Transendothelial Migration In Response Research Articles

Abstract 6763: A human-relevant vascularized organ-on-a chip platform for anti-tumor drug efficacy and liver injury assessment

Abstract Cancer global burden accounts for 19.3 million new cases and 10 million deaths in 2020. Despite advances on the understanding of disease progression and efforts to find successful therapeutic strategies, most drugs fail in clinical stages. Lack of models with clinical mimicry recapitulating the in vivo pathophysiology and the organ microenvironment where the tumor forms is a key driver reducing the success rate of anti-cancer drugs. Animal models remain the gold standard for preclinical research but fail to recapitulate the complex human tumor microenvironment, a critical component determining tumor development, progression, and response to treatments. Organ-on-a-chip models allow to control the environment and organization of cells in an in vivo-like arrangement by culturing multiple cell types to better replicate human conditions, and by implementing different biochemical and physical stimuli, such as fluid shear stress, chemical/nutrient gradients, and perfusion/flow to recapitulate the human vascular system physiology. We leverage our 3D bioprinting technologies to develop complex vascularized organ-on-a-chip models aimed to recapitulate the tumor and liver niche. Both organ-systems are interconnected by our h-VIOS™ platform, allowing dynamic perfusion for 21 days and enabling the simultaneous assessment of anti-tumor therapeutic efficacy and drug-induced liver injury. Our hydrogels feature a complex perfusable vasculature that interfaces with an interstitial chamber where tissues of interest are grown. The vasculature allows for continued and controlled delivery of nutrients, oxygen, and small molecule drugs. Dextran diffusion studies revealed diffusion coefficients ranging from 43.2 μm2/s (4kDa) to 8 μm2/s (70kDa). Vascular architectures are lined with endothelial cells forming a functional barrier expressing the junctional markers ZO-1 and PECAM-1. Moreover, we can modulate the permeability of our vasculature architecture, allowing the extravasation of immune and cancer cells. PBMC-derived T cells and Jurkat cells undergo transendothelial migration in response to a CXCL12 gradient, highlighting the applicability of our model for immunotherapeutic efficacy studies. The tumor interstitial chamber incorporates co-culture of cancer spheroids or tumoroids, fibroblasts, endothelial and immune cells. Colon carcinoma spheroids effectively integrate into the tumor niche with no growth arrest. The liver model consists of an interstitial chamber with a lobular microstructure supporting the monoculture of primary human hepatocytes, and sustaining their viability and function for 9 days as demonstrated by high albumin production, urea synthesis and CYP activity. Our organ-on-a-chip models are fully customizable and anticipated to contribute to advance personalized medicine and improve clinical translation rates. Citation Format: Juliana Navarro-Yepes, Queeny Dasgupta, Purboja Purkayastha, Kevin Janson, Cassio Mello, Ameya Narkar, Soon Seng Ng, Taci Pereira. A human-relevant vascularized organ-on-a chip platform for anti-tumor drug efficacy and liver injury assessment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6763.

Apremilast effectively inhibits TNFα-induced vascular inflammation in human endothelial cells.

Patients with chronic inflammatory diseases (e.g. psoriasis and rheumatoid arthritis) are at increased risk for the development of atherosclerosis and cardiovascular diseases (CVD). Previous studies have suggested that phosphodiesterase 4 (PDE4) inhibitors possess anti-inflammatory properties. Here we examined the effect of the PDE4 inhibitor apremilast, a well-established anti-psoriatic drug, on pro-inflammatory responses in TNFα-activated endothelial cells. Human umbilical vein endothelial cells (HUVEC) were treated with tumour necrosis factor-α (TNFα) in the presence or absence of apremilast. Expression levels of pro-inflammatory cytokines, chemokines and adhesion molecules were assessed by ELISA, western blot and RT-PCR. Effects of apremilast on adhesion and transendothelial migration (TEM) of THP-1 monocytic cells were analysed in transwell assays. Apremilast suppressed TNFα-induced expression and secretion of important endothelial and monocytic pro-inflammatory factors, including granulocyte-macrophage colony-stimulating factor (GM-CSF), C-X-C motif chemokine ligand 10 (CXCL10), chemokine (C-C motif) ligand 2 (CCL2), vascular cell adhesion molecule 1 (VCAM-1), E-selectin and matrix metalloproteinase-9 (MMP9). Functionally, apremilast reduced adhesion of THP-1 cells to activated HUVECs and TEM in response to TNFα. Mechanistically, apremilast suppressed activation of nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPK) signalling in activated HUVECs. Furthermore, inhibition of p38, C-Jun-N-terminale Kinase (JNK) and NFκB in activated HUVECs decreased expression of GM-CSF, VCAM-1 and E-selectin. Additionally, apremilast decreased IL-17A-induced secretion of IL-6 and CCL2. We demonstrate that apremilast has distinct anti-inflammatory effects in activated HUVECs, indicating that apremilast could have the therapeutic potential to prevent higher risk for CVD in patients with chronic inflammatory diseases.

PKD regulates actin polymerization, neutrophil deformability, and transendothelial migration in response to fMLP and trauma.

Neutrophils are important mediators of the innate immune defense and of the host response to a physical trauma. Because aberrant infiltration of injured sites by neutrophils was shown to cause adverse effects after trauma, we investigated how neutrophil infiltration could be modulated at the cellular level. Our data indicate that protein kinase D (PKD) is a vital regulator of neutrophil transmigration. PKD phosphorylates the Cofilin-phosphatase Slingshot-2L (SSH-2L). SSH-2L in turn dynamically regulates Cofilin activity and actin polymerization in response to a chemotactic stimulus for neutrophils, for example, fMLP. Here, we show that inhibition of PKD by two specific small molecule inhibitors results in broad, unrestricted activation of Cofilin and strongly increases the F-actin content of neutrophils even under basal conditions. This phenotype correlates with a significantly impaired neutrophil deformability as determined by optical stretcher analysis. Consequently, inhibition of PKD impaired chemotaxis as shown by reduced extravasation of neutrophils. Consequently, we demonstrate that transendothelial passage of both, neutrophil-like NB4 cells and primary PMNs recovered from a hemorrhagic shock trauma model was significantly reduced. Thus, inhibition of PKD may represent a promising modulator of the neutrophil response to trauma.

Protective Role of Insulin-Like Growth Factor-1 Receptor in Endothelial Cells against Unilateral Ureteral Obstruction–Induced Renal Fibrosis

Insulin-like growth factor-1 receptor (IGF-1R) can regulate vascular homeostasis and endothelial function. We studied the role of IGF-1R in oxidative stress-induced endothelial dysfunction. Unilateral ureteral obstruction (UUO) was performed in wild-type (WT) mice and mice with endothelial cell (EC)-specific IGF-1R knockout (KO). After UUO in endothelial IGF-1R KO mice, endothelial barrier dysfunction was more severe than in WT mice, as seen by increased inflammatory cell infiltration and vascular endothelial (VE)-cadherin phosphorylation. UUO in endothelial IGF-1R KO mice increased interstitial fibroblast accumulation and enhanced extracellular protein deposition as compared with the WT mice. Endothelial barrier function measured by transendothelial migration in response to hydrogen peroxide (H2O2) was impaired in ECs. Silencing IGF-1R enhanced the influence of H2O2 in disrupting the VE-protein tyrosine phosphatase/VE-cadherin interaction. Overexpression of IGF-1R suppressed H2O2-induced endothelial barrier dysfunction. Furthermore, by using the piggyBac transposon system, we expressed IGF-1R in VE cells in mice. The expression of IGF-1R in ECs also suppressed the inflammatory cell infiltration and renal fibrosis induced by UUO. IGF-1R KO in the VE-cadherin lineage of bone marrow cells had no significant effect on the UUO-induced fibrosis, as compared with control mice. Our results indicate that IGF-1R in the endothelium maintains the endothelial barrier function by stabilization of the VE-protein tyrosine phosphatase/VE-cadherin complex. Decreased expression of IGF-1R impairs endothelial function and increases the fibrosis of kidney disease.

Control of TNF-Induced Dendritic Cell Maturation by Hybrid-Type N-Glycans

The activity of α-1,2-mannosidase I is required for the conversion of high-mannose to hybrid-type (ConA reactive) and complex-type N-glycans (Phaseolus vulgaris-leukoagglutinin [PHA-L] reactive) during posttranslational protein N-glycosylation. We recently demonstrated that α-1,2-mannosidase I mRNA decreases in graft-infiltrating CD11c(+) dendritic cells (DCs) prior to allograft rejection. Although highly expressed in immature DCs, little is known about its role in DC functions. In this study, analysis of surface complex-type N-glycan expression by lectin staining revealed the existence of PHA-L(low) and PHA-L(high) subpopulations in murine splenic conventional DCs, as well as in bone marrow-derived DC (BMDCs), whereas plasmacytoid DCs are nearly exclusively PHA-L(high). Interestingly, all PHA-L(high) DCs displayed a strongly reduced responsiveness to TNF-α-induced p38-MAPK activation compared with PHA-L(low) DCs, indicating differences in PHA-L-binding capacities between DCs with different inflammatory properties. However, p38 phosphorylation levels were increased in BMDCs overexpressing α-1,2-mannosidase I mRNA. Moreover, hybrid-type, but not complex-type, N-glycans are required for TNF-α-induced p38-MAPK activation and subsequent phenotypic maturation of BMDCs (MHC-II, CD86, CCR7 upregulation). α-1,2-mannosidase I inhibitor-treated DCs displayed diminished transendothelial migration in response to CCL19, homing to regional lymph nodes, and priming of IFN-γ-producing T cells in vivo. In contrast, the activity of α-1,2-mannosidase I is dispensable for LPS-induced signaling, as well as the DCs' general capability for phenotypic and functional maturation. Systemic application of an α-1,2-mannosidase I inhibitor was able to significantly prolong allograft survival in a murine high-responder corneal transplantation model, further highlighting the importance of N-glycan processing by α-1,2-mannosidase I for alloantigen presentation and T cell priming.

MLCK210 deficiency ameliorates atherosclerosis in apoE knockout mice

Endothelial barrier dysfunction is a key event in the pathogenesis of atherosclerosis. The objective of this study was to explore the role and mechanisms of MLCK210 (non‐muscle myosin light‐chain kinase) in the development of atherosclerosis in mice with an apoE knockout background. Histopathological examination demonstrated that MLCK210 deficiency reduced atherosclerotic lesion and macrophage deposit. Western blot and RT‐PCR analyses revealed MLCK210 expression in the aortic endothelium and peripheral monocytes. By measuring transendothelial electric resistance, we found that MLCK210 deficiency attenuated aorta endothelial hyperpermeability to pro‐inflammatory stimuli, including thrombin, ox‐LDL and TNF‐alpha. In monocytes, MLCK210 deficiency reduced their transendothelial migration in response to murine MCP‐1. Further mechanistic studies showed that these inflammatory mediators induced Src phosphorylation in endothelial cells and monocytes. The phosphorylation response was attenuated in MLCK210 deficient cells, corresponding to reduced endothelial permeability and monocytes transmigration. Taken together, the data suggest that MLCK210 contributes to the development of atherosclerosis by regulating endothelial barrier function and monocyte migration via a mechanism involving Src signaling. Supported by NIH HL61507, HL84542, HL70752, and HL73324.

Leukocyte Extravasation: An Immunoregulatory Role for α-l-Fucosidase?

Fucosylated oligosaccharides and glycoconjugates have been implicated in several biological events, including the cell-cell adhesion processes that mediate inflammation. Alpha-L-fucosidase (ALF) is an exoglycosidase that is involved in the hydrolytic degradation of alpha-L-fucose from glycoconjugates. In this study, we investigated the potential role of ALF in regulation of leukocyte migration. Measurement of transendothelial migration in response to CCL5 demonstrated that pretreatment of monocytic cells with ALF reduced migration (p = 0.0004) to a greater extent than treatment of the endothelial monolayer (p = 0.0374). Treatment with ALF significantly reduced the adhesion of monocytic cells to immobilized P-selectin.Fc. A murine model of experimental autoimmune uveitis was then used to show that treatment of splenic cells with ALF produced an 8.6-fold decrease in rolling and a 3.2-fold decrease in cell migration across the retinal vasculature. Further in vitro studies demonstrated that treatment of monocytes with the chemokines CCL3 or CCL5 increased the level of mRNA encoding ALF; this was accompanied by the detection of significant increases in both the 51- and 56-kDa components of ALF by Western blotting. Treatment of monocytic cells with ALF for 2 h significantly reduced the cell surface expression of CD31, with a further decrease in expression observed after 5 h (p = 0.002). Thus, CD31 and fucosylated ligands of P-selectin seem to be the candidates through which ALF mediates its effect in vitro. These data identify a previously unrecognized immunoregulatory role for ALF in late stages of inflammation.

The Par polarity complex regulates Rap1- and chemokine-induced T cell polarization

Cell polarization is required for virtually all functions of T cells, including transendothelial migration in response to chemokines. However, the molecular pathways that establish T cell polarity are poorly understood. We show that the activation of the partitioning defective (Par) polarity complex is a key event during Rap1- and chemokine-induced T cell polarization. Intracellular localization and activation of the Par complex are initiated by Rap1 and require Cdc42 activity. The Rac activator Tiam1 associates with both Rap1 and components of the Par complex, and thereby may function to connect the Par polarity complex to Rap1 and to regulate the Rac-mediated actin remodelling required for T cell polarization. Consistent with these findings, Tiam1-deficient T cells are impaired in Rap1- and chemokine-induced polarization and chemotaxis. Our studies implicate Tiam1 and the Par polarity complex in polarization of T cells, and provide a mechanism by which chemokines and Rap1 regulate T cell polarization and chemotaxis.

Effects of the very late adhesion molecule 4 antagonist WAY103 on human peripheral blood eosinophil vascular cell adhesion molecule 1–dependent functions

Eosinophil infiltration to the lung in allergic inflammation can be initiated by the tethering of circulating cells through very late adhesion molecule 4 (VLA-4; alpha4beta1, CD49d/CD29) to vascular cell adhesion molecule 1 (VCAM-1) expressed on pulmonary vascular endothelium. Small-molecule VLA-4 antagonists have been proposed as a therapeutic mechanism to prevent eosinophil infiltration in asthma; however, they might affect other eosinophil functions. The small-molecule VLA-4 antagonist (2S)-3-(4-Dimethylcarbamoyloxyphenyl)-2-{[(4R)-5,5-dimethyl-3-(1-methyl-1H-pyrazole-4 sulfonyl)thiazolidine-4-carbonyl]amino}propionic acid (WAY103) was assessed for its effects on eosinophil VLA-4-dependent functions, including adhesion, migration, respiratory burst, and degranulation. Human peripheral blood eosinophils were preincubated with WAY103, anti-alpha4, and/or anti-beta2 integrin mAbs and then assessed for adhesion to recombinant VCAM-1, intercellular adhesion molecule 1, and endothelial cell monolayers. Transmigration was measured by using human pulmonary microvascular endothelial cell monolayers and Transwell filters. Superoxide anion generation was determined by means of cytochrome C reduction and degranulation by means of eosinophil-derived neurotoxin release. WAY103 inhibition of eosinophil adhesion to recombinant VCAM-1 was dose dependent (63% inhibition with 100 nM WAY103, P < .04) and comparable with inhibition caused by anti-alpha4 mAb (60.1% inhibition). Although pretreatment with WAY103 also decreased eosinophil adhesion to TNF-alpha plus IL-4-activated human pulmonary microvascular endothelial cell monolayers, it did not prevent eosinophil transendothelial migration in response to RANTES. Finally, WAY103 inhibited VCAM-1-stimulated superoxide generation but enhanced cytokine-activated eosinophil-derived neurotoxin degranulation. Although small-molecule VLA-4 antagonists, such as WAY103, might reduce eosinophil adhesion, this approach might not be sufficient to eliminate this cell from in vivo allergic airway inflammatory participation and could even promote specific cell activation.

Effect of the Cysteinyl Leukotriene Antagonist Pranlukast on Transendothelial Migration of Eosinophils

Background:Evidence shows that leukotriene receptor antagonist (LTRA) can cause a partial reduction of eosinophils in the asthmatic airway. Although cysteinyl leukotrienes (CysLTs) can regulate the development of eosinophilic inflammation, LTRA might modulate the eosinophilic response to other inflammatory molecules involved in allergic inflammation. Montelukast is an LTRA that inhibits eosinophil transendothelial migration (TEM) in response to platelet-activating factor (PAF). The present study evaluates whether pranlukast (an LTRA) modifies eosinophil TEM in response to chemoattractants including PAF and C-C chemokines. Methods: Eosinophils isolated from the blood of healthy individuals were incubated with or without pranlukast. We then evaluated eosinophil transmigration across human umbilical vein endothelial cells in response to LTD₄, eotaxin, RANTES and PAF. Results: Pranlukast did not modify the spontaneous transmigration of eosinophils (n = 5). As reported, eosinophil TEM was significantly augmented by 0.1 µM LTD₄ and this enhancement was blocked by 1 µM pranlukast (p < 0.001; n = 6). On the other hand, pranlukast did not modify eosinophil transmigration in response to eotaxin, RANTES, or PAF (p > 0.1; n = 5). Conclusion: The inhibitory effect of pranlukast on eosinophil transmigration is highly specific for the CysLT1-dependent pathway.

CCR2 expression by brain microvascular endothelial cells is critical for macrophage transendothelial migration in response to CCL2

While the expression of chemokine receptors by endothelial cells is now well established, little is known of the function of these receptors at this cellular locale. However, given that chemokines are instrumental in directing leukocytes to specific parenchymal sites, one possibility is that endothelial chemokine receptors play a role in the process of leukocyte extravasation. To test this hypothesis, we investigated the contribution of CCR2, the major cognate receptor for the chemokine CCL2 (formerly known as MCP-1), to CCL2-stimulated transendothelial migration of macrophages (mØ) across cultured brain microvascular endothelial cells (BMEC). Specifically, we prepared both BMEC and mØ from wild-type (WT) mice and mice deficient in CCR2; i.e., CCR2 (−/−), and compared the ability of WT and CCR2 (−/−) BMEC to support CCL2-stimulated transendothelial migration of WT and CCR2 (−/−) mØ. In response to CCL2, WT mØ, but not CCR2 (−/−) mØ, were stimulated to migrate across WT BMEC, consistent with the recognized obligatory role for CCR2 in mediating CCL2-stimulated responses. Remarkably, however, neither WT nor CCR2 (−/−) mØ were stimulated by CCL2 to migrate across CCR2 (−/−) BMEC. In contrast, both types of mØ were able to migrate similarly across both types of BMEC in response to another chemokine – CCL3 (formerly known as MIP-1α) – which utilizes receptors other than CCR2. Lastly, CCL2-induced mØ transendothelial migration was blocked by treatment of WT BMEC with pertussis toxin, suggesting that CCR2 is functionally coupled to the inhibitory G protein Gα i, much as it is in other cell types. These results highlight a heretofore-unrecognized role for endothelial CCR2 in mediating transendothelial migration.

Effect of pranlukast, a cysteinyl leukotriene-receptor 1-antagonist, on eosinophil tranendothelial migration

Rationale There is increasing evidence that leukotriene receptor antagonist (LTRA) possesses anti-inflammatory effects on the airway disease of asthma. Although cysteinyl-leukotrienes (Cys LTs) by themselves regulates the development of eosinophilic inflammation, LTRA may modulate the eosinophilic response to another inflammatory molecules involved in allergic inflammation. The objective of this study is to evaluate whether pranlukast, a LTRA which has been widely used in Japan, modifies eosinophil transendothelial migration (TEM) in response to chemoattractants including C-C chemokines. Methods Eosinophils were isolated from the peripheral blood of healthy subjects and incubated in the presence or absence of pranlukast. Following the pretreatment, eosinophil TEM across human umbilical vein endothelial cells was examined by an EPO assay. Results Pranlukast did not modify the spontaneous TEM of eosinophils (P>0.1; n=5). As previously reported, the eosinophil TEM was significantly augmented by 0.1 μM LTD 4 and this enhancement was blocked by 1μM pranlukast (p<0.001; n=6). On the other hand, pranlukast did not modify eosinophil TEM in response to eotaxin, RANTES or PAF (P>0.1; n=5). Conclusions The inhibitory effect of pranlukast on eosinophil TEM is highly specific for Cys-LT1-dependent pathway and LTRA is unlikely to modulate TEM elicited by C-C chemokines. Such selectivity on eosinophil migrative response may determine the individual response to LTRA in reduction of eosinophil accumulation in the asthmatic airways.

Fractalkine preferentially mediates arrest and migration of CD16+ monocytes.

CD16+ monocytes represent 5–10% of peripheral blood monocytes in normal individuals and are dramatically expanded in several pathological conditions including sepsis, human immunodeficiency virus 1 infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines and may represent dendritic cell precursors in vivo. The mechanisms that mediate the recruitment of CD16+ monocytes into tissues remain unknown. Here we investigate molecular mechanisms of CD16+ monocyte trafficking and show that migration of CD16+ and CD16− monocytes is mediated by distinct combinations of adhesion molecules and chemokine receptors. In contrast to CD16− monocytes, CD16+ monocytes expressed high CX3CR1 and CXCR4 but low CCR2 and CD62L levels and underwent efficient transendo-thelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1α (CXCL12) but not monocyte chemoattractant protein 1 (CCL2). CD16+ monocytes arrested on cell surface–expressed FKN under flow with higher frequency compared with CD16− monocytes. These results demonstrate that FKN preferentially mediates arrest and migration of CD16+ monocytes and suggest that recruitment of this proinflammatory monocyte subset to vessel walls via the CX3CR1-FKN pathway may contribute to vascular and tissue injury during pathological conditions.

Migration of human blood dendritic cells across endothelial cell monolayers: adhesion molecules and chemokines involved in subset-specific transmigration.

Distinct subsets of dendritic cells (DCs) are present in blood, probably "en route" to different tissues. We have investigated the chemokines and adhesion molecules involved in the migration of myeloid (CD11c(+)) and plasmacytoid (CD123(+)) human peripheral blood DCs across vascular endothelium. Among blood DCs, the CD11c(+) subset vigorously migrated across endothelium in the absence of any chemotactic stimuli, whereas spontaneous migration of CD123(+) DCs was limited. In bare cell migration assays, myeloid DCs responded with great potency to several inflammatory and homeostatic chemokines, whereas plasmacytoid DCs responded poorly to all chemokines tested. In contrast, the presence of endothelium greatly favored transmigration of plasmacytoid DCs in response to CXCL12 (stromal cell-derived factor-1) and CCL5 (regulated on activation, normal T expressed and secreted). Myeloid DCs exhibited a very potent transendothelial migration in response to CXCL12, CCL5, and CCL2 (monocyte chemoattractant protein-1). Furthermore, we explored whether blood DCs acutely switch their pattern of migration to the lymph node-derived chemokine CCL21 (secondary lymphoid-tissue chemokine) in response to microbial stimuli [viral double-stranded (ds)RNA or bacterial CpG-DNA]. A synthetic dsRNA rapidly enhanced the response of CD11c(+) DCs to CCL21, whereas a longer stimulation with CpG-DNA was needed to trigger CD123(+) DCs responsive to CCL21. Use of blocking monoclonal antibodies to adhesion molecules revealed that both DC subsets used platelet endothelial cell adhesion molecule-1 to move across activated endothelium. CD123(+) DCs required beta(2) and beta(1) integrins to transmigrate, whereas CD11c(+) DCs may use integrin-independent mechanisms to migrate across activated endothelium.

MRL/lpr lupus-prone mice show exaggerated ICAM-1-dependent leucocyte adhesion and transendothelial migration in response to TNF-alpha.

Endothelial activation and dysfunctional leucocyte-endothelial interactions are thought to play key roles in the pathogenesis of systemic lupus erythematosus (SLE). The object of this study was to investigate directly the effect of increased endothelial adhesion molecule expression on leucocyte-endothelial cell interactions, using the MRL/lpr mouse model. Leucocyte rolling, arrest and transendothelial migration were quantified in the cremaster muscle microcirculation of 20-week-old MRL/lpr mice, using intravital microscopy. Endothelial adhesion molecule expression was quantified using intravenously injected radiolabelled monoclonal antibodies. Basal expression of intercellular adhesion molecule 1 (ICAM-1) by cremaster endothelium was 2-fold greater in MRL/lpr than in MRL/++ mice (P<0.05). There was a 1.6-fold increase in expression of vascular adhesion molecule 1 (VCAM-1), but no increase in E-selectin or P-selectin expression. Following intrascrotal injection of saline, no difference was detected in leucocyte-endothelial interactions between MRL/lpr and control MRL/++ mice. In contrast, intrascrotal injection of tumour necrosis factor alpha (TNF-alpha) (2 h test period) led to significantly increased numbers of adherent and extravasated leucocytes in MRL/lpr (5.98+/-0.71 and 5.45+/-0.34 leucocytes per 100 micro m vessel segment respectively) compared with MRL/++ mice (3.63+/-0.26 and 2.97+/-0.24 respectively, each P<0.05). Treatment of TNF-alpha-stimulated mice with anti-ICAM-1 F(ab')2 (YN1) abolished the difference between MRL/lpr and MRL/++ mice, whereas a negative control anti-DNP F(ab')2 had no effect. MRL/lpr lupus-prone mice show exaggerated ICAM-1-dependent leucocyte-endothelial interactions in response to TNF-alpha. Increased leucocyte-endothelial interactions due to endothelial priming could contribute to the clinical link between infection and flares of lupus disease activity.

Annexin 1 modulates monocyte-endothelial cell interaction in vitro and cell migration in vivo in the human SCID mouse transplantation model.

The effect of the glucocorticoid inducible protein annexin 1 (ANXA1) on the process of monocytic cell migration was studied using transfected U937 cells expressing variable protein levels. An antisense (AS) (36.4AS; approximately 50% less ANXA1) and a sense (S) clone (15S; overexpressing the bioactive 24-kDa fragment) together with the empty plasmid CMV clone were obtained and compared with wild-type U937 cells in various models of cell migration in vitro and in vivo. 15S-transfected U937 cells displayed a reduced (50%) degree of trans-endothelial migration in response to stromal cell-derived factor-1alpha (CXC chemokine ligand 12 (CXCL12)). In addition, the inhibitory role of endogenous ANXA1 on U937 cell migration in vitro was confirmed by the potentiating effect of a neutralizing anti-ANXA1 serum. Importantly, overexpression of ANXA1 in clone 15S inhibited the extent of cell migration into rheumatoid synovial grafts transplanted into SCID mice. ANXA1 inhibitory effects were not due to modifications in adhesion molecule or CXCL12 receptor (CXCR4) expression as shown by the similar amounts of surface molecules found in transfected and wild-type U937 cells. Likewise, an equal chemotactic response to CXCL12 in vitro excluded an intrinsic defect in cell motility in clones 15S and 36.4AS. These data strongly support the notion that ANXA1 critically interferes with a leukocyte endothelial step essential for U937 cell, and possibly monocyte, transmigration both in vitro and in vivo.

In vitro characterization and micromechanics of tumor cell chemotactic protrusion, locomotion, and extravasation.

The objective of this paper is to introduce some novel in vitro applications in characterizing human melanoma cell protrusion and migration in response to soluble extracellular matrix protein stimulation. Specifically, we describe two assay systems: (1) dual-micropipette manipulation and (2) flow-migration chamber. Applications of the dual-micropipet technique provided kinetic measure of cell movement, cyclic pseudopod protrusion, and subsequent cell locomotion governed by chemotactic molecular transport dynamics. Chemotactic concentration gradient was found to influence significantly pseudopod protrusion frequency and locomotion speed, but not the protrusion extension. To further characterize active tumor cell extravasation, a process that involves dynamic tumor cell adhesion to vascular endothelium under flow conditions and subsequent transendothelial migration in response to chemotactic signals from the interstitial space, we developed a flow-migration chemotaxis system. This assay enabled characterization of tumor cell transcellular migration in terms of chemotactic signal gradients, shear forces, and cell-substrate adhesion. Results suggest that shear flow plays significant roles in tumor cell extravasation that is regulated by both tumor cell motility and tumor cell adhesion to endothelial molecules in a cooperative process.

Growth factor regulation of neutrophil-endothelial cell interactions

The effects of the angiogenic factors basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) on human polymorphonuclear leukocyte (PMNL)-endothelial cell adhesion and transendothelial migration (TEM) were investigated. Stimulation of human umbilical vein endothelial cells by VEGF or bFGF for 18 h up-regulated intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 expression and significantly increased PMNL adhesion and TEM in response to complement fragment 5a (C5a) or interleukin (IL)-8. In contrast, continued exposure to bFGF (24 h-6 days) down-regulated basal and IL-1- or tumor necrosis factor (TNF)-induced intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin expression as well as PMNL adhesion and TEM. These effects could be reversed by introduction of high concentrations of TNF-alpha, C5a, or IL-8. None of these inhibitory effects was observed with VEGF. The acute effects of bFGF and VEGF may facilitate PMNL emigration during acute inflammation, but continued bFGF production may have anti-inflammatory actions during chronic inflammation, angiogenesis, and tumor defense by inhibition of endothelial activation for leukocyte recruitment.

Migration of neutrophils across human pulmonary endothelial cells is not blocked by matrix metalloproteinase or serine protease inhibitors.

It has long been speculated that neutrophils deploy proteases to digest subendothelial matrix as they migrate from the bloodstream. Direct evidence for the involvement of proteases in neutrophil transendothelial migration is, however, lacking. To address this issue we used transmission electron microscopy to verify the presence of continuous basal lamina beneath pulmonary endothelial cells grown on microporous filters, and then examined the effects of protease inhibitors on neutrophil migration through the endothelial cells and their associated subcellular matrix. Inhibitors of the two major matrix-degrading protease groups present in neutrophils, the matrix metalloproteinases (MMPs) and serine proteases, were assessed for their ability to modulate neutrophil transendothelial migration in response to the chemoattractant n-formylmethionyl leucylphenylalanine (FMLP). Neither the naturally occurring MMP inhibitor, tissue inhibitor of metalloproteinase-1, nor the hydroxamic acid-based inhibitors GM-6001, BB-3103, or Ro 31-9790 had any significant effect on FMLP-stimulated neutrophil migration across endothelial cells and associated basal lamina, with >/= 80% of neutrophils migrating through the system, even in the presence of inhibitors, at concentrations that totally inhibited all the gelatinase B (MMP-9) released upon stimulation with FMLP. Similarly, with serine protease inhibitors no significant inhibition of neutrophil migration was observed with a naturally occurring inhibitor, secretory leukocyte protease inhibitor, or a low molecular-weight synthetic inhibitor, Pefabloc SC. These results indicate that neither MMP nor serine protease digestion of sub-endothelial matrix is required for successful neutrophil transendothelial migration.

Adherent neutrophils activate endothelial myosin light chain kinase: role in transendothelial migration.

Increased vascular endothelial cell (EC) permeability and neutrophilic leukocyte (PMN) diapedesis through paracellular gaps are cardinal features of acute inflammation. Activation of the EC contractile apparatus is necessary and sufficient to increase vascular permeability in specific models of EC barrier dysfunction. However, it is unknown whether EC contraction with subsequent paracellular gap formation is required for PMN transendothelial migration in response to chemotactic factors. To test this possibility, we assessed migration of human PMNs across confluent bovine pulmonary arterial EC monolayers. Transendothelial PMN migration in the absence of a chemotactic gradient was minimal, whereas abluminal addition of leukotriene B4 (LTB4; 5 microM) resulted in significantly increased PMN migration. Reductions in EC myosin light chain kinase (MLCK) activity by EC monolayer pretreatment with specific MLCK inhibitors (KT-5926 or ML-7) or by increases in cAMP-protein kinase A activity (cholera toxin) significantly reduced PMN transmigration (30-70% inhibition). In contrast, pretreatment with the myosin-associated phosphatase inhibitor calyculin resulted in the accumulation of phosphorylated myosin light chains, EC contraction, and significantly enhanced PMN migration. Finally, the interaction of PMNs with 32P-labeled EC monolayers was shown to directly increase EC myosin phosphorylation in a time-dependent fashion. Taken together, these results are consistent with the hypothesis that the phosphorylation status of EC myosin regulates PMN migration and further indicate that EC MLCK is activated by chemoattractant-stimulated PMNs. Neutrophil-dependent activation of the EC contractile apparatus with subsequent paracellular gap formation may be a key determinant of transendothelial PMN migration responses to chemotactic agents.