Synergistic effect of TNF-alpha in soluble VCAM-1-induced angiogenesis through alpha 4 integrins

In our present study we focused on soluble VCAM-1 (sVCAM-1)/alpha(4) integrin-induced angiogenesis and found that this type of angiogenesis was mediated through p38 mitogen-activated protein kinase and focal adhesion kinase (FAK). HUVEC expressed both alpha(4) and beta(1) integrins, and it was reported that expression of alpha(4) integrin and its counterreceptor, sVCAM-1/VCAM-1, was enhanced in response to an inflammatory cytokine, TNF-alpha. In endothelial cells phosphorylation of p38 and FAK, but not that of extracellular signal-regulated kinase 1/2 was induced by sVCAM-1. Migration of endothelial cells was stimulated in response to sVCAM-1 at similar levels as those induced by vascular endothelial growth factor, and sVCAM-1-induced migration was almost completely blocked by neutralizing Ab against alpha(4) integrin, by either an inhibitor of p38 (SB203580), or by adenovirus containing FAK-related nonkinase. sVCAM-1 also induced the formation of blood vessels in Matrigel plug assay in vivo, and this neovascularization was blocked by SB203580 or neutralizing Ab against alpha(4) integrin. Moreover, we also confirmed that both TNF-alpha and sVCAM-1 could synergistically induce angiogenesis in the corneas of mice when each factor at used dose could not induce. This angiogenesis by TNF-alpha and sVCAM-1 was almost completely blocked by coadministration of SB203580 and also by neutralizing Ab against alpha(4) integrin. These results suggest that sVCAM-1/alpha(4) integrin induces angiogenesis through p38 and FAK signaling pathways.     

Molecular mechanisms underlying IL-4-induced leukocyte recruitment in vivo: a critical role for the alpha 4 integrin.

IL-4 is known to induce recruitment of eosinophils and mononuclear leukocytes. In vitro this occurs in part by selective expression of VCAM-1, the ligand for the alpha 4 integrin. The objective of this study was to determine the molecular mechanisms that underlie IL-4-induced leukocyte recruitment in vivo. Mice received an intrascrotal injection of IL-4 (100 ng). Twenty-four hours later, leukocyte rolling, adhesion, and emigration in cremasteric postcapillary venules were examined via intravital microscopy, and expression of VCAM-1 and P- and E-selectin was quantitated using a radiolabeled mAb technique. IL-4 increased VCAM-1 expression, but P-selectin and E-selectin remained at constitutive levels. IL-4 induced significant increases in leukocyte adhesion and emigration, with 50% of the emigrated cells being eosinophils and the remainder being mononuclear leukocytes. Leukocyte rolling in IL-4-treated mice was >95% inhibitable using an anti-P-selectin Ab. However, IL-4-induced leukocyte recruitment was unaltered in mice treated chronically with P-selectin Ab or mice deficient in either P-selectin or P- and E-selectin, suggesting that the residual rolling supported all of the IL-4-induced recruitment. In IL-4-treated mice following P-selectin blockade, tethering and rolling were not dependent on L-selectin, but were abolished by alpha 4 integrin blockade. These findings show that the alpha 4 integrin can initiate leukocyte-endothelial cell interactions in the absence of selectins under shear conditions in vivo, and that the absence of selectins does not affect recruitment of eosinophils and mononuclear cells to IL-4-treated tissue.     

Alpha 4 integrin-dependent leukocyte recruitment does not require VCAM-1 in a chronic model of inflammation

Rats immunized with Mycobacterium butyricum in Freund's adjuvant develop a chronic vasculitis, with large increases in leukocyte rolling and adhesion in mesenteric postcapillary venules that are significantly inhibited with an alpha 4 integrin Ab. Using intravital microscopy to visualize chronically inflamed microvessels, we demonstrated that alpha 4 integrin-dependent leukocyte rolling and adhesion was inhibited with a beta 1 integrin, but not a beta 7 integrin Ab. To date, VCAM-1 has been presumed to be the primary ligand for alpha 4 beta 1 integrin in the vasculature. However, alpha 4 beta 1 integrin-dependent interactions were not reduced by monoclonal or polyclonal VCAM-1 Abs or a VCAM-1 antisense oligonucleotide despite increased VCAM-1 expression in the mesenteric vasculature. To ensure that the VCAM-1 Abs were functional and used at saturating concentrations, blood from Ab-treated rats was perfused over monolayers of CHO cells transfected with rat VCAM-1. Sufficient alpha 4 integrin or VCAM-1 Ab was present to inhibit leukocyte interactions with rat VCAM-1 by 95-100%. Under in vitro flow conditions, only mononuclear leukocytes were recruited from blood of control rats onto purified VCAM-1. However, neutrophils were also recruited onto VCAM-1 from whole blood of adjuvant-immunized animals via alpha 4 integrin. Another ligand for alpha 4 beta 1 integrin is the connecting segment-1 (CS-1) region of fibronectin. An Ab to the CS-1 portion of fibronectin, which did not reduce rolling and adhesion in adjuvant arthritis animals, completely inhibited leukocyte adhesion to CS-1 under static conditions. These findings provide the first evidence that alpha 4 beta 1 integrin-dependent leukocyte rolling and adhesion can occur in vivo via a mechanism other than VCAM-1.     

Evidence of IL-18 as a novel angiogenic mediator

Angiogenesis, or new blood vessel growth, is a key process in the development of synovial inflammation in rheumatoid arthritis (RA). Integral to this pathologic proliferation are proinflammatory cytokines. We hypothesized a role for IL-18 as an angiogenic mediator in RA. We examined the effect of human IL-18 on human microvascular endothelial cell (HMVEC) migration. IL-18 induced HMVEC migration at 1 nM (p < 0.05). RA synovial fluids potently induced endothelial cell migration, but IL-18 immunodepletion resulted in a 68 +/- 5% decrease in HMVEC migration (p < 0.05). IL-18 appears to act on HMVECs via alpha(v)beta(3) integrin. To test whether IL-18 induced endothelial cell tube formation in vitro, we quantitated the degree of tube formation on Matrigel matrix. IL-18, 1 or 10 nM, resulted in a 77% or 87% increase in tube formation compared with control (p < 0.05). To determine whether IL-18 may be angiogenic in vivo, we implanted IL-18 in Matrigel plugs in mice, and IL-18 at 1 and 10 nM induced angiogenesis (p < 0.05). The angiogenesis observed appears to be independent of the contribution of local TNF-alpha, as evidenced by adding neutralizing anti-TNF-alpha Ab to the Matrigel plugs. In an alternative in vivo model, sponges embedded with IL-18 or control were implanted into mice. IL-18 (10 nM) induced a 4-fold increase in angiogenesis vs the control (p < 0.05). These findings support a novel function for IL-18 as an angiogenic factor in RA and may elucidate a potential therapeutic target for angiogenesis-directed diseases.     

Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis.

Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.     

TNF-alpha and IL-1 sequentially induce endothelial ICAM-1 and VCAM-1 expression in MRL/lpr lupus-prone mice.

Dysfunctional leukocyte-endothelial interactions are thought to play a key role in systemic lupus erythematosus pathogenesis. We questioned the importance of TNF-alpha and IL-1 for endothelial activation in MRL/lpr lupus-prone mice. Endothelial ICAM-1 and VCAM-1 expression increased significantly with disease evolution in kidney, heart, and brain, as shown by i.v. injected radiolabeled Ab uptake. Lung endothelial VCAM-1 also increased, while lung endothelial ICAM-1 did not rise above a high basal level. Immunoassays showed a significantly raised circulating level of TNF-alpha by 14 wk, with levels of circulating IL-1alpha and IL-1beta being additionally raised by 20 wk. With 14-wk-old MRL/lpr, anti-TNF-alpha antiserum inhibited expression of ICAM-1 and VCAM-1 by endothelial cells cultured with sera in vitro, and uptake of anti-ICAM-1 and anti-VCAM-1 mAb in lung, kidney, brain, and heart in vivo. In contrast, both anti-TNF-alpha and anti-IL-1 antisera were required for maximal inhibition in vitro and in vivo at 20 wk. These data indicate that TNF-alpha is largely responsible for the early up-regulation of endothelial ICAM-1 and VCAM-1, but that IL-1 enhances expression in late disease. Our observations provide novel insights of possible relevance to understanding endothelial activation in systemic lupus erythematosus, and highlight an approach that can be extended to dissecting other chronic inflammatory diseases.     

Regulation of integrin alpha vbeta 3-mediated endothelial cell migration and angiogenesis by integrin alpha5beta1 and protein kinase A

Recent studies indicate that angiogenesis depends, in part, on ligation of integrin alpha(5)beta(1) by fibronectin. Evidence is now provided that integrin alpha(5)beta(1) regulates the function of integrin alpha(v)beta(3) on endothelial cells during their migration in vitro or angiogenesis in vivo. Secretion of fibronectin by endothelial cells leads to the ligation of integrin alpha(5)beta(1), which potentiates alpha(v)beta(3)-mediated migration on vitronectin without influencing alpha(v)beta(3)-mediated cell adhesion. Endothelial cell attachment to vitronectin suppresses protein kinase A (PKA) activity, while addition of soluble anti-alpha(5)beta(1) restores this activity. Moreover, agents that activate intracellular PKA, such as forskolin, dibutyryl cAMP or alpha(5)beta(1) antagonists, suppress endothelial cell migration on vitronectin in vitro or angiogenesis in vivo. In contrast, inhibitors of PKA reverse the anti-migratory or anti-angiogenic effects mediated by alpha(5)beta(1) antagonists. Therefore, alpha(v)beta(3)-mediated endothelial cell migration and angiogenesis can be regulated by PKA activity, which depends on the ligation state of integrin alpha(5)beta(1).     

IL-12 inhibition of endothelial cell functions and angiogenesis depends on lymphocyte-endothelial cell cross-talk

In vivo IL-12-dependent tumor inhibition rests on the ability of IL-12 to activate a CD8-mediated cytotoxicity, inhibit angiogenesis, and cause vascular injury. Although in vivo studies have shown that such inhibition stems from complex interactions of immune cells and the production of IFN-gamma and other downstream angiostatic chemokines, the mechanisms involved are still poorly defined. Here we show that IL-12 activates an anti-angiogenic program in Con A-activated mouse spleen cells (activated spc) or human PBMC (activated PBMC). The soluble factors they release in its presence arrest the cycle of endothelial cells (EC), inhibit in vitro angiogenesis, negatively modulate the production of matrix metalloproteinase-9, and the ability of EC to adhere to vitronectin and up-regulate ICAM-1 and VCAM-1 expression. These effects do not require direct cell-cell contact, yet result from continuous interaction between activated lymphoid cells and EC. We used neutralizing Abs to show that the IFN-inducible protein-10 and monokine-induced by IFN-gamma chemokines are pivotal in inducing these effects. Experiments with nu/nu mice, nonobese diabetic-SCID mice, or activated spc enriched in specific cell subpopulations demonstrated that CD4(+), CD8(+), and NK cells are all needed to mediate the full anti-angiogenetic effect of IL-12.     

IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium. Association with expression of VCAM-1.

The present studies were performed to explore potentially selective mechanisms of leukocyte adhesion in an attempt to understand how preferential recruitment of eosinophils and basophils might occur during allergic and other inflammatory reactions. Stimulation of human vascular endothelial cells for 24 h with IL-4 (30 to 1,000 U/ml) induced adhesion for eosinophils (up to approximately four-fold of control) and basophils (up to approximately twofold of control) but not neutrophils (less than 125% of control). Analysis of endothelial expression of adhesion molecules by flow cytometry revealed that IL-4 treatment induced vascular cell adhesion molecule-1 (VCAM-1) expression without significantly affecting the expression of other adhesion molecules, namely endothelial-leukocyte adhesion molecule-1 (ELAM-1) or intercellular adhesion molecule-1 (ICAM-1). The concentration-response curve for IL-4-induced VCAM-1 expression paralleled that for adhesion. Endothelial cells stimulated with IL-4 expressed adhesive properties for eosinophils by 3 h; the response increased steadily during a 24-h time course study. Eosinophils and basophils adhered to plates coated with a recombinant form of VCAM-1. This adhesion was blocked with antibodies to VCAM-1 but not ELAM-1. mAb directed against either VCAM-1 or VLA-4 inhibited (by approximately 75%) the binding of eosinophils and basophils to IL-4-stimulated endothelial cells. Because VLA-4 and VCAM-1 have been demonstrated to bind to each other in other adhesion systems, these results suggest that IL-4 stimulates eosinophil and basophil adhesion by inducing endothelial cell expression of VCAM-1 which binds to eosinophil and basophil VLA-4. The lack of expression of VLA-4 on neutrophils and the failure of IL-4 to stimulate neutrophil adherence support this conclusion. It is proposed that local release of IL-4 in vivo in allergic diseases or after experimental allergen challenge may partly explain the enrichment of eosinophils and basophils (vs neutrophils) observed in these situations.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.     

Induction of cardiac angiogenesis requires killer cell lectin-like receptor 1 and α4β7 integrin expression by NK cells

Recent findings indicate that NK cells are involved in cardiac repair following myocardial infarction. The aim of this study is to investigate the role NK cells in infarct angiogenesis and cardiac remodeling. In normal C57BL/6 mice, myelomonocytic inflammatory cells invaded infarcted heart within 24 h followed by a lymphoid/NK cell infiltrate by day 6, accompanied by substantial expression of IL-2, TNF-α, and CCL2. In contrast, NOD SCID mice had virtually no lymphoid cells infiltrating the heart and did not upregulate IL-2 levels. In vitro and in vivo, IL-2-activated NK cells promoted TNF-α-stimulated endothelial cell proliferation, enhanced angiogenesis and reduced fibrosis within the infarcted myocardium. Adoptive transfer of IL-2-activated NK cells to NOD SCID mice improved post-myocardial infarction angiogenesis. RNA silencing technology and neutralizing Abs demonstrated that this process involved α4β7 integrin/VCAM-1 and killer cell lectin-like receptor 1/N-cadherin-specific binding. In this study, we show that IL-2-activated NK cells reduce myocardial collagen deposition along with an increase in neovascularization following acute cardiac ischemia through specific interaction with endothelial cells. These data define a potential role of activated NK cells in cardiac angiogenesis and open new perspectives for the treatment of ischemic diseases.     

Regulation of found in inflammatory zone 1 expression in bleomycin-induced lung fibrosis: role of IL-4/IL-13 and mediation via STAT-6

Found in inflammatory zone (FIZZ)1, also known as resistin-like molecule alpha, belongs to a novel class of cysteine-rich secreted protein family, named FIZZ/resistin-like molecule, with unique tissue expression patterns. FIZZ1 is induced in alveolar type II epithelial cells (AECs) in bleomycin (BLM)-induced lung fibrosis, and found to induce myofibroblast differentiation in vitro. The objective of this study was to elucidate the regulation of AEC FIZZ1 expression in pulmonary fibrosis. AECs were isolated from rat lungs and the effects of a number of cytokines on FIZZ1 expression were evaluated by RT-PCR. Of all cytokines examined, only IL-4 and IL-13 were effective in stimulating FIZZ1 expression in AECs. Stimulation by IL-4/IL-13 was accompanied by increases in phosphorylated STAT6 and JAK1. FIZZ1 expression was also stimulated by transfection with a STAT6 expression plasmid, but was inhibited by antisense oligonucleotides directed against STAT6. In vivo studies showed that compared with wild-type controls, both IL-4- and IL-13-deficient mice showed reduced BLM-induced lung FIZZ1 expression and fibrosis, which were essentially abolished in IL-4 and IL-13 doubly deficient mice. Furthermore, STAT6-deficient mice showed marked reduction in BLM-induced lung FIZZ1 expression. Thus, IL-4 and IL-13 are potent inducers of AEC FIZZ1 expression via STAT6 and play key roles in BLM-induced lung FIZZ1 expression and fibrosis. This represents a potential mechanism by which IL-4/IL-13 could play a role in the pathogenesis of lung fibrosis.     

VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site.

Cytokine-activated human endothelial cells express vascular cell adhesion molecule-1 (VCAM-1), which binds lymphocytes. We now identify the integrin VLA-4 as a receptor for VCAM-1 because VLA-4 surface expression on K-562 cells (following transfection of the VLA alpha 4 subunit cDNA) resulted in specific cell adhesion to VCAM-1, and anti-VLA-4 antibodies completely inhibited VCAM-1-dependent cell-cell attachment. In addition, VLA-4 expression allowed K-562 cells to attach to the heparin II binding region (FN-40) of fibronectin. However, VLA-4/VCAM-1 and VLA-4/FN-40 interactions are readily distinguishable: only the former was inhibited by the anti-VLA-4 monoclonal antibody HP1/3, and only the latter was inhibited by soluble FN-40. The VCAM-1/VLA-4 ligand-receptor pair may play a major role in the recruitment of mononuclear leukocytes to inflammatory sites in vivo.     

Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.     

Endostatin inhibits adhesion of endothelial cells to collagen I via alpha(2)beta(1) integrin, a possible cause of prevention of chondrosarcoma growth

Endostatin derived from collagen XVIII is a potent endogenous anti-angiogenic factor that induces regression of various tumors of epithelial origin. Endostatin has been shown to inhibit endothelial cell functions, however, its effect remains controversial. We first attempted here to apply the inhibitory effect of recombinant human endostatin on chondrosarcomas, which originate from the mesenchyme, in nude mice. Endostatin induced reduction of chondrosarcoma growth and tumor angiogenesis in vivo. However, endostatin showed no effect on the proliferation and migration of chondrosarcoma cells in vitro. Next, we investigated the interactions between endostatin and endothelial cells in detail. Endostatin inhibited the migration on and attachment to collagen I but did not affect the proliferation of endothelial cells. Although the migration of endothelial cells was stimulated by angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor, endostatin showed similar inhibitory effects on it in the presence and absence of the stimulants. Moreover, the inhibitory effect against endothelial cell attachment to collagen I was attenuated or modulated in the presence of neutralizing antibodies of alpha(2), alpha(5)beta(1), and alpha(V)beta(3) integrins but not that of alpha(1) integrin. Our results suggest that endostatin might suppress the alpha(2)beta(1) integrin function of endothelial cells via alpha(5)beta(1) or alpha(V)beta(3) integrin. We propose here that endostatin might be effective for anti-angiogenic therapy for human chondrosarcomas through the suppression of alpha(2)beta(1) integrin functions in endothelial cells.     

Application of plasmid DNA encoding IL-18 diminishes development of herpetic stromal keratitis by antiangiogenic effects

HSV-1 infection of the eye can cause a blinding immunoinflammatory stromal keratitis (SK) lesion. Using the mouse model, we have demonstrated that angiogenesis is an essential step in lesion pathogenesis because its inhibition results in diminished severity. The molecules involved in causing corneal angiogenesis are multiple and include the vascular endothelial growth factor (VEGF) family of proteins. In this report we show that application of plasmid DNA encoding IL-18 to the cornea of mice before HSV-1 ocular infection resulted in reduced angiogenesis and diminished SK immunoinflammatory lesions. The antiangiogenic effects of IL-18 treatment appeared to be mediated by inhibition of VEGF production in the cornea. We also showed that IL-18 controlled VEGF expression in vitro and also decreased CpG oligodeoxynucleotide induced VEGF-dependent neovascularization. In addition the administration of IL-18-binding protein, an IL-18 antagonist, into the inflammatory eye resulted in elevated angiogenesis and increased VEGF expression. Our results indicate that IL-18 is an important endogenous negative regulator of HSV-induced angiogenesis resulting in reduced SK lesion severity. Our results could mean that IL-18 administration may represent a useful approach to manage unwanted angiogenesis.     

Invariant NKT cells biased for IL-5 production act as crucial regulators of inflammation

Although invariant NKT (iNKT) cells play a regulatory role in the pathogenesis of autoimmune diseases and allergy, an initial trigger for their regulatory responses remains elusive. In this study, we report that a proportion of human CD4+ iNKT cell clones produce enormous amounts of IL-5 and IL-13 when cocultured with CD1d+ APC in the presence of IL-2. Such IL-5 bias was never observed when we stimulated the same clones with alpha-galactosylceramide or anti-CD3 Ab. Suboptimal TCR stimulation by plate-bound anti-CD3 Ab was found to mimic the effect of CD1d+ APC, indicating the role of TCR signaling for selective induction of IL-5. Interestingly, DNA microarray analysis identified IL-5 and IL-13 as the most highly up-regulated genes, whereas other cytokines produced by iNKT cells, such as IL-4 and IL-10, were not significantly induced. Moreover, iNKT cells from BALB/c mice showed similar IL-5 responses after stimulation with IL-2 ex vivo or in vivo. The iNKT cell subset producing IL-5 and IL-13 could play a major role in the development of allergic disease or asthma and also in the immune regulation of Th1 inflammation.     

Tumor cell surface alpha 4 beta 1 integrin mediates adhesion to vascular endothelium: demonstration of an interaction with the N-terminal domains of INCAM-110/VCAM-1.

Hematogenous metastasis involves adhesive interactions between blood-borne tumor cells and the vessel wall. By the use of in vitro assays, the adhesion of human melanoma, osteosarcoma, and kidney carcinoma (but not colon carcinoma) cell lines was shown to involve the cytokine-inducible endothelial cell surface protein inducible cell adhesion molecule 110 (INCAM-110) and the alpha 4 beta 1 integrin, molecules normally involved in endothelial-leukocyte interactions. Tumor adhesion to human endothelial cell monolayers was increased 1.9- to 8.2-fold by endothelial activation with the cytokine tumor necrosis factor (TNF) and inhibited by the anti-INCAM-110 monoclonal antibody (mAb) E1/6. Each of these tumor cells expressed members of the beta 1 integrin family of adhesion molecules, and antibodies to the alpha 4 and beta 1 integrin subunits inhibited tumor-endothelial adhesion (48-87% inhibition). A cDNA encompassing the three N-terminal Ig-like domains of vascular cell adhesion molecule 1 (VCAM-1) encoded a protein recognized by the anti-INCAM-110 mAb E1/6 and, when captured onto plastic, supported melanoma cell adhesion by an alpha 4 integrin-dependent mechanism. In contrast to mAb E1/6, a second anti-INCAM-110 mAb Hu8/4 neither inhibited adhesion to activated endothelium nor bound the first three Ig-like domains of INCAM-110/VCAM-1. These data indicate that the adherence of several human tumors to activated endothelium is mediated by an interaction of alpha 4 beta 1 integrin and the N-terminal Ig-like domains of endothelial INCAM-110/VCAM-1. Tumor acquisition of the alpha 4 integrin subunit and endothelial expression of INCAM-110 may affect the frequency and distribution of metastasis.     

A monoclonal antibody to beta 1 integrin (CD29) stimulates VLA- dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components

The leukocyte beta 1 integrin receptor very late activation antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) binds to vascular cell adhesion molecule-1 (VCAM-1) expressed on cytokine-activated endothelium. A mAb designated 8A2 was identified that stimulated the binding of U937 cells to CHO cells transfected with VCAM-1 cDNA but not endothelial-leukocyte adhesion molecule or CD4 cDNA. mAb 8A2 also rapidly stimulated the adherence of peripheral blood lymphocytes (PBLs) to VCAM-1-transfected CHO cells or recombinant human tumor necrosis factor-treated human umbilical vein endothelial cells. mAb 8A2-stimulated binding of PBL was inhibited by mAbs to VLA-4 or VCAM-1. Surface expression of VLA-4 was not altered by mAb 8A2 treatment and monovalent Fab fragments of mAb 8A2 were active. Immunoprecipitation studies reveal that mAb 8A2 recognizes beta 1-subunit (CD29) of integrin receptors. In contrast to mAbs directed to VLA-4 alpha-subunit (alpha 4, CD49d), mAb 8A2 did not induce homotypic aggregation of PBL. Additionally, mAb 8A2 stimulated adherence of PBL and hematopoietic cell lines to purified matrix components laminin and fibronectin. This binding was blocked by mAbs to the VLA alpha-subunits alpha 6 (CD49f), or alpha 5 (CD49e) and alpha 4 (CD49d), respectively. We conclude that mAb 8A2 modulates the affinity of VLA-4 and other leukocyte beta 1 integrins, and should prove useful in studying the regulation of beta 1 integrin function.