Rotavirus-neutralizing antibodies inhibit virus binding to integrins α2β1 and α4β1

Summary Rotavirus outer capsid proteins VP5^*, VP8^* and VP7 elicit neutralizing, protective antibodies. The α2β1 integrin is a cellular receptor for rotavirus that is bound by VP5^*. Some rotaviruses also recognize the α4β1 integrin. In this study, the effects of antibodies to rotavirus on virus binding to recombinant α2β1 and α4β1 expressed on K562 cells were determined. All neutralizing monoclonal antibodies to VP5^* tested (YO-2C2, 2G4, 1A10) and two to VP7 (RV-3:2, RV-4:2) inhibited rotavirus binding to α2β1. Rotavirus binding to α4β1 was reduced by 2G4 and neutralizing antibody F45:2, directed to VP7. However, a neutralizing antibody to VP8^* (RV-5:2) and one to VP7 (RV-3:1) did not affect rotavirus binding to these integrins. Virus-cell binding was unaffected by non-neutralizing antibody RVA to the rotavirus inner capsid protein VP6. The attachment of human rotavirus strain Wa to these integrins was inhibited by infection sera with neutralizing activity collected from two children hospitalised with severe rotavirus gastroenteritis. A negative reference serum did not affect rotavirus-cell attachment. As the binding of rotaviruses to α2β1 and α4β1 is inhibited by neutralizing antibodies to VP5^* and VP7, and serum from children with rotavirus disease, rotavirus recognition of these integrins may be important for host infection.     

Hepatitis C virus-induced furin and thrombospondin-1 activate TGF-β1: role of TGF-β1 in HCV replication

In this study, we demonstrated the molecular mechanisms of TGF-β1 induction as well as proteolytic activation in HCV (JFH-1)-infected cells. Our studies showed the synthesis and secretion of TGF-β1 in HCV-infected cells which was reduced in the presence of Ca²⁺ chelators, an inhibitor of mitochondrial Ca²⁺ uptake, and antioxidants. We also showed that the expression of HCV NS proteins NS3/4A, and NS5A can induce TGF-β1 by cell-based luciferase assay. Furthermore, mutational analysis revealed that the functionally active protease domain of NS3 and N-terminus domain of NS5A are required for TGF-β1 activity. Using siRNA approach we demonstrated that HCV-induced furin and thrombospondin-1 (TSP-1) are involved in the proteolytic activation of TGF-β1. Our results also suggest that TGF-β1 positively regulates HCV RNA replication. Collectively, these observations provide insight into the mechanism of TGF-β1 activation, which likely manifest in liver fibrosis associated with hepatitis C infection.     

α4β1 and α5β1 Control Cell Migration on Fibronectin by Differentially Regulating Cell Speed and Motile Cell Phenotype

Computer-aided time lapse fluorescence videomicroscopy was used to study single cell migration behavior of human aortic endothelial cells on fibronectin coated substrates of varying protein surface density. The role of receptors _₅ _₁, __v _₃, and ₄₁ in mediating cell adhesion and migration on fibronectin was characterized using integrin specific monoclonal antibodies. Matrix density had a direct effect on controlling the proportion of migrating cells and the directional persistence of cell movement (p < 0.01). While there was relatively little influence of fibronectin surface density on absolute migration speed, the ability of endothelial cells to disperse over a surface, as measured by the dispersion coefficient, was biphasic with respect to the surface density of this matrix protein (p < 0.005). Both cell speed and the proportion of migrating cells was controlled by ₄₁ (p < 0.01). However, ₅₁ selectively regulated the transformation of stationary cells to those exhibiting motile behavior (p <0.05). Migratory responses on fibronectin were not influenced by blockade of the _v3 receptor. It is noteworthy that cell surface adhesive receptors which control commitment to a motile phenotype are not necessarily the same as those that control migration speed. © 1998 Biomedical Engineering Society. PAC98: 8722-q     

Peripheral blood neutrophil production of interleukin-1 receptor antagonist and interleukin-1β

Journal of Clinical Immunology - The objective of this study was to characterize interleukin-1 receptor antagonist (IL-1ra) and interleukin-1β (IL-1β) production by human peripheral blood...     

Expression of TGF-β1 and Bax and their significance in intervertebral disc degeneration

目的 探讨退行性变椎间盘组织中TGF-β1和Bax的表达及其意义.方法 收集正常与退变椎间盘组织,并根据病理改变将退变椎间盘组织分成4级,采用HE、免疫组化、TUNEL染色和RT-PCR法进行研究.结果 免疫组化和RT-PCR均显示在正常组织中有TGF-β1表达,Bax只有微量表达;在病变组织中随病理分级加大TGF-β1随之增加,与正常组相比,差异有显著性;Bax也逐步增加,与正常组相比,差异有显著性,Bax表达升高与凋亡指数(AI)呈正相关性.结论 退变椎间盘的病理分级与TGF-β1的表达增高相关,由此调控了Bax的表达,导致了细胞凋亡,促进了椎间盘的退变.     

Macrophage motility requires distinct α5β1/FAK and α4β1/paxillin signaling events

Macrophages function as key inflammatory mediators at sites of infection and tissue damage. Integrin and growth factor receptors facilitate recruitment of monocytes/macrophages to sites of inflammation in response to numerous extracellular stimuli. We have shown recently that FAK plays a role in regulating macrophage chemotaxis and invasion. As FAK is an established downstream mediator of integrin signaling, we sought to define the molecular circuitry involving FAK and the predominant β1 integrin heterodimers expressed in these cells-α4β1 and α5β1. We show that α4β1 and α5β1 integrins are required for efficient haptotactic and chemotactic invasion and that stimulation of these integrin receptors leads to the adoption of distinct morphologies associated with motility. FAK is required downstream of α5β1 for haptotaxis toward FN and chemotaxis toward M-CSF-1 and downstream of α4β1 for the adoption of a polarized phenotype. The scaffolding molecule paxillin functions independently of FAK to promote chemotaxis downstream of α4β1. These studies expand our understanding of β1 integrin signaling networks that regulate motility and invasion in macrophages and thus, provide important new insights into mechanisms by which macrophages perform their diverse functions.     

Artificial extracellular matrix composed of collagen I and highly sulfated hyaluronan interferes with TGFβ1 signaling and prevents TGFβ1-induced myofibroblast differentiation

Sulfated glycosaminoglycans are promising components for functional biomaterials since sulfate groups modulate the binding of growth factors and thereby influence wound healing. Here, we have investigated the influence of an artificial extracellular matrix (aECM) consisting of collagen I (coll) and hyaluronan (HA) or highly sulfated HA (hsHA) on dermal fibroblasts (dFb) with respect to their differentiation into myofibroblasts (MFb). Fibroblasts were cultured on aECM in the presence of aECM-adsorbed or soluble transforming growth factor β1 (TGFβ1). The synthesis of α-smooth muscle actin (αSMA), collagen and the ED-A splice variant of fibronectin (ED-A FN) were analyzed at the mRNA and protein levels. Furthermore, we investigated the bioactivity and signal transduction of TGFβ1 in the presence of aECM and finally made interaction studies of soluble HA or hsHA with TGFβ1. Artificial ECM composed of coll and hsHA prevents TGFβ1-stimulated αSMA, collagen and ED-A FN expression. Our data suggest an impaired TGFβ1 bioactivity and downstream signaling in the presence of aECM containing hsHA, shown by massively reduced Smad2/3 translocation to the nucleus. These data are explained by in silico docking experiments demonstrating the occupation of the TGFβ-receptor I binding site by hsHA. Possibly, HA sulfation has a strong impact on TGFβ1-driven differentiation of dFb and thus could be used to modulate the properties of biomaterials.     

Sulfated hyaluronan and chondroitin sulfate derivatives interact differently with human transforming growth factor-β1 (TGF-β1)

This study demonstrates that the modification of hyaluronan (hyaluronic acid; Hya) and chondroitin sulfate (CS) with sulfate groups leads to different binding affinities for recombinant human transforming growth factor-β1 (TGF-β1) for comparable average degrees of sulfation (DS). In general, Hya derivates showed higher binding strength than CS derivatives. In either case, a higher degree of sulfation leads to a stronger interaction. The high-sulfated hyaluronan sHya3 (average DS≈3) exhibited the tightest interaction with TGF-β1, as determined by surface plasmon resonance and enzyme-linked immunosorbent assay. The binding strength was significantly weakened by carboxymethylation. Unmodified Hya and low-sulfated, native CS showed weak or no binding affinity. The interaction characteristics of the different sulfated glycosaminoglycans are promising for incorporation into bioengineered coatings of biomaterials to modulate growth factor binding in medical applications.     

The influence of the HLA-DRB, HLA-DQB and polymorphic positions of the HLA-DRβ1 and HLA-DQβ1 molecules on risk of Iranian type 1 diabetes mellitus patients

Type 1 Diabetes mellitus (T1D) is an autoimmune and multifactorial disease. HLA-DRB1 and DQB1 loci have the strongest association with T1D. This study aimed at investigating (i) susceptibility or protection of alleles, genotypes and haplotypes of HLA-DRB1 and DQB1 loci; and (ii) highly polymorphic amino acid residues of HLA-DRβ1 and DQβ1 in 105 Iranian T1D patients and 100 controls. The results indicated that DRB1*04:01, 03:01, DQB1*03:02, 02:01 alleles, DRB1*03:01/04:01, 03:01/13:03, DQB1*02:01/03:02 genotypes, DRB1*04:01-DQB1*03:02, DRB1*03:01-DQB1*02:01, DRB1*07:01-DQB1*03:03 haplotypes had positive association with T1D. In contrast, HLA-DRB1*15:01, 13:01, DQB1*03:01, 06:01 alleles, DRB1*11:01/15:01, DQB1*03:01/06:01, 03:01/05:01 genotypes and DRB1*15:01-DQB1*06:01, DRB1*11:01-DQB1*03:01 haplotypes had negative association with T1D. Analysis of amino acid sequence of HLA-DRβ1 and DQβ1 revealed that DRβ1(Lys71+) and DQβ1(Asp57-) were significantly more frequent in patients than in controls and had a positive effect in the development of T1D. Haplotype analysis demonstrated that HLA-DRB1(Lys71+) allele provided major susceptibility for T1D, and DQβ1(Asp57-) had an additive effect. We designed an allele-specific primer to develop an easy, quick and cost-benefit method to detect the DRβ1(Lys71+) . This method can identify all 114 DRB1 alleles encoding DRβ1(Lys71+) by three PCR reactions. The PcPPV and PcNPV were also calculated to determine the impact of HLA genotype testing at amino acid positions. It showed that the DRβ1(Lys71+/+) genotype carrier had 1% absolute risk of developing T1D.     

Integrin α1/β1 and α2/β1 as a receptor for IgA1 in human glomerular mesangial cells in IgA nephropathy

IgA nephropathy (IgAN) is characterized by mesangial deposition of IgA1 and galactose-deficient IgA1 is expected to play a pathogenic role. However, the identity of the receptor for IgA1 is still controversial. Hence, the aim of this study was to explore the receptor for galactose-deficient IgA1. Human monoclonal IgA1 was treated with exoglycosidase and FITC-conjugated control, asialo- and agalactosyl-IgA1 was used as a probe to detect the receptor in cultured human mesangial cells. Tumor necrosis factor-α or transforming growth factor-β1 treatment accelerated IgA1-binding on mesangial cells, and these effects were diminished by the addition of dexamethasone, whereas these changes were not dependent on galactose-deficiency of IgA1. According to comprehensive gene expression analysis, we focused on integrin β1. Pre-treatment by Mn(2+), which activates integrin by changing its structure, enhanced the binding of IgA1 in cultured mesangial cells. Furthermore, pre-incubation with collagens specifically enhanced binding of IgA1 in the cultured human mesangial cells without activation by Mn(2+). Collagen type IV distributed in the mesangial region of the glomeruli as well as Bowman's capsule and tubular basal membrane in IgAN patients, and the IgA1 with collagen type IV induced proliferative signals on mesangial cells by phosphorylating extracellular signal-regulated kinase more effectively than the IgA1 alone. Immunoprecipitation assay revealed the binding of IgA1 and integrin α1/β1 and α2/β1 heterodimer and down-regulation of integrin α1, α2 and β1 expression in human mesangial cells induced by each specific small interfering RNA diminished the ability to bind IgA1 probe. Integrin α1/β1 and α2/β1 would be a candidate receptor for IgA1.     

Vitamin D3 and Calcipotriol Enhance the Secretion of Transforming Growth Factor-β1 and -β2 in Cultured Murine Keratinocytes

Abstract

Vitamin D₃ and its analogue calcipotriol (MC 903) inhibit the proliferation of cultured keratinocytes and induce their differentiation. Since TGFβs are very potent inhibitors of keratinocyte growth we studied the effects of vitamin D₃ and calcipotriol on the secretion of TGFβ in cultured murine keratinocytes. Vitamin D, and calcipotriol (10^?6 - 10^?9 M) inhibited the DNA-synthesis of mouse keratinocytes by 50–80% in a time and dose-dependent manner as measured by [³H]-thymidine incorporation. Analysis of the conditioned medium of the keratinocytes indicated that the cells secreted into their medium activity that inhibited the growth of indicator Mv1Lu mink lung epithelial cells. Neutralizing antibodies against TGFβ1 and TGFβ2 decreased, and when used together, prevented the observed growth inhibition of the indicator cells. Heat treatment of the conditioned medium, which activates latent forms of TGFβ, revealed higher levels of growth inhibitory activity in the medium from vitamin D₃ and calcipotriol treated than from control cultures indicating that a fraction of TGFβ was in a latent form. Active TGFβ was, however, detected considerably more in vitamin D₃ and calcipotriol treated cultures than in control cultures. Immunoblotting analysis of the medium revealed enhanced secretion of TGFβ protein. These results indicate that enhanced TGFβ1 and TGFβ2 secretion and activity is associated with vitamin D₃-mediated growth inhibition of cultured keratinocytes.

This work was presented in part at the Keystone Symposium “Negative Growth Control”, Keystone, CO, Jan. 26-Feb. 2, 1992 (Koli and Keski-Oja 1992).     

11β-hydroxysteroid dehydrogenase type 1, brain atrophy and cognitive decline

Excess cortisol levels are linked with brain atrophy and cognitive decline in older people. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) potently amplifies intracellular glucocorticoid action by converting inert cortisone to active cortisol, but any causal importance in brain aging is unexplored. We tested the hypotheses that higher systemic 11β-HSD1 activity predicts brain atrophy and cognitive decline in older men. In a longitudinal study of 41 men (65-70 years old at baseline) we measured baseline systemic 11β-HSD1 activity, the urinary 5alpha- and 5beta-tetrahydrocortisol to tetrahydrocortisone ratio (ratio of tetrahydrometabolites of cortisol (THFs)/ratio of tetrahydrometabolites of cortisol (THE)), and assessed change in brain atrophy, white matter lesions and cognitive function over 6 years. Baseline THFs/THE correlated negatively with baseline hippocampal volumes (left: r = -0.37; right: r = -0.34; p < 0.05) and positively with ventricular volumes (r = 0.43, p = 0.006) and periventricular white matter lesions (rho = 0.31, p = 0.047). Importantly, baseline THFs/THE but not cortisol predicted increase in ventricular volumes (r = 0.33, p = 0.037) and decline in processing speed (r = -0.55, p = 0.0002) over 6 years. The predictive link between systemic 11β-HSD1 activity and progressive brain atrophy and cognitive decline suggests 11β-HSD1 inhibition as a plausible therapy for brain aging.     

The involvement of TGF-β1 and CTGF in regional gingival overgrowth

Gingival overgrowth is a multifactorial and invalidating condition. Our research is about gingival overgrowth caused by gingival plaque, its purpose being the evaluation of the presence of gingivitis and/or parodontitis in patients with gingival growth and the extent in which there is a connection between gingival overgrowth and the inflammatory process that can contribute to an exceedingly stimulation of the overgrowth. Immunohistological study was conducted on human material--gingival mucosa that came from patients with ages between 20-65 years, divided into three groups: group I--control group, group II--patients with gingivitis, group III--patients with local or general periodontitis. The intensity of immunohistochemical staining of TGF-β1 and CTGF varies from one group to another, and also depends on the area of gingival mucosa that was observed. TGF-β1 has a crucial role in periodontal disease fibrogenesis by intensifying the action of CTGF.     

Endothelial α3β1-Integrin Represses Pathological Angiogenesis and Sustains Endothelial-VEGF

Integrin α3β1 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of α3β1 can affect angiogenesis either positively or negatively, either a direct in vivo role for α3β1 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of α3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where α3 is deleted specifically in endothelial cells (ec-α3−/−). Here we show that ec-α3−/− mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that α3β1 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial α3β1 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.Angiogenesis, the formation of new blood vessels from pre-existing ones, is enhanced in various pathological conditions including rheumatoid arthritis, diabetic retinopathy, and cancer development. Angiogenic processes are regulated both by growth factors, such as vascular endothelial growth factor (VEGF), and adhesion molecules, such as integrins.^1,2,3 Inhibitors of VEGF signaling can extend progression-free survival in colorectal, lung, and breast cancers when used in combination with chemotherapy^4,5 and in renal cancer when used as a monotherapy.⁶ However, recent data suggest that complications with acquired resistance to these agents and how they affect metastasis may compromise their efficacy.^7,8 Thus, understanding the molecular mechanisms that underlie the regulation of angiogenesis, and especially VEGF function, is essential for the development of safe and effective antiangiogenic therapies.Endothelial integrins include the fibronectin receptor α5β1, collagen receptors α2β1 and α1β1, vitronectin receptors αvβ3 and αvβ5, and laminin receptors α6β1, α6β4, and α3β1. Although many integrins have been shown to be involved in tumor angiogenesis,¹ the role of endothelial-α3β1 in this process is unknown. In addition, integrin α3β1 is a binding partner for several other ligands, and together these have been implicated in either promoting or inhibiting angiogenesis. For example, deletion of CD151 or embryonic laminin α4-subunit, molecules that can both bind α3β1, results in impaired angiogenesis,^9,10,11 whereas adult laminin-8–deficient mice display increased tumor vascularization.¹² In addition, it has also been proposed that the antitumorigenic effects of recombinant α3(IV)NC1 can, in part, be attributed to its binding to αvβ3 and α3β1-integrin,^13,14 whereas the antiangiogenic functions of thrombospondin-1 are reversed by binding αvβ3 and α3β1.^15,16 And finally, the binding of VEGF to α3β1 in vitro is thought to enhance angiogenic responses,¹⁷ whereas the interaction of TIMP-2 with α3β1-integrin has been reported to inhibit VEGF receptor-2 function.¹⁸ Taken together, these contradictory results indicate that a precise and, more importantly, direct role of α3β1-integrin in pathological angiogenesis is not well understood. Accordingly, because α3β1-directed inhibitors are being designed to either block tumor cell growth or angiogenesis,^19,20 the requirement to test directly the role of α3β1 in pathological angiogenesis becomes a priority.Genetic ablation of the α3-integrin-subunit in mice results in a lethal phenotype where mice die within hours after birth,²¹ rendering them inappropriate for pathological angiogenesis studies. We therefore have generated mice where the α3-integrin-subunit is deleted in endothelial cells (ec-α3−/−). These mice are viable and fertile, and here we report that mice deficient in endothelial-α3β1-integrin display enhanced tumor growth and elevated tumor angiogenesis. In addition we show that the deletion of α3-integrin in endothelial cells results in enhanced VEGF-mediated angiogenic responses both ex vivo and in vivo. Moreover, we present evidence for a novel and unexpected mechanism by which deficiency in α3β1 regulates angiogenesis by the regulation of endothelial VEGF. Although autocrine signaling by endothelial VEGF has been shown to regulate endothelial cell survival,²² its role in angiogenesis or a mechanism by which it is regulated has not been demonstrated. We show that α3β1 reduces the expression of endothelial VEGF, which surprisingly, at low levels, elevates VEGFR2 expression and thus enhances VEGF-induced angiogenic responses.     

Inhibitors of apoptosis proteins and IL-1β: a tangled relationship

Inhibitors of apoptosis proteins (IAPs) are important regulators of both cell death and inflammation. In this issue of Immunity, Vince et al. (2012) report that inhibition of IAPs results in the processing and secretion of IL-1β through RIP3-mediated caspase-1- and caspase-8-dependent pathways.     

Interleukin-1β-induced neutrophil recruitment and acute lung injury in hamsters

Administering recombinant interleukin-1 (IL-1) intratracheally caused lung neutrophil accumulation and lung injury in hamsters. The percentage of leukocytes that were neutrophils increased progressively in lavages from lungs of hamsters given 25, 50, or 100 ng IL-1 intratracheally 2 h before. Lung injury, reflected by increased lung lavage protein concentrations and lung lavage hemoglobin concentrations, increased 2 h after administering 100 ng IL-1. Lung injury, reflected by lung wet weight/body weight ratios, followed similar patterns, with significant increases occurring 2 h after insufflating 50 or 100 ng IL-1. Our results indicate that increased concentrations of IL-1 in lung airways can cause neutrophil recruitment and lung injury in hamsters. This mechanism may contribute to the development of lung neutrophil accumulation and lung injury that characterizes ARDS patients who have increased airway levels of IL-1.