Integrin activation by P-Rex1 is required for selectin-mediated slow leukocyte rolling and intravascular crawling.

Integrin activation is essential for the function of leukocytes. Impaired integrin activation on leukocytes is the hallmark of the leukocyte adhesion deficiency syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. In inflammation, leukocytes collect different signals during the contact with the microvasculature, which activate signaling pathways leading to integrin activation and leukocyte recruitment. We report the role of P-Rex1, a Rac-specific guanine nucleotide exchanging factor, in integrin activation and leukocyte recruitment. We find that P-Rex1 is required for inducing selectin-mediated lymphocyte function-associated antigen-1 (LFA-1) extension that corresponds to intermediate affinity and induces slow leukocyte rolling, whereas P-Rex1 is not involved in the induction of the high-affinity conformation of LFA-1 obligatory for leukocyte arrest. Furthermore, we demonstrate that P-Rex1 is involved in Mac-1-dependent intravascular crawling. In vivo, both LFA-1-dependent slow rolling and Mac-1-dependent crawling are defective in P-Rex1(-/-) leukocytes, whereas chemokine-induced arrest and postadhesion strengthening remain intact in P-Rex1-deficient leukocytes. Rac1 is involved in E-selectin-mediated slow rolling and crawling. In vivo, in an ischemia-reperfusion-induced model of acute kidney injury, abolished selectin-mediated integrin activation contributed to decreased neutrophil recruitment and reduced kidney damage in P-Rex1-deficient mice. We conclude that P-Rex1 serves distinct functions in LFA-1 and Mac-1 activation.

Inducing apoptosis in rolling cancer cells: a combined therapy with aspirin and immobilized TRAIL and E-selectin.

Though metastasis is considered an inefficient process, over 90% of cancer related deaths are attributed to the formation of secondary tumors. Thus, eliminating circulating cancer cells could lead to improved patient survival. This study was aimed at exploiting the interactions of cancer cells with selectins under flow to selectively kill captured colon cancer cells. Microtubes functionalized with E-selectin and TRAIL were perfused with colon cancer cell line Colo205 either treated with 1 mM aspirin or untreated for 1 or 2 h. Cells were collected from the microtube and analyzed by flow cytometry. Aspirin treatment alone killed only 3% cells in culture. A 95% difference in the number of cells killed between control and TRAIL + ES surfaces was seen when aspirin treated cells were perfused over the functionalized surface for 2 h. We have demonstrated a novel biomimetic method to capture and neutralize cancer cells in flow, thus reducing the chances for the formation of secondary tumors.

Variability in chemokine-induced adhesion of human mesenchymal stromal cells.

BACKGROUND AIMS. Intravenously applied mesenchymal stromal cells (MSC) are under investigation for numerous clinical indications. However, their capacity to activate shear stress-dependent adhesion to endothelial ligands is incompletely characterized. METHODS. Parallel-plate flow chambers were used to induce firm adhesion of MSC to integrin ligand vascular cell adhesion molecule (VCAM)-1. Human MSC were stimulated by chemokine (C-C motif) ligand (CCL15)/macrophage inflammatory protein (MIP-5), CCL19/MIP-3ß chemokine (C-X-C motif) ligand (CXCL8)/interleukin (IL)-8, CXCL12/ stromal derived factor (SDF-1) or CXCL13/B lymphocyte chemoattractant (BLC). RESULTS. Two MSC isolates responded to three chemokines (either to CCL15, CCL19 and CXCL13, or to CCL19, CXCL12 and CXCL13), two isolates responded to two chemokines (to CCL15 and CCL19, or to CCL19 and CXCL13), and one isolate responded to CCL19 only. In contrast, all tested MSC isolates responded to selectins (P-selectin and E-selectin) or integrin ligand VCAM-1, as visualized by a velocity reduction under flow. CONCLUSIONS. Inter-individual variability of chemokine-induced integrin activation should be considered when evaluating human MSC as cellular therapies.

Mucosal tolerization to E-selectin protects against memory dysfunction and white matter damage in a vascular cognitive impairment model.

Vascular cognitive impairment (VCI) is the second most prevalent type of dementia in the world. The white matter damage that characterizes the common subcortical ischemic form of VCI can be modeled by ligating both common carotid arteries in the Wistar rat to induce protracted cerebral hypoperfusion. In this model, we find that repetitive intranasal administration of recombinant E-selectin to induce mucosal tolerance and to target immunomodulation to activating blood vessels potently suppresses both white matter (and possibly gray matter) damage and markers of vessel activation (tumor necrosis factor and E-selectin); it also preserves behavioral function in T-maze spontaneous alternation, T-maze spatial discrimination memory retention, and object recognition tests. Immunomodulation may be an effective novel strategy to prevent progression of VCI.

E-selectin regulates gene expression in metastatic colorectal carcinoma cells and enhances HMGB1 release.

Extravasation of cancer cells is a pivotal step in the formation of hematogenous metastasis. Extravasation is initiated by the loose adhesion of cancer cells to endothelial cells via an interaction between endothelial selectins and selectin ligands expressed by the tumor cells. The present study shows that the interaction between recombinant E-selectin (rE-selectin) and colorectal cancer (CRC) cells alters the gene expression profile of the cancer cells. A DNA microarry analysis indicated that E-selectin-mediated alterations were significantly more pronounced in the metastatic CRC variants SW620 and KM12SM than in the corresponding non-metastatic local SW480 and KM12C variants. The number of genes altered by E-selectin in the metastatic variants was about 10-fold higher than the number of genes altered in the corresponding local variants. Aiming to identify genes involved in CRC metastasis, we focused, by using a DNA microarry analysis, on genes that were altered by E-selectin in a similar fashion exclusively in both metastatic variants. This analysis indicated that E-selectin down regulated (at least by 1.6-folds) the expression of 7 genes in a similar fashion, in both metastatic cells. The DNA microarry analysis was validated by real time PCR or by RT-PCR. HMGB1 was among these genes. Confocal microscopy indicated that E-selectin down regulated the cellular expression of the HMGB1 protein and enhanced the release of HMGB1 into the culture medium. The released HMGB1 in turn, activated endothelial cells to express E-selectin.

Endothelial E-selectin potentiates neovascularization via endothelial progenitor cell-dependent and -independent mechanisms.

BACKGROUND: Although potential participation of bone marrow-derived circulating endothelial progenitor cells (EPCs) to neoangiogenesis has been proposed, the precise molecular mechanisms of EPC recruitment to vascular endothelium has not been fully elucidated. METHODS AND RESULTS: Peripheral blood mononuclear cells were isolated from healthy volunteers and cultured for 7 days to obtain EPCs. Tumor necrosis factor-alpha-activated human umbilical vein endothelial cells (HUVECs) supported significantly more rolling and adhesion of EPCs compared with inactivated HUVEC monolayer. Pretreatment of activated HUVEC with an adhesion-blocking mAb to E-selectin significantly reduced EPCs adhesion to HUVECs. When HUVECs were transduced with a recombinant adenovirus of E-selectin (AdRSVE-sel) or that of beta-galactosidase (AdRSVLacZ), E-selectin-transduced but not LacZ-transduced HUVECs exhibited significantly more EPC rolling as well as adhesion. Further, effect of AdRSVE-sel or AdRSVLacZ was examined in mouse hind limb ischemic model. AdRSVE-sel-transduced mice showed significantly less limb necrosis and higher laser Doppler ratio when compared with AdRSVLacZ-transduced mice. Interestingly, blood flow recovery of ischemic limb observed in AdRSVE-sel-transduced mice was more prominent when combined with EPC administration compared with that of AdRSVLacZ-transduced mice. CONCLUSIONS: Endothelial E-selectin plays a crucial role in EPC-endothelial interaction in vitro. The importance of E-selectin was also confirmed in vivo even in the absence of exogenous EPC. These data provide molecular background for novel cell-based therapy for ischemic atherosclerosis.

Intranasal administration of E-selectin to induce immunological tolerization can suppress subarachnoid hemorrhage-induced vasospasm implicating immune and inflammatory mechanisms in its genesis.

Evidence that inflammatory and immune mechanisms may have a critical role in the development of vasospasm after subarachnoid hemorrhage is accumulating. We examined, therefore, whether induction of immunological tolerance to the adhesion molecule that is uniquely expressed on activated endothelium, E-selectin, could inhibit the vasospasm provoked by subarachnoid blood in a rat subarachnoid hemorrhage model. We found that intranasal instillation of E-selectin every other day for 10 days on a mucosal tolerization schedule suppressed delayed type hypersensitivity to E-selectin confirming tolerance to that molecule and markedly suppressed basilar artery spasm after subarachnoid hemorrhage. The results of this proof-of-concept study suggest that agents that can mimic the local effects of the mediators of mucosal tolerance could have therapeutic potential for the management of post-subarachnoid hemorrhage vasospasm.

Contrasting effects of P-selectin and E-selectin on the differentiation of murine hematopoietic progenitor cells.

OBJECTIVE: The two endothelial selectins, P- and E-selectin, are critically important for adhesion and homing of hematopoietic progenitor cells (HPC) into the bone marrow. Little is known, however, about the roles of these two selectins in hematopoiesis. Here, we demonstrate that the most primitive HPC capable of long-term in vivo repopulation express P-selectin glycoprotein ligand-1/CD162 (PSGL-1), a receptor common to both P- and E-selectin. In addition, we demonstrate that P-selectin delays the differentiation of HPC whereas E-selectin enhances their differentiation along the monocyte/granulocyte pathway, describing different roles for these selectins in the regulation of hematopoiesis. MATERIALS AND METHODS: Murine bone marrow HPC were isolated according to their expression of c-kit and PSGL-1, transplanted into lethally irradiated congenic recipients, and chimerism analyzed 6 months posttransplant. Bone marrow lineage-negative (Lin(-)) Sca-1(+)c-kit(+) cells were then cultured on immobilized P- or E-selectin for 4 weeks in the presence of cytokines. Hematopoietic potential was assessed using in vitro phenotyping and colony-forming assays and in vivo spleen colony-forming unit (CFU-S) and long-term competitive repopulation assays. RESULTS: Long-term competitive repopulating HSCs were Lin(-)c-kit(bright) and expressed intermediate levels of PSGL-1. Both P- and E-selectin slowed the proliferation of Lin(-)Sca-1(+)c-kit(+) cells during the first two weeks of liquid culture. After two weeks, however, cells cultured on immobilized P-selectin showed increased proliferation with increased production of both colony-forming cells (CFC) and CFU-S(12) compared to the other cultures. In contrast, E-selectin enhanced the differentiation of Lin(-)Sca-1(+)c-kit(+) cells into cells that expressed the granulocyte maturation marker, Gr-1, accompanied by loss of CFC potential from these cultured cells. Finally, the long-term repopulation potential of these cells was not maintained following culture on either selectin. CONCLUSION: These results suggest that the two endothelial selectins, E-selectin and P-selectin, have very different effects on HPC. E-selectin accelerates the differentiation of maturing HPC towards granulocyte and monocyte lineages while maintaining the production of more immature CFU-S(12) in ex vivo liquid suspension culture. In marked contrast, P-selectin delays the differentiation of Lin(-)Sca-1(+)c-kit(+) cells, allowing enhanced ex vivo expansion of CFC and CFU-S(12) but not HSCs.

Nitric oxide reduces T lymphocyte adhesion to human brain microvessel endothelial cells via a cGMP-dependent pathway.

The entry of lymphocytes into the brain is normally limited by the blood-brain barrier, however, during inflammation prominent lymphocytic infiltration occurs. In this study, we investigated the effects of nitric oxide (NO) on the adhesion of T cells to cultured human brain microvessel endothelial cells. T cell adhesion to unstimulated or tumor necrosis factor-alpha (TNF-alpha)-treated cells was quantified by counting the number of lymphocytes bound to the monolayer by light microscopy. TNF-alpha increased T cell adhesion in a time-dependent manner. Incubation of monolayers with NO donors decreased adhesion. This effect was blocked by a guanylyl cyclase inhibitor and mimicked by a cGMP agonist, and was thus dependent on the generation of cGMP. NO did not modulate adhesion molecule expression in the endothelial cells, suggesting an action on the T cells. Pre-treatment of T cells with NO or a cGMP agonist decreased binding to recombinant endothelial adhesion molecules. These findings suggest that NO can modulate the adhesion of T cells to human brain microvessel endothelial cells via a cGMP-dependent mechanism, and may thus regulate lymphocyte traffic during central nervous system inflammation.

Autoperfused mouse flow chamber reveals synergistic neutrophil accumulation through P-selectin and E-selectin.

To study rolling of mouse neutrophils on P- and E-selectins in whole blood and without cell isolation, we constructed an autoperfused flow chamber made from rectangular microslides (0.2x2 mm) perfused from a carotid artery catheter. A differential pressure transducer served to measure wall shear stress. Green fluorescent neutrophils rolled on P-selectin but not E-selectin coated at 50 ng/ml, with some rolling on E-selectin at 150 ng/ml. However, when P- and E-selectins were coimmobilized, the resulting number of rolling neutrophils was sixfold and fourfold higher than on P- or E-selectin alone. Velocity and flux analysis shows that P-selectin initiates neutrophil rolling, and a small amount of E-selectin, unable to capture many neutrophils, reduces the rolling velocity of all neutrophils by more than 90%. The unexpected synergism between E- and P-selectins explains why neutrophil recruitment is enhanced when both selectins are expressed.

Microenviromental change after synthetic E-selectins interfere in ischemia-reperfusion in rats and its contribution to endogenetic/exogenous never stem cells.

OBJECTIVE: To explore the special significances in advantages of using anti-inflammatory drugs, such as amelioration of growing conditions and the promotion of cell growth. METHODS: Utilizing anti-adhesive effects of synthetic E-selectins, we observed the changes of inflammatory cytokines (TNF-α, IL-1ß) contented in brain tissues and rat serums in rats hind cerebral ischemia-reperfusion models. Both growth and expression of endogenetic/exogenous neurological stem cells were detected after ameliorated local microenvironment. RESULTS: The contents of TNF-α and IL-1ß were decreased in brain tissues and rat serums after applying synthetic E-selectins. Expression of exogenous neurological stem cells was enhanced. Animal neurological functions improved. CONCLUSION: Anti-inflammatory therapy in early stage could enhance proliferation of stem cells so that it has vital significations in treating cerebrovascular diseases.

E-selectin can mediate the arrest type of adhesion of colon cancer cells under physiological shear flow.

The aim of this study was to determine whether colon cancer cells flowing in blood exhibit the same adhesion pattern to the vascular bed as leucocytes using a flow adhesion system. In shear flow conditions, five colon cancer cell lines showed less tethering to E-selectin substrates than polymorphonuclear cells (PMN). However, some of the Colo201 cells formed complete arrest on E-selectin in continuous shear flow which was never observed in PMN cells. Colo201 cells expressed both sialyl Le-x and sialyl Le-a at similar levels in flow cytometry. However, the staining pattern showed marked contrast under the fluorescein microscope. The cell membrane of Colo201 cells was uniformly stained with anti-sialyl Le-a MAb, whereas anti-sialyl Le-x MAb only stained in the patchy areas. Pretreatment of Colo201 cells with anti-sLe-a decreased tethering, while anti-sLe-x significantly inhibited the arrest formation. Our data suggest that E-selectin alone can mediate colon cancer cell lodgement and subsequent metastasis without the contribution of integrin molecules and that the different distribution of E-selectin ligands may affect the adhesion behaviour of colon cancer cells in flow conditions.

Inhibition of E-selectin-, ICAM-1-, and VCAM-1-mediated cell adhesion by benzo[b]thiophene-, benzofuran-, indole-, and naphthalene-2-carboxamides: identification of PD 144795 as an antiinflammatory agent.

It was previously reported that 3-alkoxybenzo[b]thiophene-2-carboxamides exemplified by 1, 5-methoxy-3-(1-methylethoxy)benzo[b]thiophene-2-carboxamide, decreased the adherence of neutrophils to activated endothelial cells by inhibiting the upregulation of the adhesion molecules E-selectin and ICAM-1 on the surface of the endothelium. This finding is extended here to a series of 3-thiobenzo[b]thiophene-2-carboxamides and also heterocyclic analogs of 1, including benzofurans, indoles, and napthalenes. The compounds that inhibited the expression of E-selectin and ICAM-1 had the same effect on the expression of VCAM-1. PD 144795, 5-methoxy-3-(1-methylethoxy)benzo[b]thiophene-2-carboxamide 1-oxide (44), the sulfoxide analog of 1, was orally active in several models of inflammation. The in vitro and in vivo activity of PD 144795 resided predominately in the S-enantiomer.

Activation of human endothelial cells by mobilized porcine leukocytes in vitro: implications for mixed chimerism in xenotransplantation.

BACKGROUND: The induction of immunologic tolerance to pig antigens in primates may facilitate the development of successful clinical xenotransplantation protocols. The infusion of mobilized porcine peripheral blood leukocytes (PBPC, consisting of approximately 2% peripheral blood progenitor cells) into preconditioned baboons, intended to induce mixed hematopoietic cell chimerism, however, results in a severe thrombotic microangiopathy (TM) that includes vascular injury, microvascular thrombosis, and pronounced thrombocytopenia. Because the mechanisms responsible for TM are unclear, we have explored the effects of PBPC on human umbilical vein endothelial cell (HUVEC) activation. METHODS: Confluent HUVEC monolayers were established in 96-well cell culture clusters. PBPC were mobilized from miniature swine with porcine interleukin 3 (pIL-3), porcine stem cell factor (pSCF), and human granulocyte-colony stimulating factor (hG-CSF) and were collected by leukapheresis. PBPC were added to HUVEC (0-1x10(7) PBPC/well) for 3- to 24-hr periods and, with cell-based ELISA techniques, surface levels of E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) were measured. In some cases, peripheral blood leukocytes (PBL) were collected from pigs that did not receive pIL-3, pSCF, or hG-CSF and were added to HUVEC. PBPC were also sorted into subsets of CD2- cells, CD2+ cells, and cellular debris, each of which were added separately to HUVEC. Transwell permeable membrane inserts were placed over HUVEC to prevent direct cell-cell contact with PBPC in some instances. RESULTS: PBPC from different pigs (n=6) induced an increase in the expression of E-selectin, VCAM-1, and ICAM-1 to levels 5, 4, and 2 times greater than baseline, respectively. ICAM-1 expression reached maximum levels after the addition of 6x10(5) PBPC/well. Expression of E-selectin and VCAM-1 increased further with the addition of greater numbers of PBPC, reaching maximum levels after the addition of 1x10(7) PBPC/well. PBPC-induced up-regulation of E-selectin, VCAM-1, and ICAM-1 had a maximum effect after approximately 6 hr, 12 hr, and 6 to 9 hr, respectively (n=3). The effects of fresh and frozen PBPC on HUVEC were similar (n=2). Compared to PBPC, PBL induced higher levels of E-selectin, VCAM-1, and ICAM-1 on HUVEC (n=2). The addition of CD2- cells to HUVEC induced an increase in E-selectin and VCAM-1 to levels 4 times greater than baseline, whereas the addition of CD2+ cells or debris did not elicit a substantial effect (n=2). Transwell permeable membranes prevented PBPC-induced up-regulation of E-selectin, VCAM-1, and ICAM-1 on HUVEC (n=2), suggesting that the mechanism of activation requires direct cell-cell contact. CONCLUSIONS: Porcine PBPC activate HUVEC, as suggested by an increase in surface E-selectin, VCAM-1, and ICAM-1 levels, and have a maximum effect after 9 hr. Freezing of PBPC does not affect PBPC-induced activation of HUVEC. PBL induce greater activation of HUVEC than do PBPC. CD2- cells are primarily responsible for PBPC-induced activation of HUVEC and direct cell-cell contact is required. Removal of CD2- cells before the administration of PBPC or the use of agents that interrupt PBPC-endothelial cell interactions may prevent or treat TM in baboons.

Anti-inflammatory activity of creatine supplementation in endothelial cells in vitro.

1 Creatine (CR) supplementation augments muscle strength in skeletal muscle cells by increasing intracellular energy pools. However, the effect of CR supplementation on endothelial cells remains to be clarified. 2 In this study, we investigated whether CR supplementation had any anti-inflammatory activity against human pulmonary endothelial cells in culture. 3 We confirmed that supplementation with 0.5 mM CR significantly increased both intracellular CR and phosphocreatine (PC) through a CR transporter while keeping intracellular ATP levels constant independent of CR supplementation and a CR transporter antagonist. 4 In the assay system of endothelial permeability, supplementation with 5 mM CR significantly suppressed the endothelial permeability induced by serotonin and H(2)O(2). 5 In cell adhesion experiments, supplementation with 5 mM CR significantly suppressed neutrophil adhesion to endothelial cells. 6 In the measurement of adhesion molecules, CR supplementation with more than 0.5 mM CR significantly inhibited the expressions of ICAM-1 and E-selectin on endothelial cells, and the inhibition was significantly suppressed by an adenosine A(2A) receptor antagonist. 7 The present study suggests that CR supplementation has anti-inflammatory activities against endothelial cells.

Role of selectins on IgE-mediated skin reaction.

Selectins play an important role on leukocytes infiltration into inflammatory tissues. To understand the role of selectins, we investigated the effects of selectin-IgG chimeras and anti selectin antibodies on the murine IgE-mediated skin inflammation model. Biphasic skin reactions were induced by intradermal challenge with ovalbumin (OA) to ears of actively sensitized mice. This reaction was characterized by immediate and late phase responses observed as which were induced via a rapid increase in capillary permeability and leukocyte infiltration, respectively. The expression of E-selectin mRNA was significantly increased to reach its highest level at 2 h after OA challenge. E-, P-, and L-selectin-IgG chimeras inhibited the late phase responses, i.e. ear swelling, neutrophil infiltration and eosinophil infiltration at 24 h after OA challenge in a dose-dependent manner at dose range of 0.1 - 10 mg kg(-1), i.v. Antiselectin antibodies did not inhibit the increase of ear swelling. But anti E- and P-selectin antibodies significantly inhibited neutrophil infiltration and eosinophil infiltration. These results indicate that selectins play an important role on the late phase response of the murine IgE-mediated skin inflammation model by mediating inflammatory cell adhesion to endothelium.

Role of adhesion molecules and platelets in TNF-induced adhesion of tumor cells to endothelial cells: implications for experimental metastasis.

Mechanisms for TNF-enhanced adhesion of tumor cells to endothelial cells were investigated for their in vivo relevance in a model of experimental metastasis. Mouse fibrosarcoma and thymoma cells were used to analyze TNF-modified adherence to three different mouse endothelioma cell lines and the results were compared to the in vivo colonization behavior of the tumor cells. TNF enhanced tumor cell adhesion in vitro and extravasation in vivo with similar characteristics. The role of different adhesion molecules in these experimental systems was tested. Blocking of ICAM-1, LFA-1, VCAM-1, E-selectin, and P-selectin did not reduce TNF-enhanced metastasis even though tumor cell adhesion in vitro was reduced. However, the correlation between inhibition of integrin binding and inhibition of metastasis achieved with competing peptides indicated an important role for extracellular matrix components in tumor cell attachment. Platelets play a dual role: although in vitro platelets prevented tumor cell adhesion to endothelial cells, in vivo platelet-depletion of mice reduced metastasis.

C-C and C-X-C chemokines trigger firm adhesion of monocytes to vascular endothelium under flow conditions.

In summary, our findings indicate that specific chemokines that are elaborated by endothelial cells after cytokine or endotoxin activation can play an essential role in monocyte recruitment beyond their chemoattractant activities. We show that this action is to translate initial monocyte tethering into firm adhesion via rapid leukocyte integrin activation. The in vitro model presented here provides a sensitive system for investigating the modulating ability of chemokines and reveals an important biological effect that is not predicted by results in simpler in vitro assays, such as measurement of calcium transients or chemotaxis. The surprising finding that the C-X-C chemokine IL-8 can trigger monocyte firm adhesion to vascular endothelium suggests a potential role for this chemokine in monocyte recruitment and underscores the biological complexity of the chemokine family.

Enhanced neutrophil and eosinophil adhesion in patients with primary Sjögren's syndrome.

OBJECTIVE: To study granulocyte adhesion to E-selectin, VCAM-1 and ICAM-1 in patients with primary Sjögren's syndrome (pSS). In previous studies diminished neutrophil adhesion has been shown as measured by the nylon fiber method. METHODS: Neutrophil and eosinophil adhesion to the adhesion molecules E-selectin, VCAM-1 and ICAM-1 were measured using transfected fibroblasts. The cell surface expression of the integrin proteins CD11a, CD11b, CD18 and CD29 on neutrophils was assayed by means of flow cytometry. RESULTS: Neutrophils and eosinophils from patients with pSS had elevated basal adhesion in the presence of Mn2+ as compared with controls (basal adhesion was considered to be the adhesion to untransfected fibroblasts). Granulocyte adhesion to E-selectin was also elevated. No differences were seen between patients and controls in cell surface expression of the integrin proteins CD11a, CD11b, CD18 and CD29 on neutrophils, nor was there any difference in these parameters between patients with and without extra glandular symptoms. CONCLUSIONS: These results suggest that blood neutrophils and eosinophils are activated in pSS. Accordingly they do not confirm results from earlier studies of impaired neutrophil adhesion in pSS.

Local hemodynamics affect monocytic cell adhesion to a three-dimensional flow model coated with E-selectin.

Monocyte adhesion to the endothelium depends on concentrations of receptors/ligands, local concentrations of chemoattractants, monocyte transport to the endothelial surface and hemodynamic forces. Monocyte adhesion to the inert surface of a three-dimensional perfusion model was shown to correlate inversely with wall shear stress, but was also affected by flow patterns which influenced the near-wall cell availability. We hypothesized that (a) under the same flow conditions, insolubilized E-selectin on the model's surface may mediate adhesive interactions at higher wall shear stresses, compared to an uncoated model, and (b) pulsatile flow may modify the adhesion profile obtained under steady flow. An axisymmetric flow model with a stenosis and a sudden expansion produced a range of wall shear stresses and a separated flow with recirculation and reattachment. Pre-activated U937 cells were perfused through the model under either steady (Re = 100, 140) or pulsatile (Remean = 107) flow. The velocity field was characterized through computational fluid dynamics and validated by inert particle tracking. Surface E-selectin greatly increased cell adhesion in all regions at Re = 100 and 140, compared to an uncoated model under the same flow conditions. In regions where the cells near the wall were abundant (taper and stenosis), adhesion to E-selectin correlated with the reciprocal of local wall shear stress when flow was steady. Pulsatile flow distributed the adherent cells more evenly throughout the coated model. Hence, characterizing both the local hemodynamics and the biological activity on the vessel wall is important in leukocyte adhesion.