T cell-specific overexpression of TGFß1 fails to influence atherosclerosis in ApoE-deficient mice.

Clinical data have indicated a negative correlation between plasma TGFß1 concentrations and the extent of atherosclerosis and have thus led to the hypothesis that the pleiotropic cytokine may have anti-atherogenic properties. T-cells are currently discussed to significantly participate in atherogenesis, but the precise role of adaptive immunity in atherogenesis remains to be elucidated. TGFß1 is known to strongly modulate the function of T-cells, however, inhibition of TGFß1 signalling in T-cells of atherosclerosis-prone knock-out mice failed to unequivocally clarify the role of the cytokine for the development of atherosclerosis. In the present study, we thus tried to specify the role of TGFß1 in atherogenesis by using the murine CD2-TGFß1 transgenic strain which represents a well characterized model of T-cell specific TGFß1 overexpression. The CD2-TGFß1 transgenic mice were crossed to ApoE knock-out mice and quantity and quality of atherosclerosis regarding number of macrophages, smooth muscle cells, CD3 positive T-cells and collagen was analyzed in CD2-TGFß1 ApoE double mutants as well as non-transgenic ApoE controls on both normal and atherogenic diet of a duration of 8, 16 or 24 weeks, respectively. In all experimental groups investigated, we failed to detect any influence of TGFß1 overexpression on disease. Total number of CD3-positive T-lymphocytes was not significantly different in atherosclerotic lesions of CD2-TGFß1 ApoE(-/-) females and isogenic ApoE(-/-) controls, even after 24 weeks on the atherogenic diet. The synopsis of these data and our previous study on TGFß1 overexpressing macrophages suggests that potential effects of TGFß1 on atherosclerosis are most probably mediated by macrophages rather than T-cells.

Overexpression of TGF-ß1 in macrophages reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.

Although macrophages represent the hallmark of both human and murine atherosclerotic lesions and have been shown to express TGF-ß1 (transforming growth factor ß1) and its receptors, it has so far not been experimentally addressed whether the pleiotropic cytokine TGF-ß1 may influence atherogenesis by a macrophage specific mechanism. We developed transgenic mice with macrophage specific TGF-ß1 overexpression, crossed the transgenics to the atherosclerotic ApoE (apolipoprotein E) knock-out strain and quantitatively analyzed both atherosclerotic lesion development and composition of the resulting double mutants. Compared with control ApoE(-/-) mice, animals with macrophage specific TGF-ß1 overexpression developed significantly less atherosclerosis after 24 weeks on the WTD (Western type diet) as indicated by aortic plaque area en face (p<0.05). Reduced atherosclerotic lesion development was associated with significantly less macrophages (p<0.05 after both 8 and 24 weeks on the WTD), significantly more smooth muscle cells (SMCs; p<0.01 after 24 weeks on the WTD), significantly more collagen (p<0.01 and p<0.05 after 16 and 24 weeks on the WTD, respectively) without significant differences of inner aortic arch intima thickness or the number of total macrophages in the mice pointing to a plaque stabilizing effect of macrophage-specific TGF-ß1 overexpression. Our data shows that macrophage specific TGF-ß1 overexpression reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.

Transgenic mice for a tamoxifen-induced, conditional expression of the Cre recombinase in osteoclasts.

BACKGROUND: Studies on osteoclasts, the bone resorbing cells, have remained limited due to the lack of transgenic mice allowing the conditional knockout of genes in osteoclasts at any time during development or adulthood. METHODOLOGY/PRINCIPAL FINDING: We report here on the generation of transgenic mice which specifically express a tamoxifen-inducible Cre recombinase in osteoclasts. These mice, generated on C57BL/6 and FVB background, express a fusion Cre recombinase-ERT2 protein whose expression is driven by the promoter of cathepsin K (CtsK), a gene highly expressed in osteoclasts. We tested the cellular specificity of Cre activity in CtsKCreERT2 strains by breeding with Rosa26LacZ reporter mice. PCR and histological analyses of the CtsKCreERT2LacZ positive adult mice and E17.5 embryos show that Cre activity is restricted largely to bone tissue. In vitro, primary osteoclasts derived from the bone marrow of CtsKCreERT2+/-LacZ+/- adult mice show a Cre-dependent ß-galactosidase activity after tamoxifen stimulation. CONCLUSIONS/SIGNIFICANCE: We have generated transgenic lines that enable the tamoxifen-induced, conditional deletion of loxP-flanked genes in osteoclasts, thus circumventing embryonic and postnatal gene lethality and avoiding gene deletion in other cell types. Such CtsKCreERT2 mice provide a convenient tool to study in vivo the different facets of osteoclast function in bone physiology during different developmental stages and adulthood of mice.

Aldosterone/NaCl-induced renal and cardiac fibrosis is modulated by TGF-ß responsiveness of T cells.

We examined the contribution of transforming growth factor (TGF)-ß-T-cell signaling to aldosterone (aldo)/salt-induced fibrosis in the kidneys and the hearts of FVB/N wild-type (WT) or transgenic (Tg) mice expressing a dominant-negative TGF-ß type II receptor in T cells (hCD2-ΔkTßRII). Animals received aldo through osmotic minipumps and had access to either 1% NaCl (aldo/NaCl group) or tap water (vehicle group) for 4 weeks. Systolic blood pressure was measured during this period via a tail cuff. The animals were then killed, and urine, blood, kidneys and hearts were collected. Systolic blood pressure did not differ between the groups. Aldo/NaCl enhanced renal, cardiac and left ventricular weight in WT animals slightly, but only renal weight was increased in Tg animals. Urinary protein excretion was enhanced in Tg animals (fourfold) and increased further in WT (twofold) and Tg (1.8-fold) mice on aldo/NaCl treatment. Aldo/NaCl increased interstitial fibrosis in the kidneys (1.5-fold) and the hearts of WT (2.5-fold) animals. Under control conditions, Tg mouse cardiac (3.2-fold) and renal (1.7-fold) tissues were slightly more fibrotic compared with WT, and this condition was not further aggravated by aldo/NaCl. Aldo/NaCl-induced mRNA expression of renal fibronectin (10.7-fold in WT) but not of renal collagen mRNA expression (WT: Col1a1 7.7-fold; Col3a1, 3.1-fold; and Col4a1 3.3-fold) was abrogated in Tg animals. In hearts, aldo/NaCl-induced plasminogen activator inhibitor-1 mRNA (twofold) expression depended on TGF-ß-T-cell signaling. Our results indicate that (i) aldo/NaCl can induce renal and cardiac damage in the absence of blood pressure changes, (ii) the elimination TGF-ß-T-cell cross-talk leads to renal and cardiac fibrosis but does not exacerbate aldo/NaCl-induced damage and (iii) the pathological aldo/NaCl effect is modified, in part, by TGF-ß-T-cell cross-talk.

The vinculin-DeltaIn20/21 mouse: characteristics of a constitutive, actin-binding deficient splice variant of vinculin.

BACKGROUND: The cytoskeletal adaptor protein vinculin plays a fundamental role in cell contact regulation and affects central aspects of cell motility, which are essential to both embryonal development and tissue homeostasis. Functional regulation of this evolutionarily conserved and ubiquitously expressed protein is dominated by a high-affinity, autoinhibitory head-to-tail interaction that spatially restricts ligand interactions to cell adhesion sites and, furthermore, limits the residency time of vinculin at these sites. To date, no mutants of the vinculin protein have been characterized in animal models. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigate vinculin-DeltaEx20, a splice variant of the protein lacking the 68 amino acids encoded by exon 20 of the vinculin gene VCL. Vinculin-DeltaEx20 was found to be expressed alongside with wild type protein in a knock-in mouse model with a deletion of introns 20 and 21 (VCL-DeltaIn20/21 allele) and shows defective head-to-tail interaction. Homozygous VCL-DeltaIn20/21 embryos die around embryonal day E12.5 showing cranial neural tube defects and exencephaly. In mouse embryonic fibroblasts and upon ectopic expression, vinculin-DeltaEx20 reveals characteristics of constitutive head binding activity. Interestingly, the impact of vinculin-DeltaEx20 on cell contact induction and stabilization, a hallmark of the vinculin head domain, is only moderate, thus allowing invasion and motility of cells in three-dimensional collagen matrices. Lacking both F-actin interaction sites of the tail, the vinculin-DeltaEx20 variant unveils vinculin's dynamic binding to cell adhesions independent of a cytoskeletal association, and thus differs from head-to-tail binding deficient mutants such as vinculin-T12, in which activated F-actin binding locks the protein variant to cell contact sites. CONCLUSIONS/SIGNIFICANCE: Vinculin-DeltaEx20 is an active variant supporting adhesion site stabilization without an enhanced mechanical coupling. Its presence in a transgenic animal reveals the potential of splice variants in the vinculin gene to alter vinculin function in vivo. Correct control of vinculin is necessary for embryonic development.

Activin a promotes the TGF-beta-induced conversion of CD4+CD25- T cells into Foxp3+ induced regulatory T cells.

TGF-beta induces the conversion of CD4(+)CD25(-) T cells into CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg). Activin A is a pleiotropic TGF-beta family member and is expressed in response to inflammatory signals. In this study, we report on the effects of activin A on the conversion of CD4(+)CD25(-) T cells into Foxp3-expressing induced Treg (iTreg). Activin A was able to promote the conversion of CD4(+)CD25(-) T cells into iTreg in a dose-dependent manner in vitro. Activin A together with TGF-beta1 had synergistic effects on the rate of iTreg conversion in vitro. Intact TGF-beta1 signaling seemed to be essential for the effects of activin A on iTreg generation because cells overexpressing a dominant negative TGF-beta type II receptor could not be converted by activin A in vitro. In vivo, the frequency of peripheral, but not central, Treg was increased in transgenic mice with elevated activin A serum levels and the in vivo conversion rate of CD4(+)CD25(-) T cells into Foxp3-expressing iTreg was increased as compared with wild type mice. These data suggest a role for activin A as a promoter of the TGF-beta dependent conversion of CD4(+)CD25(-) T cells into iTreg in vitro and in vivo. Therefore, besides promoting inflammation, activin A may contribute to the regulation of inflammation via the expansion of peripheral Treg.

IL-4/IL-13-dependent alternative activation of macrophages but not microglial cells is associated with uncontrolled cerebral cryptococcosis.

Both interleukin (IL)-4- and IL-13-dependent Th2-mediated immune mechanisms exacerbate murine Cryptococcus neoformans-induced bronchopulmonary disease. To study the roles of IL-4 and IL-13 in cerebral cryptococcosis, IL-4 receptor alpha-deficient (IL-4Ralpha(-/-)), IL-4-deficient (IL-4(-/-)), IL-13-deficient (IL-13(-/-)), IL-13 transgenic (IL-13(T/+)), and wild-type mice were infected intranasally. IL-13(T/+) mice displayed a higher fungal brain burden than wild-type mice, whereas the brain burdens of IL-4Ralpha(-/-), IL-4(-/-), and IL-13(-/-) mice were significantly lower as compared with wild-type mice. On infection, 68% of wild-type mice and 88% of IL-13-overexpressing IL-13(T/+) mice developed significant cerebral lesions. In contrast, only a few IL-4Ralpha(-/-), IL-4(-/-), and IL-13(-/-) mice had small lesions in their brains. Furthermore, IL-13(T/+) mice harbored large pseudocystic lesions in the central nervous system parenchyma, bordered by voluminous foamy alternatively activated macrophages (aaMphs) that contained intracellular cryptococci, without significant microglial activation. In wild-type mice, aaMphs tightly bordered pseudocystic lesions as well, and these mice, in addition, showed microglial cell activation. Interestingly, in resistant IL-4(-/-), IL-13(-/-), and IL-4Ralpha(-/-) mice, no aaMphs were discernible. Microglial cells of all mouse genotypes neither internalized cryptococci nor expressed markers of alternative activation, although they displayed similar IL-4Ralpha expression levels as macrophages. These data provide the first evidence of the development of aaMphs in a central nervous system infectious disease model, pointing to distinct roles of macrophages versus microglial cells in the central nervous system immune response against C. neoformans.

Coexpression of TGF-beta1 and IL-10 enables regulatory T cells to completely suppress airway hyperreactivity.

In allergic airway disease, Treg may play an important role in the modulation of airway hyperreactivity (AHR) and inflammation. We therefore investigated the therapeutic potential of Treg in an Ag-dependent murine asthma model. We here describe that AHR can be completely suppressed by adoptive transfer of Treg overexpressing active TGF-beta1. Using mice with impaired TGF-beta signaling in T cells, we could demonstrate that TGF-beta signaling in recipient effector T cells or transferred Treg themselves is not required for the protective effects on AHR. However, the expression of IL-10 by Treg was found to be essential for the suppression of AHR, since Treg overexpressing active TGF-beta1 but deficient in IL-10 lacked protective effects. Airway inflammation could not be significantly suppressed by wild-type or transgenic Treg. In conclusion, modulation of cytokine expression by Treg may have therapeutic potential for the treatment of AHR in asthma. The mechanisms of the effects of Treg on airway inflammation require further clarification.

P38 MAP kinase signaling is required for the conversion of CD4+CD25- T cells into iTreg.

CD4+CD25+ regulatory T cells (Treg) are important mediators of immune tolerance. A subset of Treg can be generated in the periphery by TGF-beta dependent conversion of conventional CD4+CD25- T cells into induced Treg (iTreg). In chronic viral infection or malignancy, such induced iTreg, which limit the depletion of aberrant or infected cells, may be of pathogenic relevance. To identify potential targets for therapeutic intervention, we investigated the TGF-beta signaling in Treg. In contrast to conventional CD4+ T cells, Treg exhibited marked activation of the p38 MAP kinase pathway. Inhibition of p38 MAP kinase activity prevented the TGF-beta-dependent conversion of CD4+CD25- T cells into Foxp3+ iTreg in vitro. Of note, the suppressive capacity of nTreg was not affected by inhibiting p38 MAP kinase. Our findings indicate that signaling via p38 MAP kinase seems to be important for the peripheral generation of iTreg; p38 MAP kinase could thus be a therapeutic target to enhance immunity to chronic viral infection or cancer.

A gene-dosage effect for interleukin-4 receptor alpha-chain expression has an impact on Th2-mediated allergic inflammation during bronchopulmonary mycosis.

Interleukin (IL)-4 and IL-13 are key factors in the pathogenesis of bronchopulmonary mycosis induced in mice by infection with Cryptococcus neoformans. Both cytokines use the IL-4 receptor alpha-chain (IL-4Ralpha). In this study, we investigated the role played by IL-4Ralpha expression in susceptibility to pulmonary C. neoformans infection. IL-4Ralpha(-/-) mice were extremely resistant. To characterize the effect of IL-4Ralpha expression level on disease outcome, we generated IL-4Ralpha(+/-) first-generation (F1) mice. IL-4Ralpha(+/-) mice showed intermediate levels of IL-4Ralpha expression, in contrast to higher levels in wild-type mice and no expression in IL-4Ralpha(-/-) mice, indicating biallelic expression of the gene for IL-4Ralpha (Il4ra). Concomitant with intermediate IL-4Ralpha expression, F1 mice showed intermediate susceptibility associated with altered Th2/Th17 cytokine production, decreased immunoglobulin E levels, and reduced allergic inflammation. This indicates a gene-dosage effect of IL-4Ralpha expression on susceptibility to bronchopulmonary mycosis. These data provide the basis for novel therapies antagonizing IL-4Ralpha in Th2-related pulmonary infection and possibly also in asthma.

Spontaneous hepatic fibrosis in transgenic mice overexpressing PDGF-A.

Platelet derived growth factor (PDGF) plays a central role in repair mechanisms after acute and chronic tissue damage. To further evaluate the role of PDGF-A in liver fibrogenesis in vivo, we generated transgenic mice with hepatocyte-specific overexpression of PDGF-A using the CRP-gene promoter. Transgenic but not wildtype mice showed expression of PDGF-A mRNA in the liver. Hepatic PDGF-A overexpression was accompanied by a significant increase in hepatic procollagen III mRNA expression as well as TGF-beta1 expression. Liver histology showed increased deposition of extracellular matrix in transgenic but not in wildtype mice. PDGF-A-transgenic mice showed positive sinusoidal staining for alpha-SMA indicating an activation of hepatic stellate cells. Since the profibrogenic effect of PDGF-A was accompanied by increased TGF-beta1 protein concentration in the liver of transgenic mice, it can be postulated that PDGF-A upregulates expression of TGF-beta1 which is a strong activator of hepatic stellate cells. Thus, these results point towards a fibrosis induction by PDGF-A via the TGF-beta1 signalling pathway. In conclusion, expression and functional analysis of PDGF-A in the liver of transgenic mice suggest a relevant profibrogenic role of PDGF-A via TGF-beta1 induction. Counteracting PDGF-A may therefore be one of the effects of tyrosine kinase inhibitors which showed protective effects in animal models of liver fibrosis.

IL-13 induces disease-promoting type 2 cytokines, alternatively activated macrophages and allergic inflammation during pulmonary infection of mice with Cryptococcus neoformans.

In the murine model of Cryptococcus neoformans infection Th1 (IL-12/IFN-gamma) and Th17 (IL-23/IL-17) responses are associated with protection, whereas an IL-4-dependent Th2 response exacerbates disease. To investigate the role of the Th2 cytokine IL-13 during pulmonary infection with C. neoformans, IL-13-overexpressing transgenic (IL-13Tg(+)), IL-13-deficient (IL-13(-/-)), and wild-type (WT) mice were infected intranasally. Susceptibility to C. neoformans infection was found when IL-13 was induced in WT mice or overproduced in IL-13Tg(+) mice. Infected IL-13Tg(+) mice had a reduced survival time and higher pulmonary fungal load as compared with WT mice. In contrast, infected IL-13(-/-) mice were resistant and 89% of these mice survived the entire period of the experiment. Ag-specific production of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with a significant type 2 cytokine shift but only minor changes in IFN-gamma production. Consistent with enhanced type 2 cytokine production, high levels of serum IgE and low ratios of serum IgG2a/IgG1 were detected in susceptible WT and IL-13Tg(+) mice. Interestingly, expression of IL-13 by susceptible WT and IL-13Tg(+) mice was associated with reduced IL-17 production. IL-13 was found to induce formation of alternatively activated macrophages expressing arginase-1, macrophage mannose receptor (CD206), and YM1. In addition, IL-13 production led to lung eosinophilia, goblet cell metaplasia and elevated mucus production, and enhanced airway hyperreactivity. This indicates that IL-13 contributes to fatal allergic inflammation during C. neoformans infection.

TGF-beta superfamily signaling is essential for tooth and hair morphogenesis and differentiation.

Members of the transforming growth factor beta (TGF-beta) superfamily of signaling molecules are involved in the regulation of many developmental processes that involve the interaction between mesenchymal and epithelial tissues. Smad7 is a potent inhibitor of many members of the TGF-beta family, notably TGF-beta and activin. In this study, we show that embryonic overexpression of Smad7 in stratified epithelia using a keratin 5 promoter, results in severe morphogenetic defects in skin and teeth and leads to embryonic and perinatal lethality. To further analyze the functions of Smad7 in epithelial tissues of adult mice, we used an expression system that allowed a controlled overexpression of Smad7 in terms of both space and time. Skin defects in adult mice overexpressing Smad7 were characterized by hyper-proliferation and missing expression of early markers of keratinocyte differentiation. Upon Smad7-mediated blockade of TGF-beta superfamily signaling, ameloblasts failed to produce an enamel layer in incisor teeth. In addition, TGF-beta blockade in adult mice altered the pattern of thymic T cell differentiation and the number of thymic T cells was significantly reduced. This study shows that TGF-beta superfamily signaling is essential for development of hair, tooth and T-cells as well as differentiation and proliferation control in adult tissues.

Granulocyte-macrophage colony-stimulating factor is essential for normal wound healing.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent growth factor, which plays an important role during the process of wound healing. In clinical settings it has occasionally been employed in the treatment of cutaneous wounds of diverse etiologies. In a previous study, we have shown the positive influence of GM-CSF on full thickness excisional wounds in transgenic mice overexpressing GM-CSF in the basal layer of the epidermis. Direct GM-CSF action as well as indirect processes through the induction of secondary cytokines were proposed to contribute towards the beneficial effects. In this study, we analyzed the process of wound healing in transgenic mice overexpressing a GM-CSF antagonist in the epidermis. These mice not only exhibited a delayed scab rejection and reepithelialization but also neovascularization was reduced. The newly formed tissue was of poor quality as exhibited by the presence of extensive fibrosis. We suggest that the presence of GM-CSF in the repair process is of basic importance and its absence leads not only to delayed wound healing but it is also detrimental for the quality of the newly formed tissue.

Cutting edge: TGF-beta signaling is required for the in vivo expansion and immunosuppressive capacity of regulatory CD4+CD25+ T cells.

Data regarding the role of TGF-beta for the in vivo function of regulatory CD4(+)CD25(+) T cells (Treg) are controversial. A transgenic mouse model with impaired TGF-beta signaling specifically in T cells was used to assess the role of endogenous TGF-beta for the in vivo function of CD4(+)CD25(+) Treg in a murine model of colitis induced by dextran sulfate. Transfer of wild-type, but not transgenic CD4(+)CD25(+) Treg was found to suppress colitis in wild-type mice. In addition, by transferring CFSE-labeled CD4(+)CD25(+) Treg we could demonstrate that endogenous TGF-beta promotes the expansion of CD4(+)CD25(+) Treg in vivo. Transgenic mice themselves developed reduced numbers of peripheral CD4(+)CD25(+) Treg and were more susceptible to the induction of colitis, which could be prevented by the transfer of wild-type Treg. These data indicate that TGF-beta signaling in CD4(+)CD25(+) Treg is required for their in vivo expansion and suppressive capacity.

TGF-beta suppresses tumor progression in colon cancer by inhibition of IL-6 trans-signaling.

Alterations of TGF-beta signaling have been described in colorectal cancer, although the molecular consequences are largely unknown. By using transgenic mice overexpressing TGF-beta or a dominant-negative TGF-betaRII, we demonstrate that TGF-beta signaling in tumor infiltrating T lymphocytes controls the growth of dysplastic epithelial cells in experimental colorectal cancer, as determined by histology and a novel system for high-resolution chromoendoscopy. At the molecular level, TGF-beta signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell-derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-beta-dependent IL-6 trans-signaling prevented tumor progression in vivo. Taken together, our data provide novel insights into TGF-beta signaling in colorectal cancer and suggest novel therapeutic approaches for colorectal cancer based on inhibition of TGF-beta-dependent IL-6 trans-signaling.

TGFbeta regulates the CD4+CD25+ T-cell pool and the expression of Foxp3 in vivo.

Factors influencing the development of CD4+CD25+ T-cells in vivo are poorly understood. In order to investigate the contribution of TGFbeta1 to the development and function of CD4+CD25+ T-cells, we generated a gain of function mutation resulting in the overexpression of an active form of TGFbeta1 in T-cells under control of the human CD2 promoter. In peripheral lymphoid organs and in the thymus, the frequency of CD4+CD25+ T-cells was increased in transgenic mice. This appeared to be due to an autocrine effect of TGFbeta on T-cells, since concomitant impairment of TGFbeta-signaling in double transgenic mice resulted in a phenotype similar to wild type. In contrast, in single transgenic mice with impaired TGFbeta-signaling in T-cells, CD4+CD25+ T-cell numbers were reduced in peripheral lymphoid organs but not in the thymus. In addition, TGFbeta was found to regulate the expression of Foxp3 in vivo, a transcription factor essential for the generation and function of regulatory T-cells. In CD4+CD25+ T-cells, TGFbeta1 increased the expression of Foxp3, whereas a decreased expression was seen in CD4+CD25+ T-cells with impaired TGFbeta-signaling. TGFbeta1 induced the expression of IL-10 in transgenic T-cells, but the increased in vitro suppressive capacity observed in transgenic CD4+CD25+ T-cells was due to the secretion of TGFbeta and not IL-10. Therefore, our study provides in vivo evidence for a role of TGFbeta in the homeostasis of CD4+CD25+ T-cells.

A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model.

Alzheimer disease (AD) is characterized by excessive deposition of amyloid beta-peptides (A beta peptides) in the brain. In the nonamyloidogenic pathway, the amyloid precursor protein (APP) is cleaved by the alpha-secretase within the A beta peptide sequence. Proteinases of the ADAM family (adisintegrin and metalloproteinase) are the main candidates as physiologically relevant alpha-secretases, but early lethality of knockout animals prevented a detailed analysis in neuronal cells. To overcome this restriction, we have generated transgenic mice that overexpress either ADAM10 or a catalytically inactive ADAM10 mutant. In this report we show that a moderate neuronal overexpression of ADAM10 in mice transgenic for human APP([V717I]) increased the secretion of the neurotrophic soluble alpha-secretase-released N-terminal APP domain (APPs alpha), reduced the formation of A beta peptides, and prevented their deposition in plaques. Functionally, impaired long-term potentiation and cognitive deficits were alleviated. Expression of mutant catalytically inactive ADAM10 led to an enhancement of the number and size of amyloid plaques in the brains of double-transgenic mice. The results provide the first in vivo evidence for a proteinase of the ADAM family as an alpha-secretase of APP, reveal activation of ADAM10 as a promising therapeutic target, and support the hypothesis that a decrease in alpha-secretase activity contributes to the development of AD.

Susceptibility to collagen-induced arthritis is modulated by TGFbeta responsiveness of T cells.

The objective of our study was to determine the regulatory effects that endogenous transforming growth factor beta (TGFbeta) exerts on T cells in the pathogenesis of collagen-induced arthritis (CIA). CIA was induced in transgenic mice expressing a dominant negative TGFbeta type II receptor in T cells under the control of the human CD2 promoter. Clinical and histological arthritis scores were determined and experiments on disease induction and the healing phase of disease were performed. The proliferation and cytokine production of draining lymph node cells in vitro were analyzed. Transgenic mice were more susceptible to induction of CIA. The overall incidence was higher in transgenic mice than in wild-type mice (57% vs 35%, P < 0.05). Affected transgenic animals displayed a significantly higher clinical (4.5 +/- 0.6 vs 1.67 +/- 0.19, P = 0.001) and histological arthritis score (8.01 +/- 0.9 vs 4.06 +/- 1.1, P < 0.05). Draining lymph node cells of transgenic mice secreted more tumor necrosis factor alpha and IFNgamma and proliferated more vigorously in response to collagen type II and upon CD3/CD28 costimulation in vitro. Therefore, the regulation of T cells by endogenous TGFbeta is important for the maintenance of joint integrity after arthritis induction. Defects in TGFbeta-signalling as a susceptibility factor for rheumatoid arthritis may warrant further investigation.

Placenta-derived CD95 ligand causes liver damage in hemolysis, elevated liver enzymes, and low platelet count syndrome.

BACKGROUND & AIMS: The HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome is a life-threatening complication during pregnancy. The associated liver disease may be severe, and maternal hepatic complications may progress to the point that transplantation becomes necessary. CD95 (APO-1, Fas)-mediated apoptosis of liver cells is one of the major pathogenic mechanisms during liver disease. The interaction of CD95 with its ligand, CD95L(FasL), induces apoptosis and thus the source of the death-inducing ligand is critical for understanding the pathomechanism of liver damage involving the CD95-system. METHODS: Sera from HELLP patients were analyzed and used in cell culture experiments to study CD95-mediated apoptosis. We established a mouse model for placenta-induced liver damage and used a new therapeutical agent, LY498919, to block CD95 apoptosis. RESULTS: We describe apoptosis in the liver of HELLP patients and cytotoxic activity for primary human hepatocytes in HELLP serum. Blocking of CD95 signaling reduced the cytotoxic activity of HELLP serum. In addition, cytotoxic activity increased as HELLP syndrome developed. Furthermore, CD95L was found to be produced in the placenta and extracts of placenta were cytotoxic for human hepatocytes. Injection of mouse placenta extract in mice induces liver damage that could be prevented by blocking CD95L. CONCLUSIONS: Taken together, these data suggest that CD95L derived from the placenta acts systemically and is a primary cause of liver damage in HELLP syndrome. Our results also show that blocking of CD95L can reduce liver cell apoptosis, indicating that such a strategy may have therapeutic advantages.