p38 signaling was required for poly I:C‐induced expression of B7‐H1. Plasmacytoid DC responded only to CpG with upregulation of B7‐H1 and in addition to p38 also utilized the PI3K and ERK pathways to regulate B7‐H1 expression. As a functional consequence of B7‐H1 expression on DC, we demonstrate downmodulation of IFN‐γ production in T cells. Thus, the differential regulation of B7‐H1 on DC subsets may suppress immune responses variably, depending on the target DC population. Further analysis of the regulatory mechanisms may facilitate the development of new immunosuppressive therapies, utilizing the regulation of B7‐H1 expression on DC.