Processing of amyloid beta-peptides by neutral cysteine protease bleomycin hydrolase

We studied the processing of amyloid beta-peptides (Abetas) including Abeta(1-40), Abeta(1-42) and pAbeta(3-42) by rat neutral cysteine protease bleomycin hydrolase (BH) according to the methods of SDS-PAGE, HPLC and matrix-assisted laser desorption/inonization time-of-flight mass spectrometry (MALDI-TOF MS). BH significantly processed them by novel features of its diverse activities. It initially cleaved at two sites, His(14)-Gln(15) and Phe(19)-Phe(20) degraded to short intermediates then to amino acids by aminopeptidase and/or carboxypeptidase activities. Also, full-length Abetas were clipped at the carboxyl(C)-terminal region. On the other hand, BH cleaved at only the His(14)-Gln(15) bond in pbetaA(3-42) within a short period of the reaction by endopeptidase activity, and processed the intermediates in order by carboxypeptidase activity. On processing by BH, it found that both fibrillar Abeta(1-40) and Abeta(1-42) were more resistant than non-fibrillar peptides. These results indicate that the processing specificity of BH depends upon the structure and sequence of Abetas.     

Implication of the hypoxia response element of the Vegf promoter in mouse models of retinal and choroidal neovascularization, but not retinal vascular development

Retinal neovascularization (NV) and macular edema, resulting from blood-retinal barrier (BRB) breakdown, are major causes of visual loss in ischemic retinopathies. Choroidal NV (CNV) occurs in diseases of the retinal pigmented epithelium/Bruch's membrane complex and is another extremely prevalent cause of visual loss. We used mice in which the hypoxia response element (HRE) is deleted from the vascular endothelial growth factor (vegf) promoter (Vegf(delta/delta) mice) to explore the role of induction of VEGF through the HRE in these disease processes. Compared to wild type (Vegf+/+) mice with oxygen-induced ischemic retinopathy (OIR) in which vegf mRNA levels were increased and prominent retinal NV and BRB breakdown occurred, Vegf(delta/delta) littermates with OIR failed to increase vegf mRNA levels in the retina and had significantly less retinal NV and BRB breakdown, but showed prominent dilation of some superficial retinal vessels. Vegf(+/delta) littermates with ischemic retinopathy developed comparable retinal NV to Vegf+/+ mice, exhibited intermediate levels of BRB breakdown, and did not show vasodilation. In a mouse model of CNV, due to laser-induced rupture of Bruch's membrane, the area of CNV at Bruch's membrane rupture sites was more than tenfold greater in Vegf+/+ mice than in Vegf(delta/delta) littermates. In contrast to these dramatic differences in pathologic ocular NV, Vegf(delta/delta) mice showed subtle differences in retinal vascular development compared to Vegf+/+ mice; it was slightly delayed, but otherwise normal. These data suggest that induction of VEGF through the HRE in its promoter is critical for retinal and CNV, but not for retinal vascular development.     

Not lipoteichoic acid but lipoproteins appear to be the dominant immunobiologically active compounds in Staphylococcus aureus

Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of TLR2. However, we previously demonstrated that LTA fraction prepared from bacterial cells contains lipoproteins, which activate cells via TLR2. In this study, we investigated the immunobiological activity of LTA fraction obtained from S. aureus wild-type strain, lipoprotein diacylglycerol transferase deletion (delta lgt) mutant, which lacks palmitate-labeled lipoproteins, and its complemented strain and evaluated the activity of LTA molecule. LTA fraction was prepared by butanol extraction of the bacteria followed by hydrophobic interaction chromatography. Although all LTA fractions activated cells through TLR2, the LTA from delta lgt mutant was 100-fold less potent than those of wild-type and complemented strains. However, no significant structural difference in LTA was observed in NMR spectra. Further, alanylation of LTA molecule showed no effect in immunobiological activity. These results showed that not LTA molecule but lipoproteins are dominant immunobiologically active TLR2 ligand in S. aureus.     

IL-15 regulates CD8+ T cell contraction during primary infection

During the course of acute infection with an intracellular pathogen, Ag-specific T cells proliferate in the expansion phase, and then most of the T cells die by apoptosis in the following contraction phase, but the few that survive become memory cells and persist for a long period of time. Although IL-15 is known to play an important role in long-term maintenance of memory CD8+ T cells, the potential roles of IL-15 in CD8+ T cell contraction are not known. Using an adoptive transfer system of OT-I cells expressing OVA257-264/Kb-specific TCR into control, IL-15 knockout (KO) and IL-15 transgenic (Tg) mice followed by challenge with recombinant Listeria monocytogenes expressing OVA, we found that the survival of CD44+CD62L-CD127- effector OT-I cells during the contraction phase is critically dependent on IL-15. In correlation with the expression level of Bcl-2 in OT-I cells, the number of OT-I cells was markedly reduced in IL-15 KO mice but remained at a high level in IL-15 Tg mice during the contraction phase, compared with control mice. In vivo administration of rIL-15 during the contraction phase in IL-15 KO mice inhibited the contraction of effector OT-I cells accompanied by up-regulation of Bcl-2 expression. Furthermore, enforced expression of Bcl-2 protected the majority of effector OT-I cells from death in IL-15 KO mice after infection. These results suggest that IL-15 plays a critical role in protecting effector CD8+ T cells from apoptosis during the contraction phase following a microbial infection via inducing antiapoptotic molecules.     

Genetic dissection of vasculitis, myeloperoxidase-specific antineutrophil cytoplasmic autoantibody production, and related traits in spontaneous crescentic glomerulonephritis-forming/Kinjoh mice

The spontaneous crescentic glomerulonephritis-forming/Kinjoh (SCG/Kj) mouse is a model of human crescentic glomerulonephritis and vasculitis associated with the production of the myeloperoxidase (MPO)-specific antineutrophil cytoplasmic autoantibody (MPO-ANCA). Although the disease is mediated initially by mutation of the Fas gene (lpr), SCG/Kj mice also have non-Fas predisposing genetic factors. To define these factors, genome-wide quantitative trait locus (QTL) mapping was performed on female (B(6)x SCG/Kj) F(2) intercross mice. Fourteen non-Fas QTLs were identified. QTLs of glomerulonephritis were located on chromosomes 1, 10, 13, 16, and 17, vasculitis on chromosomes 1 and 17, splenomegaly on chromosome 1, hypergammaglobulinemia on chromosomes 1, 2, 4, 6, 7, 11, 13, and 17, antinuclear Ab on chromosomes 1, 8, 10, and 12, and MPO-ANCA production on chromosomes 1 and 10. Significant QTLs derived from SCG/Kj on chromosomes 1, 2, 7, and 13 were designated Scg-1 to Scg-5, respectively, and those derived from B(6) on chromosomes 4, 6, 17, and 10 were designated Sxb-1 to Sxb-4, respectively. Two loci linked to MPO-ANCA production on chromosomes 1 and 10 were designated Man-1 and Man-2 (for MPO-ANCA), respectively. Although both Scg-1 and Scg-2 were on chromosome 1 and shared several functions, it was of interest that aberrant MPO-ANCA production was exclusively controlled by Man-1, the centromeric half region of the Scg-2 chromosomal segment. We also examined the epistatic effects between the lpr mutation and non-Fas susceptibility genes. QTLs are discussed in relation to previously described loci, with emphasis on their candidate genes.     

Regulation of Th2 cell development by Polycomb group gene bmi-1 through the stabilization of GATA3

The Polycomb group (PcG) gene products regulate the maintenance of the homeobox gene expression in Drosophila and vertebrates and also the cell cycle progression in thymocytes and Th2 cell differentiation in mature T cells. We herein studied the role of PcG gene bmi-1 product in Th1/Th2 cell differentiation and found that Bmi-1 facilitates Th2 cell differentiation in a Ring finger-dependent manner. Biochemical studies indicate that Bmi-1 interacts with GATA3 in T cells, which is dependent on the Ring finger of Bmi-1. The overexpression of Bmi-1 resulted in a decreased ubiquitination and an increased protein stability of GATA3. In bmi-1-deficient Th cells, the levels of Th2 cell differentiation decreased as the degradation and ubiquitination on GATA3 increased. Therefore, Bmi-1 plays a crucial role in the control of Th2 cell differentiation in a Ring finger-dependent manner by regulating GATA3 protein stability.     

Prostaglandin D2 plays an essential role in chronic allergic inflammation of the skin via CRTH2 receptor

PGD(2) plays roles in allergic inflammation via specific receptors, the PGD receptor designated DP and CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells). We generated mutant mice carrying a targeted disruption of the CRTH2 gene to investigate the functional roles of CRTH2 in cutaneous inflammatory responses. CRTH2-deficent mice were fertile and grew normally. Ear-swelling responses induced by hapten-specific IgE were less pronounced in mutant mice, giving 35-55% of the responses of normal mice. Similar results were seen in mice treated with a hemopoietic PGD synthase inhibitor, HQL-79, or a CRTH2 antagonist, ramatroban. The reduction in cutaneous responses was associated with decreased infiltration of lymphocytes, eosinophils, and basophils and decreased production of macrophage-derived chemokine and RANTES at inflammatory sites. In models of chronic contact hypersensitivity induced by repeated hapten application, CRTH2 deficiency resulted in a reduction by approximately half of skin responses and low levels (63% of control) of serum IgE production, although in vivo migration of Langerhans cells and dendritic cells to regional lymph nodes was not impaired in CRTH2-deficient mice. In contrast, delayed-type hypersensitivity to SRBC and irritation dermatitis in mutant mice were the same as in wild-type mice. These findings indicate that the PGD(2)-CRTH2 system plays a significant role in chronic allergic skin inflammation. CRTH2 may represent a novel therapeutic target for treatment of human allergic disorders, including atopic dermatitis.     

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.