Heparin II domain of fibronectin uses alpha4beta1 integrin to control focal adhesion and stress fiber formation, independent of syndecan-4

Co-signaling events between integrins and cell surface proteoglycans play a critical role in the organization of the cytoskeleton and adhesion forces of cells. These processes, which appear to be responsible for maintaining intraocular pressure in the human eye, involve a novel cooperative co-signaling pathway between alpha5beta1 and alpha4beta1 integrins and are independent of heparan sulfate proteoglycans. Human trabecular meshwork cells isolated from the eye were plated on type III 7-10 repeats of fibronectin (alpha5beta1 ligand) in the absence or presence of the heparin (Hep) II domain of fibronectin. In the absence of the Hep II domain, cells had a bipolar morphology with few focal adhesions and stress fibers. The addition of the Hep II domain increased cell spreading and the numbers of focal adhesions and stress fibers. Cell spreading and stress fiber formation were not mediated by heparan sulfate proteoglycans because treatment with chlorate, heparinase, or soluble heparin did not prevent Hep II domain-mediated cell spreading. Cell spreading and stress fiber formation were mediated by alpha4beta1 integrin because soluble anti-alpha4 integrin antibodies inhibited Hep II domain-mediated cell spreading and soluble vascular cell adhesion molecule-1 (alpha4beta1 ligand)-induced cell spreading. This is the first demonstration of the Hep II domain mediating cell spreading and stress fiber formation through alpha4beta1 integrin. This novel pathway demonstrates a cooperative, rather than antagonistic, role between alpha5beta1 and alpha4beta1 integrins and suggests that interactions between the Hep II domain and alpha4beta1 integrin could modulate the strength of cytoskeleton-mediated processes in the trabecular meshwork of the human eye.     

Role of the programmed death-1 pathway in regulation of alloimmune responses in vivo

Programmed death-1 (PD-1), an inhibitory receptor up-regulated on activated T cells, has been shown to play a critical immunoregulatory role in peripheral tolerance, but its role in alloimmune responses is poorly understood. Using a novel alloreactive TCR-transgenic model system, we examined the functions of this pathway in the regulation of alloreactive CD4+ T cell responses in vivo. PD-L1, but not PD-1 or PD-L2, blockade accelerated MHC class II-mismatched skin graft (bm12 (I-Abm12) into B6 (I-Ab)) rejection in a similar manner to CTLA-4 blockade. In an adoptive transfer model system using the recently described anti-bm12 (ABM) TCR-transgenic mice directly reactive to I-Abm12, PD-1 and PD-L1 blockade enhanced T cell proliferation early in the immune response. In contrast, at a later time point preceding accelerated allograft rejection, only PD-L1 blockade enhanced T cell proliferation. In addition, PD-L1 blockade enhanced alloreactive Th1 cell differentiation. Apoptosis of alloantigen-specific T cells was inhibited significantly by PD-L1 but not PD-1 blockade, indicating that PD-1 may not be the receptor for the apoptotic effect of the PD-L1-signaling pathway. Interestingly, the effect of PD-L1 blockade was dependent on the presence of CD4+ CD25+ regulatory T cells in vivo. These data demonstrate a critical role for the PD-1 pathway, particularly PD-1/PD-L1 interactions, in the regulation of alloimmune responses in vivo.     

Analysis of the role of negative T cell costimulatory pathways in CD4 and CD8 T cell-mediated alloimmune responses in vivo

Negative costimulatory signals mediated via cell surface molecules such as CTLA-4 and programmed death 1 (PD-1) play a critical role in down-modulating immune responses and maintaining peripheral tolerance. However, their role in alloimmune responses remains unclear. This study examined the role of these inhibitory pathways in regulating CD28-dependent and CD28-independent CD4 and CD8 alloreactive T cells in vivo. CTLA-4 blockade accelerated graft rejection in C57BL/6 wild-type recipients and in a proportion of CD4(-/-) but not CD8(-/-) recipients of BALB/c hearts. The same treatment led to prompt rejection in CD28(-/-) and a smaller proportion of CD4(-/-)CD28(-/-) mice with no effect in CD8(-/-)CD28(-/-) recipients. These results indicate that the CTLA-4:B7 pathway provides a negative signal to alloreactive CD8(+) T cells, particularly in the presence of CD28 costimulation. In contrast, PD-1 blockade led to accelerated rejection of heart allografts only in CD28(-/-) and CD8(-/-)CD28(-/-) recipients. Interestingly, PD-1 ligand (PD-L1) blockade led to accelerated rejection in wild-type mice and in all recipients lacking CD28 costimulation. This effect was accompanied by expansion of IFN-gamma-producing alloreactive T cells and enhanced generation of effector T cells in rejecting allograft recipients. Thus, the PD-1:PD-L1 pathway down-regulates alloreactive CD4 T cells, particularly in the absence of CD28 costimulation. The differential effects of PD-1 vs PD-L1 blockade support the possible existence of a new receptor other than PD-1 for negative signaling through PD-L1. Furthermore, PD-1:PD-L1 pathway can regulate alloimmune responses independent of an intact CD28/CTLA-4:B7 pathway. Harnessing physiological mechanisms that regulate alloimmunity should lead to development of novel strategies to induce durable and reproducible transplantation tolerance.     

Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis

Heme oxygenase-1 (HO-1) is emerging as an important cytoprotective enzyme system in a variety of injury models. To optimize future therapeutic applications of HO-1, it is necessary to delineate the precise functions and mechanisms as well as modes of externally regulating HO-1 expression. Investigations have been limited by difficulties with the generation of HO-1 null mice and the lack of specific HO-1 inhibitors. Lung ischemia-reperfusion (I-R) injury is the inciting event in acute lung failure following transplantation, surgery, and shock. To study the function of HO-1 in I-R-induced lung injury, we designed small interfering RNA (siRNA) sequences that effectively suppress HO-1 expression both in vitro and in vivo in an organ-specific manner. In this study we show that there is enhanced apoptosis, via increased Fas expression and caspase 3 activity, in the presence of HO-1 siRNA in endothelial cells and mouse lung during I-R injury, whereas HO-1 overexpression attenuates apoptosis. To the best of our knowledge, we are the first to demonstrate that lung-specific siRNA delivery can be achieved by intranasal administration without the need for viral vectors or transfection agents in vivo, thereby obviating potential concerns for toxicity if siRNA technology is to have clinical application in the future.     

Immunocytochemical localization and expression of heme oxygenase-1 in primary astroglial cell cultures during differentiation: effect of glutamate

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation releasing iron, carbon monoxide (CO), and biliverdin. We investigated subcellular localization of HO-1 using confocal laser scanning microscopy (CLSM) and the expression by Western blot in primary astroglial cells during differentiation and after exposure to glutamate (100microM). CLSM analysis of immunostained HO-1 in cultured astroglial cells during differentiation showed an increase of fluorescence between 7 and 14 days and a decrease between 14 and 21, although HO-1 peaked at 14 days it remained at high levels. The distribution of HO-1 protein undergoes modification in the various cellular compartments. Furthermore, localization of the protein in untreated astrocytes at 7 days appeared prevalently localized in the cytosol and in the perinuclear region. In contrast, at 14 and 21 days, fluorescence detection suggests that HO-1 was present also in the nucleus, and in the nucleoli. Fluorescence intensity significantly increased in glutamate-treated astrocytes during all development stages and the protein appeared in the cytosol, in the nucleus and in the nucleoli. The involvement of AMPA/Ka receptors was studied in glutamate-treated astroglial cells at 14 days by the preincubation of the cells with GYKI 52466, a specific receptor inhibitor, of AMPA/Ka receptor demonstrating the involvement of these receptors. Western blot analysis of HO-1 confirmed the CLSM results. Our results demonstrate that changes in HO-1 protein expression and localization in primary cultured astroglial cells may be part of the underlying mechanisms involved in brain development as well as in neurodegenerative diseases.     

Augmented heme oxygenase-1 induces prostaglandin uptake via the prostaglandin transporter in micro-vascular endothelial cells

Previous studies show that expression of heme oxygenase-1 (HO-1) in endothelial cells results in decreased cyclooxygenase expression and prostaglandin (PG) levels through limiting heme availability. Regulation of PGs, important inflammatory mediators, may contribute to the anti-inflammatory potential of HO-1. Here we examine the effects of HO-1 expression on PG clearance via the prostaglandin transporter (PGT). Endothelial cells expressing human HO-1 via retroviral transfer exhibit approximately 7-fold higher levels of PGT RNA and equivalently elevated uptake of [(3)H]PGE(2). The pattern and extent of uptake and the substrate inhibitory constants of PGE(2), PGF(2alpha), and thromboxane B(2) are similar to those of cloned PGT. Treatment of cells with stannous chloride, an inducer of HO-1, results in increased expression of PGT while incubation of cells expressing human HO-1 with stannic mesophorphyrin, a substrate inhibitor of HO-1, decreases PG uptake. Therefore, PG clearance via PGT may contribute to the cellular regulation of PG levels by HO-1.     

Differential role of CCR2 in islet and heart allograft rejection: tissue specificity of chemokine/chemokine receptor function in vivo

Chemokines have a pivotal role in the mobilization and activation of specific leukocyte subsets in acute allograft rejection. However, the role of specific chemokines and chemokine receptors in islet allograft rejection has not been fully elucidated. We now show that islet allograft rejection is associated with a steady increase in intragraft expression of the chemokines CCL8 (monocyte chemoattractant protein-2), CCL9 (monocyte chemoattractant protein-5), CCL5 (RANTES), CXCL-10 (IFN-gamma-inducible protein-10), and CXCL9 (monokine induced by IFN-gamma) and their corresponding chemokine receptors CCR2, CCR5, CCR1, and CXCR3. Because CCR2 was found to be highly induced, we tested the specific role of CCR2 in islet allograft rejection by transplanting fully MHC mismatched islets from BALB/c mice into C57BL/6 wild-type (WT) and CCR2-deficient mice (CCR2-/-). A significant prolongation of islet allograft survival was noted in CCR2-/- recipients, with median survival time of 24 and 12 days for CCR2-/- and WT recipients, respectively (p < 0.0001). This was associated with reduction in the generation of CD8+, but not CD4+ effector alloreactive T cells (CD62L(low)CD44(high)) in CCR2-/- compared with WT recipients. In addition, CCR2-/- recipients had a reduced Th1 and increased Th2 alloresponse in the periphery (by ELISPOT analysis) as well as in the grafts (by RT-PCR). However, these changes were only transient in CCR2-/- recipients that ultimately rejected their grafts. Furthermore, in contrast to the islet transplants, CCR2 deficiency offered only marginal prolongation of heart allograft survival. This study demonstrates the important role for CCR2 in early islet allograft rejection and highlights the tissue specificity of the chemokine/chemokine receptor system in vivo in regulating allograft rejection.     

TIM-3: a novel regulatory molecule of alloimmune activation

T cell Ig domain and mucin domain (TIM)-3 has previously been established as a central regulator of Th1 responses and immune tolerance. In this study, we examined its functions in allograft rejection in a murine model of vascularized cardiac transplantation. TIM-3 was constitutively expressed on dendritic cells and natural regulatory T cells (Tregs) but only detected on CD4(+)FoxP3(-) and CD8(+) T cells in acutely rejecting graft recipients. A blocking anti-TIM-3 mAb accelerated allograft rejection only in the presence of host CD4(+) T cells. Accelerated rejection was accompanied by increased frequencies of alloreactive IFN-γ-, IL-6-, and IL-17-producing splenocytes, enhanced CD8(+) cytotoxicity against alloantigen, increased alloantibody production, and a decline in peripheral and intragraft Treg/effector T cell ratio. Enhanced IL-6 production by CD4(+) T cells after TIM-3 blockade plays a central role in acceleration of rejection. Using an established alloreactivity TCR transgenic model, blockade of TIM-3 increased allospecific effector T cells, enhanced Th1 and Th17 polarization, and resulted in a decreased frequency of overall number of allospecific Tregs. The latter is due to inhibition in induction of adaptive Tregs rather than prevention of expansion of allospecific natural Tregs. In vitro, targeting TIM-3 did not inhibit nTreg-mediated suppression of Th1 alloreactive cells but increased IL-17 production by effector T cells. In summary, TIM-3 is a key regulatory molecule of alloimmunity through its ability to broadly modulate CD4(+) T cell differentiation, thus recalibrating the effector and regulatory arms of the alloimmune response.