Foxp3+-inducible regulatory T cells suppress endothelial activation and leukocyte recruitment

The ability of regulatory T cells (Treg) to traffic to sites of inflammation supports their role in controlling immune responses. This feature supports the idea that adoptive transfer of in vitro expanded human Treg could be used for treatment of immune/inflammatory diseases. However, the migratory behavior of Treg, as well as their direct influence at the site of inflammation, remains poorly understood. To explore the possibility that Treg may have direct anti-inflammatory influences on tissues, independent of their well-established suppressive effects on lymphocytes, we studied the adhesive interactions between mouse Treg and endothelial cells, as well as their influence on endothelial function during acute inflammation. We show that Foxp3(+) adaptive/inducible Treg (iTreg), but not naturally occurring Treg, efficiently interact with endothelial selectins and transmigrate through endothelial monolayers in vitro. In response to activation by endothelial Ag presentation or immobilized anti-CD3ε, Foxp3(+) iTreg suppressed TNF-α- and IL-1β-mediated endothelial selectin expression and adhesiveness to effector T cells. This suppression was contact independent, rapid acting, and mediated by TGF-β-induced activin receptor-like kinase 5 signaling in endothelial cells. In addition, Foxp3(+) iTreg adhered to inflamed endothelium in vivo, and their secretion products blocked acute inflammation in a model of peritonitis. These data support the concept that Foxp3(+) iTreg help to regulate inflammation independently of their influence on effector T cells by direct suppression of endothelial activation and leukocyte recruitment.     

The in vitro and in vivo effects of anti-galactose antibodies on endothelial cell activation and xenograft rejection

We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the alpha-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal(-/-) mice show a range of affinity for the alpha-Gal epitope, and that affinity was generally increased as the V(H) gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced alpha-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection.     

Nitric oxide modulates the expression of endothelial cell adhesion molecules involved in angiogenesis and leukocyte recruitment

Tumor angiogenesis and immune response have in common to be cell recognition mechanisms, which are based on specific adhesion molecules and dependent on nitric oxide (NO^•). The aim of the present study is to deepen the mechanisms of angiogenesis and inflammation regulation by NO^• to find out the molecular regulation processes that govern endothelial cell permeability and leukocyte transmigration.Effects of NO^•, either exogenous or produced in hypoxic conditions, were studied on microvascular endothelial cells from skin and lymph node because of their strong involvement in melanoma progression. We found that NO^• down-regulation of pseudo-vessel formation was linked to a decrease in endothelial cell ability to adhere to each other which can be explain, in part, by the inhibition of PECAM-1/CD31 expression. On the other hand, NO^• was shown to be able to decrease leukocyte adhesion on an endothelial monolayer, performed either in static or in rolling conditions, and to modulate differentially CD34, ICAM-1/CD54, ICAM-2/CD102 and VCAM-1/CD106 expression.In conclusion, during angiogenesis and leukocyte recruitment, NO^• regulates cell interactions by controlling adhesion molecule expression and subsequently cell adhesion. Moreover, each endothelial cell type presents its own organospecific response to NO^•, reflecting the functions of the tissue they originate from.     

The physiology of leukocyte recruitment: an in vivo perspective

The mechanisms of leukocyte recruitment have been studied extensively in vitro and have shed light on the basic molecular structure-function relationship of adhesion and signaling molecules involved in this essential immune response. This review will summarize how these in vitro observations extend to leukocyte behavior in inflamed blood vessels in the microcirculation. We highlight physiological results that might not have been predicted from in vitro systems. Special attention is placed on the physiology of rolling, adhesion, and intralumenal crawling in blood vessels. The importance of the glycocalyx, secondary tethers, shear, and the microenvironment are discussed. Docking structures forming rings of adhesion molecules together with a novel endothelial dome-like structure in vivo during transmigration are highlighted. Transcellular and paracellular emigration out of inflamed blood vessels is also discussed. The last section highlights leukocyte recruitment in some organs that do not always follow the accepted paradigm of leukocyte recruitment.     

Liver sinusoidal endothelial cells orchestrate NK cell recruitment and activation in acute inflammatory liver injury

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In vitro and in vivo expression of endothelial von Willebrand factor and leukocyte accumulation after fractionated irradiation

Previous investigations have demonstrated an increased release of von Willebrand factor (VWF; also known as vWF) in endothelial cells after high single-dose irradiation in vitro. We have also found increased levels of Vwf protein in mouse glomeruli after a high single dose of renal irradiation in vivo. In addition, increased numbers of leukocytes were observed in the renal cortex after irradiation in vivo. The aim of the present study was to investigate and quantify these biological processes after clinically relevant fractionated irradiation and to relate them to changes in renal function. A significantly greater increase in release of VWF was observed in cultured human umbilical vein endothelial cells (HUVECs) after fractionated irradiation (20 x 1.0 Gy) than after a single dose of 20 Gy (147% compared to 115% of control, respectively, P < 0.0005). In contrast with the in vitro observations, glomerular Vwf staining was lower after fractionated irradiation in vivo (20 x 2.0 Gy or 10 x 1.6 Gy +/- re-irradiation) than after a single dose of 16 Gy. The number of leukocytes accumulating in the renal cortex was also lower after fractionated in vivo irradiation than after a single radiation dose. The onset of these events preceded renal functional and histopathological changes by approximately 10 weeks. These data indicate that radiation-induced changes in endothelial VWF expression after in vivo irradiation may be distinct from the in vitro observations. Increased VWF expression may reflect pivotal processes in the pathogenesis of late radiation nephropathy and provide a clue to appropriate timing of pharmacological intervention.     

Exogenous stromal cell-derived factor-1 induces modest leukocyte recruitment in vivo

Stromal cell-derived factor-1 (SDF-1; CXCL12), a CXC chemokine, has been found to be involved in inflammation models in vivo and in cell adhesion, migration, and chemotaxis in vitro. This study aimed to determine whether exogenous SDF-1 induces leukocyte recruitment in mice. After systemic administration of SDF-1alpha, expression of the adhesion molecules P-selectin and VCAM-1 in mice was measured using a quantitative dual-radiolabeled Ab assay and leukocyte recruitment in various tissues was evaluated using intravital microscopy. The effect of local SDF-1alpha on leukocyte recruitment was also determined in cremaster muscle and compared with the effect of the cytokine TNFalpha and the CXC chemokine keratinocyte-derived chemokine (KC; CXCL1). Systemic administration of SDF-1alpha (10 microg, 4-5 h) induced upregulation of P-selectin, but not VCAM-1, in most tissues in mice. It caused modest leukocyte recruitment responses in microvasculature of cremaster muscle, intestine, and brain, i.e., an increase in flux of rolling leukocytes in cremaster muscle and intestines, leukocyte adhesion in all three tissues, and emigration in cremaster muscle. Local treatment with SDF-1alpha (1 microg, 4-5 h) reduced leukocyte rolling velocity and increased leukocyte adhesion and emigration in cremasteric venules, but the responses were much less profound than those elicited by KC or TNFalpha. SDF-1alpha-induced recruitment was dependent on endothelial P-selectin, but not P-selectin on platelets. We conclude that the exogenous SDF-1alpha enhances leukocyte-endothelial cell interactions and induces modest and endothelial P-selectin-dependent leukocyte recruitment.     

Adrenomedullin provokes endothelial Akt activation and promotes vascular regeneration both in vitro and in vivo

We previously reported that adrenomedullin (AM), a vasodilating hormone secreted from blood vessels, promotes proliferation and migration of human umbilical vein endothelial cells (HUVECs). In this study, we examined the ability of AM to promote vascular regeneration. AM increased the phosphorylation of Akt in HUVECs and the effect was inhibited by the AM antagonists and the inhibitors for protein kinase A (PKA) or phosphatidylinositol 3-kinase (PI3K). AM promoted re-endothelialization in vitro of wounded monolayer of HUVECs and neo-vascularization in vivo in murine gel plugs. These effects were also inhibited by the AM antagonists and the inhibitors for PKA or PI3K. The findings suggest that AM plays significant roles in vascular regeneration, associated with PKA- and PI3K-dependent activation of Akt in endothelial cells, and possesses therapeutic potential for vascular injury and tissue ischemia.     

Differential inhibition of polymorphonuclear leukocyte recruitment in vivo by dextran sulphate and fucoidan

The selectin-mediated rolling of leukocytes along the endothelial cells is a prerequisite step followed by firm adhesion and extravasation into the inflamed tissue. This initial contact can be suppressed by sulphated polysaccharides. We have studied the effect of sulphated polysaccharides on the ultimate polymorphonuclear leukocyte (PMN) recruitment and plasma leakage in rabbit skin in response to intradermal injection of various inflammatory mediators. PMN infiltration evoked by various PMN chemoattractants (FMLP, C5a desArg, LTB(4) and IL-8) was significantly inhibited after intravenous injection of dextran sulphate (25 mg/kg), heparin (2 x 90 mg/kg) or fucoidan (1 mg/kg). PMN-dependent plasma leakage was equally well reduced by the different sulphated polymers. Vascular permeability induced by histamine or thrombin acting via a PMN-independent mechanism was not reduced. Fucoidan was the only polysaccharide able to suppress IL-1-induced PMN infiltration for 60-70%. Local administration of dextran sulphate had no effect on PMN-dependent plasma leakage. Differential inhibition of PMN recruitment was determined after injection of dextran sulphate or fucoidan depending on the type of insult. Therefore, these results suggest that different adhesion pathways are utilized during PMN recruitment in vivo in response to chemoattractants and IL-1.     

In vitro endothelial cell activation and inflammatory responses in end-stage heart failure.

BACKGROUND: vascular endothelial cell activation and dysfunction are observed in patients with severe heart failure and may contribute to systemic manifestations of this syndrome. It remains unknown whether inflammatory activation of these cells occurs in these patients because of increased circulating proinflammatory mediators. Aim: to determine whether the serum from patients with heart failure possesses a net proinflammatory bioactivity to active proinflammatory pathways in cultured endothelial cells. METHODS: serum was obtained from stable patients with end-stage heart failure undergoing elective cardiac transplantation (Tx) and severely decompensated patients with heart failure requiring emergency left ventricular assist device (LVAD) implantation. Net proinflammatory bioactivity of serum was investigated by monitoring IkappaBalpha degradation and E-selectin expression in cultured human pulmonary artery endothelial cells (HPAEC) following incubation with serum samples. Serum cytokine concentrations were measured by ELISA and neutralizing antibodies were used to determine the role of specific factors in the observed bioactivity. RESULT: serum from both patient groups induced HPAEC IkappaBalpha degradation. Low basal HPAEC E-selectin expression significantly increased following treatment with Tx but not LVAD serum. Serum tumor necrosis factor-alpha (TNF-alpha) and IL-10 concentrations were higher in patients with LVAD than those with Tx, and soluble TNF-alpha receptor expression was high in both groups. Neither TNF-alpha nor IL-10 blocking experiments altered either bioassay result. CONCLUSION: activation of a specific profile of pro- and anti-inflammatory mediators is associated with heart failure resulting in HPAEC nuclear factor (NF)-kappaB activation. However, E-selectin expression is further regulated by unidentified factors. TNF-alpha is upregulated but appears to play no part in NFkappaB activation in these patients. These findings could have important therapeutic implications.     

Macrophage activation in vivo and in vitro

Morphology, lysosomal enzyme activity and phagocytic ability were tested in peritoneal macrophage cultures after stimulation in vivo or in vitro with endotoxin, mineral oil or latex particles, and compared to the same parameters in normal peritoneal macrophages. Treatment with latex did not give changes in the parameters tested after in vivo or in vitro stimulation. In all other types of stimulation the cells displayed varying degrees of spreading and changes in granule content. Extensive ruffling of cell membrane was obvious in endotoxin-stimulated cells. The pattern of lysosomal enzyme activity was complex and depended on the means of stimulation. Acid phosphatase showed the greatest increase after both in vivo and in vitro stimulation, N-acetyl-glucosaminidase could not be increased in vitro. Internalization of opsonized red cells mediated by the Fc receptor increased after in vivo stimulation. No such change was observed after in vitro stimulation. Normal peritoneal macrophages do not internalize significantly via the C₃ receptor. In vivo stimulation triggered the capacity to internalize up to 45% of the attached red cells. A similar reaction was obtained in vitro when both endotoxin and FCS were added to the culture medium, but not when endotoxin or FCS were used alone. We conclude that the use of the term activation of macrophages should always be based on quantitative changes in well defined parameters. Changes in one parameter will not necessarily be accompanied by the whole range of biochemical and morphological perturbations. The capacity to ingest via the C₃ receptor may be the most useful parameter.     

A functional role for circulating mouse L-selectin in regulating leukocyte/endothelial cell interactions in vivo

L-selectin mediates the initial capture and subsequent rolling of leukocytes along inflamed vascular endothelium and mediates lymphocyte migration to peripheral lymphoid tissues. Leukocyte activation induces rapid endoproteolytic cleavage of L-selectin from the cell surface, generating soluble L-selectin (sL-selectin). Because human sL-selectin retains ligand-binding activity in vitro, mouse sL-selectin and its in vivo relevance were characterized. Comparable with humans, sL-selectin was present in adult C57BL/6 mouse sera at approximately 1.7 micro g/ml. Similar levels of sL-selectin were present in sera from multiple mouse strains, despite their pronounced differences in cell surface L-selectin expression levels. Adhesion molecule-deficient mice prone to spontaneous chronic inflammation and mice suffering from leukemia/lymphoma had 2.5- and 20-fold increased serum sL-selectin levels, respectively. By contrast, serum sL-selectin levels were reduced by 70% in Rag-deficient mice lacking mature lymphocytes. The majority of serum sL-selectin had a molecular mass of 65-75 kDa, consistent with its lymphocyte origin. Slow turnover may explain the relatively high levels of sL-selectin in vivo. The t(1/2) of sL-selectin, assessed by transferring sera from wild-type mice into L-selectin-deficient mice and monitoring serum sL-selectin levels by ELISA, was >20 h, and it remained detectable for longer than 1 wk. Short-term in vivo lymphocyte migration assays demonstrated that near physiologic levels ( approximately 0.9 micro g/ml) of sL-selectin decreased lymphocyte migration to peripheral lymph nodes by >30%, with dose-dependent inhibition occurring with increasing sL-selectin concentrations. These results suggest that sL-selectin influences lymphocyte migration in vivo and that the increased sL-selectin levels present in certain pathologic conditions may adversely affect leukocyte migration.     

IL-10 inhibits vascular smooth muscle cell activation in vitro and in vivo

The anti-inflammatory cytokine IL-10 inhibits intimal hyperplasia after stent implantation via a powerful inactivation of monocytes. We tested the hypothesis that IL-10 may also inhibit vascular smooth muscle cell (SMC) activation via the inhibition of the NF-kappaB/I-kappaB system. The IL-10 receptor was detected in rat SMCs in vitro and in vivo. In LPS-stimulated rat SMCs, 1 ng/ml recombinant murine IL-10 (mIL-10) reduced I-kappaBalpha and I-kappaBbeta degradation, NF-kappaB activation, as well as the expression of the NF-kappaB-dependent gene IL-6 by 32%, 31%, 75%, and 19%, respectively (P < 0.05 for all). Similar results were obtained in vivo 6 h and 4 days after balloon abrasion of the rat aorta, a model in which intimal hyperplasia results essentially from SMC activation. Moreover, mIL-10 reduced SMC proliferation and migration in vitro (by 60% for both, P < 0.0001), resulting in reduced SMC proliferation and intimal growth 14 days after balloon abrasion of the rat aorta (by 76% and 75%, respectively; P < 0.005). In conclusion, mIL-10 has a direct inhibitory effect on SMCs in vitro and in vivo. This effect is mediated in part by NF-kappaB inactivation and may participate in the overall protective effect of IL-10 on postangioplasty restenosis.     

Inhibition of in vitro T cell activation by corneal endothelial cells.

Cells and tissues of the anterior uvea and aqueous humor express activities which inhibit immune responses. These activities include soluble factors such as TGF-beta and uncharacterized cell surface interactions. Relatively little is known regarding the immunologic activities of corneal endothelium, despite its potentially important role in contributing to the immune privilege of the anterior chamber and the high success rate of corneal transplantation. In this report, in vitro studies of cultured rat corneal endothelial (CE) cells were done using S-antigen-specific LEW rat T cell lines, or S-antigen-specific T cell hybridomas, to examine the immunologic capabilities of CE cells. Monolayers of LEW rat CE cells were unable to present antigen or a mitogen, Con A, to T cell lines or hybridomas as assessed by the lack of a proliferative response or IL-2 secretion. Furthermore, the CE cells exerted a potent inhibitory effect when added to in vitro proliferation assays of T cell lines stimulated with antigen or Con A. When T cells were preactivated on conventional antigen presenting cells and then transferred to wells containing CE cells, their proliferation was not inhibited. Although CE cells inhibited activation of T cell lines and hybridomas, they did not inhibit the growth of T cell hybridomas or CTLL cells, nor did the CE cells adversely affect the viability of resting T cells cultured on CE monolayers. The inhibitory effect was reversible as preincubation of T cells on CE cells for up to 6 days followed by washes restored T cell responsiveness when assayed on splenocytes. The inability to stimulate proliferative responses was not affected by preincubation of the CE cells with lymphokines which increase MHC antigen expression. The inhibition observed in these assays was not MHC-restricted as CE cells from both LEW and BN rats were equally inhibitory. CE cells from rabbits and cats were also potent inhibitors of T cell activation, suggesting that the mechanism is evolutionarily conserved. The mechanism of inhibition of CE cells is unknown at this time.     

Thrombin and leukocyte recruitment in endotoxemia

Because thrombin has been implicated in sepsis, it has been proposed that antithrombin III (AT III) is beneficial due to its anticoagulatory and antiadhesive effects. Using intravital microscopy, we visualized leukocyte-endothelium interactions in postcapillary venules of the feline mesentery exposed to lipopolysaccharide (LPS). At a concentration of AT III that blocks leukocyte adhesion in postischemic mesentery, we found no role for thrombin in LPS-induced rolling, adhesion and emigration, or microvascular dysfunction. Furthermore, AT III did not attenuate leukocyte-endothelial interactions after tumor necrosis factor-alpha superfusion of the mesentery. In contrast, fucoidan, a selectin inhibitor, prevented almost all LPS-induced rolling and reduced adhesion, emigration, and microvascular dysfunction. In a model of endotoxemia, leukocyte recruitment into mesentery or lungs was unaffected by AT III. Finally, in a human cell system that mimics the flow conditions in vivo, human neutrophils rolled, adhered, and emigrated similar to the feline postcapillary microvessels, and AT III had no effect on leukocyte recruitment induced by LPS. If AT III has beneficial effects in endotoxemia, it is not due to a direct effect upon leukocyte rolling, adhesion, or emigration in postcapillary venules in vivo.     

In vitro activation of leukocyte glycogen synthetase

The activity of leukocyte glycogen synthetase in a freshly prepared homogenate is almost completely in the $\text{b}$ form. Incubation of the homogenate at 30°C caused a time dependent increase in the activity measured in the absence of G-6-P ($\text{b}$ to $\text{a}$ conversion). The K_a for G-6-P decreased from 0.7 to 0.01 mM. Freezing of the homogenate resulted in a complete loss of the capacity for activation. These results demonstrate that glycogen synthetase from leukocytes of normal human subjects can be converted $\text{in vitro}$ to a form, which is almost independent of G-6-P for activity.     

Canstatin-N fragment inhibits in vitro endothelial cell proliferation and suppresses in vivo tumor growth

Type IV collagen is one of the components of vascular basement involved in regulation of angiogenesis. Canstatin, the non-collagenous 1 (NC1) domain of alpha2 chain of type IV collagen, was identified as an inhibitor of angiogenesis and tumor growth by Kamphaus et al. Our previous studies showed that canstatin-N, the N-terminal 1-89 amino acid fragment of canstatin, inhibited the neovascularization in a dose-dependent manner as tested by CAM assay. In the present study, we demonstrate that canstatin-N produced in Escherichia coli specifically inhibited in vitro the proliferation of human umbilical vein endothelial cells (ECV304) and significantly induced apoptosis. The apoptosis-inducing activity of canstatin-N was much stronger than that of canstatin, indicating that the apoptosis-inducing activity of canstatin is likely located within its N-terminal 1-89 amino acid fragment. Canstatin-N also suppressed in vivo growth of B(16) murine melanoma in BALB/c mice at a dosage of 10mg/kg/day. These results suggest that canstatin-N is a useful candidate molecule for inhibition of tumor growth.