Involvement of adhesion molecule in in vitro plaque-like formation of macrophages stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide.

BACKGROUND AND OBJECTIVE: Inflammatory agents, such as lipopolysaccharide (LPS), in periodontal pockets may promote atherogenesis by activating leukocytes. In our previous study, we developed a microchannel chip to observe the cell adhesion process in a fluid system. The objective of this investigation was to examine the mechanism by which periodontopathic bacterial LPS enhances plaque-like formation on a microchannel chip. MATERIAL AND METHODS: To evaluate the effect of Aggregatibacter actinomycetemcomitans LPS on the expression of adhesion molecules, e.g. intercellular adhesion molecule 1 (ICAM-1), lymphocyte function-associated antigen 1 (LFA-1) and L-selectin, on the surface of murine macrophage RAW264.7 cells, the expression of each adhesion molecule was examined by flow cytometry and western blot analysis. Moreover, a flow test on the microchannel chip involving anti-adhesion molecule antibodies was conducted to clarify which adhesion molecule is related to plaque-like formation of RAW264.7 cells. RESULTS: The expressions of ICAM-1 and LFA-1 on the surface of RAW 264.7 cells increased following 12 h culture with LPS; L-selectin expression was unaffected. An increase in ICAM-1 expression was also confirmed by western blot analysis. The flow test revealed that anti-ICAM-1 antibody inhibited plaque-like formation of LPS-stimulated macrophages on the micropillars of the microchannel chip. CONCLUSION: These findings indicate that ICAM-1 plays an important role in plaque-like formation of LPS-stimulated macrophages. Our microchannel chip is a suitable tool for the investigation of etiological factors of atherosclerosis, including periodontitis, in vitro.

Expression of endothelia and lymphocyte adhesion molecules in bronchus-associated lymphoid tissue (BALT) in adult human lung.

BACKGROUND: Bronchus-associated lymphoid tissue (BALT) is the secondary lymphoid tissue in bronchial mucosa and is involved in the development of bronchopulmonary immune responses. Although migration of lymphocytes from blood vessels into secondary lymphoid tissues is critical for the development of appropriate adaptive immunity, the endothelia and lymphocyte adhesion molecules that recruit specific subsets of lymphocytes into human BALT are not known. The aim of this study was to determine which adhesion molecules are expressed on lymphocytes and high endothelial venules (HEVs) in human BALT. METHODS: We immunostained frozen sections of BALT from lobectomy specimens from 17 patients with lung carcinoma with a panel of monoclonal antibodies to endothelia and lymphocyte adhesion molecules. RESULTS: Sections of BALT showed B cell follicles surrounded by T cells. Most BALT CD4+ T cells had a CD45RO+ memory phenotype. Almost all BALT B cells expressed alpha4 integrin and L-selectin. In contrast, 43% of BALT T cells expressed alpha4 integrin and 20% of BALT T cells expressed L-selectin. Almost all BALT lymphocytes expressed LFA-1. HEVs, which support the migration of lymphocytes from the bloodstream into secondary lymphoid tissues, were prominent in BALT. All HEVs expressed peripheral node addressin, most HEVs expressed vascular cell adhesion molecule-1, and no HEVs expressed mucosal addressin cell adhesion molecule-1. CONCLUSION: Human BALT expresses endothelia and lymphocyte adhesion molecules that may be important in recruiting naive and memory/effector lymphocytes to BALT during protective and pathologic bronchopulmonary immune responses.

The skin homing receptor cutaneous leucocyte-associated antigen (CLA) is up-regulated by Leishmania antigens in T lymphocytes during active cutaneous leishmaniasis.

The cutaneous leucocyte-associated antigen receptor (CLA) can direct Leishmania-specific T lymphocytes towards inflamed skin lesions. Homing receptors [CLA, lymphocyte-associated antigen 1 (LFA-1) or CD62L] were analysed in lymphocytes from blood and cutaneous leishmaniasis (CL) lesions. CL patients with active lesions (A-CL) presented lower levels of T lymphocytes expressing the CLA(+) phenotype (T CD4(+) = 10.4% +/- 7.5% and T CD8(+) = 5.8% +/- 3.4%) than did healthy subjects (HS) (T CD4(+) = 19.3% +/- 13.1% and T CD8(+) = 21.6% +/- 8.8%), notably in T CD8(+) (P < 0.001). In clinically cured patients these percentages returned to levels observed in HS. Leishmanial antigens up-regulated CLA in T cells (CLA(+) in T CD4(+) = 33.3% +/- 14.1%; CLA(+) in T CD8(+) = 22.4% +/- 9.4%) from A-CL but not from HS. An enrichment of CLA(+) cells was observed in lesions (CLA(+) in T CD4(+) = 45.9% +/- 22.5%; CLA(+) in T CD8(+) = 46.4% +/- 16.1%) in comparison with blood (CLA(+) in T CD4(+) = 10.4% +/- 7.5%; CLA(+) in T CD8(+) = 5.8% +/- 3.4%). Conversely, LFA-1 was highly expressed in CD8(+) T cells and augmented in CD4(+) T from peripheral blood of A-CL patients. In contrast, CD62L was not affected. These results suggest that Leishmania antigens can modulate molecules responsible for migration to skin lesions, potentially influencing the cell composition of inflammatory infiltrate of leishmaniasis or even the severity of the disease.

Effects of IS-741, a synthetic anti-inflammatory agent, on bleomycin-induced lung injury in mice.

Bleomycin (BLM)-induced lung injury consists of excessive inflammatory cell infiltration and fibrosis. IS-741 has been reported to be an anti-inflammatory drug through an inhibitory action on cell adhesion. In this study we investigated whether IS-741 could inhibit the progression of pulmonary fibrosis through inflammatory cell infiltration. Lung injury was induced in female C57BL/6 mice by intratracheal instillation of BLM. IS-741 was administered daily intraperitoneally. The hydroxyproline content and fluid content in the lung on Day 28 were significantly lower in the IS-741-treated mice. The histological degree of lung injury or fibrosis was reduced in IS-741-treated mice. Administration of IS-741 caused significant reduction in the absolute number of total cells, monocyte chemoattractant protein (MCP)-1, and cysteinyl leukotriene (cysLTs) levels in bronchoalveolar lavage fluid on Day 7. Furthermore, the hydroxyproline content was significantly lower in IS-741-treated mice even though IS-741 was started on Day 14 after BLM instillation. Treatment with IS-741 had an inhibitory effect on BLM-induced lung injury and fibrosis via the repression of MCP-1 or cysLTs in this murine experimental model.

Quantitation of biomolecules conjugated to nanoparticles by enzyme hydrolysis.

A novel method for quantifying biomolecules immobilised onto gold and silver nanoparticles is reported; fluorescent-labelled antibodies and DNA are hydrolysed on the surface of the nanoparticles by the addition of trypsin and DNase I, respectively, resulting in the release of the quantifiable fluorescent label into the bulk solution.

The value of leukocyte adhesion molecules in patients after ischemic stroke.

Leukocyte recruitment and inflammatory response play an important patho-physiologic role after cerebral ischemia. This study aimed to evaluate whether leukocyte adhesion molecules can predict clinical outcome in patients after ischemic stroke. We prospectively examined serial changes in p-selectin glycoprotein ligand-1 (PSGL-1), macrophage antigen-1 (Mac-1), and lymphocyte function-associated antigen-1 (LFA-1) expression by leukocyte subsets using flow cytometry at various time points in 65 acute ischemic stroke patients and 60 controls. PSGL-1 expression on neutrophils and monocytes was significantly higher from day 1 to 90 after stroke as compared with control subjects (p < 0.05). The expression of monocyte Mac-1, LFA-1, and neutrophil Mac-1 were also significantly increased on days 1 and 7 after stroke than in control subjects (p < 0.05). Neutrophil PSGL-1 expression on day 1 was significantly higher in patients with early neurologic deterioration (END) (p < 0.01). Monocyte Mac-1 expression positively correlated with National Institutes of Health Stroke Scale (NIHSS) scores on admission (p = 0.013, gamma = 0.318). Underlying disease of diabetes mellitus and NIHSS score on admission were independently associated with 3-month outcome. The expressions of leukocyte adhesion molecules on admission are significantly increased in patients with acute ischemic stroke. This study shows that higher neutrophil PSGL-1 expression on admission may imply a higher risk for END and that monocyte Mac-1 expression on admission reflects the severity of ischemic stroke on admission.

Analysis of adhesion molecules, target cells, and role of IL-2 in human FOXP3+ regulatory T cell suppressor function.

FOXP3(+) regulatory T cells (Tregs) are central to the maintenance of self-tolerance and immune homeostasis. The mechanisms of action and cellular targets for Treg-mediated suppression remain controversial. The critical adhesion molecules utilized by Tregs for the interaction with their target cells have not been well characterized. We show that human CD4(+)FOXP3(+)CD25(high) cells (hTregs) suppress the activation of mouse responders as efficiently as mouse Tregs. LFA-1 (CD11a/CD18) on the hTregs is critical for their suppressor function, since suppression can be reversed with blocking anti-hCD11a or anti-hCD18 mAb. Tregs from patients with LFA-1 deficiency fail to suppress human and mouse responders. Mouse CD4(+) T cells deficient in ICAM-1 can be suppressed by hTregs, indicating that the hTregs target mouse dendritic cells (DCs) through the binding of human LFA-1 to mouse ICAM-1. Coculture of mouse DCs with hTregs, but not hTregs from LFA-1-deficient patients, prevented the up-regulation of CD80/CD86 on the DCs and their capacity to activate responder T cells. Lastly, IL-2 is not required for hTreg suppressor function under optimal stimulatory condition and IL-2 consumption plays no role in hTreg-mediated suppression. Taken together, one of the mechanisms of Treg-mediated suppression functions across species and mediates an LFA-1/ICAM-1-dependent interaction between Tregs and DCs.

Implication of adhesion molecules in inflammation of the common bile duct in patients with secondary cholangitis due to biliary obstruction.

BACKGROUND/AIMS: The aim of the present study was to investigate the participation of the adhesion molecules and their ligands in the inflammatory process in secondary cholangitis. METHODOLOGY: Biopsy specimens were collected from the common bile duct from 29 patients with extrahepatic bile obstruction. Immunohistochemistry was used to study the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin, LFA-1, PECAM-1, Mac-1, and VLA-4). The patients were categorized into 2 groups - chronic exacerbated cholangitis and chronic sclerotic cholangitis. RESULTS: An increased ICAM-1 expression was demonstrated and also de novo VCAM-1 and E-selectin appearance on the endothelium of microvessels in chronic exacerbated cholangitis. The inflammatory cells were strongly LFA-1-, Mac-1- positive. In chronic sclerotic cholangitis the E-selectin expression persisted on vascular endothelium in the fibrous tissue and reduced inflammatory infiltrate showed LFA-1 and VLA-4 positivity. The newly formed vessels in the fibrous connective tissue were PECAM-1-positive. CONCLUSIONS: From these results, it could be concluded that in chronic exacerbated cholangitis with complete bile obstruction the firm adhesion is mediated by ICAM-1/LFA-1 and ICAM-1/Mac-1 and less by VCAM-1/VLA-4 pathways. In chronic sclerotic cholangitis, caused by incomplete obstruction, the firm adhesion was maintained by ICAM-1/LFA-1 and less by VCAM-1/VLA-4 pathways. It seems likely that PECAM-1 and VCAM-1/VLA-4 play additional roles in neutrophil recruitment.

Bosentan regulates the expression of adhesion molecules on circulating T cells and serum soluble adhesion molecules in systemic sclerosis-associated pulmonary arterial hypertension.

OBJECTIVES: To study the expression of adhesion molecules in patients with systemic sclerosis (SSc) with and without pulmonary arterial hypertension (PAH) and the effects of therapy with the endothelin-1 (ET-1) receptor antagonist, bosentan. METHODS: In all, 35 patients with SSc and 25 healthy donors (HD) were selected for this study. Of 35 patients, 10 had isolated PAH assessed by Doppler echocardiography and treated with bosentan. Peripheral blood (PB) lymphocytes were isolated by density gradient centrifugation, and the expression of lymphocyte function-associated antigen-1 (LFA-1), very late antigen-4 (VLA-4) and L-selectin on CD3 T cells was assessed by double immunofluorescence and flow-cytometry. As endothelial activation markers, serum soluble P-selectin, platelet/endothelial cell adhesion molecule (PECAM)-1, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1 and von Willebrand factor (vWF) antigen were assessed by ELISA. In patients with SSc-PAH, T cell subsets and soluble endothelial markers were assessed at baseline and after 6 and 12 months of bosentan therapy. RESULTS: In patients with SSc-PAH, serum soluble ICAM-1, VCAM-1, P-selectin and PECAM-1 levels were higher than in HD at baseline and fell to normal values after 12 months of bosentan therapy. CD3-LFA1 T cells were significantly higher in PAH-SSc at baseline than in HD or SSc and significantly decreased after therapy. CD3-L-selectin T cells were significantly lower in SSc-PAH at baseline than in HD or SSc and rose to normal levels after bosentan therapy. CONCLUSIONS: This study confirms that endothelial activation occurs in SSc, and suggests that changes in the T cell/endothelium interplay take place in SSc-associated PAH. Bosentan seems to be able to hamper these changes and restore T cell functions in these patients.

NKT cell activation mediates neutrophil IFN-gamma production and renal ischemia-reperfusion injury.

Previous work has shown that ischemia-reperfusion (IR) injury (IRI) is dependent on CD4(+) T cells from naive mice acting within 24 h. We hypothesize that NKT cells are key participants in the early innate response in IRI. Kidneys from C57BL/6 mice were subjected to IRI (0.5, 1, 3, and 24 h of reperfusion). After 30 min of reperfusion, we observed a significant increase in CD4(+) cells (145% of control) from single-cell kidney suspensions as measured by flow cytometry. A significant fraction of CD4(+) T cells expressed the activation marker, CD69(+), and adhesion molecule, LFA-1(high). Three hours after reperfusion, kidney IFN-gamma-producing cells were comprised largely of GR-1(+)CD11b(+) neutrophils, but also contained CD1d-restricted NKT cells. Kidney IRI in mice administered Abs to block CD1d, or deplete NKT cells or in mice deficient of NKT cells (Jalpha18(-/-)), was markedly attenuated. These effects were associated with a significant decrease in renal infiltration and, in activation of NKT cells, and a decrease in IFN-gamma-producing neutrophils. The results support the essential role of NKT cells and neutrophils in the innate immune response of renal IRI by mediating neutrophil infiltration and production of IFN-gamma.

LFA-1 and VLA-4 involved in human high proliferative potential-endothelial progenitor cells homing to ischemic tissue.

Cumulative evidences have revealed that endothelial progenitor cell (EPC) transplantation can promote the neovascularization in ischemic tissue, but the mechanism of EPCs homing to the site of ischemia is poorly understood. In this study, to investigate the mechanism of human umbilical cord blood-derived high proliferative potential-endothelial progenitor cells (HPP-EPCs) homing to ischemic tissue we evaluated the expression of lymphocyte function-associated antigen-1 (LFA-1, or CD11a/CD18) and very late antigen-4 (VLA-4, or CD49d/CD29) in EPCs and the changes of expression level of their ligands, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), in ischemic tissue and performed the adhesion and migration assays to analyze the interaction between the receptors and ligands. Furthermore, we studied the roles of LFA-1 and VLA-4 in EPC homing in an ischemic model of mice. The results show that LFA-1 andVLA-4 were expressed in HPPEPCs and ICAM-1 and VCAM-1 were expressed in vessel endothelium in ischemic tissues. The pre-incubation of HPP-EPCs with neutralizing antibodies against CD11a or CD49d reduced adhesion and migration of HPP-EPCs in vitro and reduced recovery of hind-limb blood flow, capillary density and incorporation of HPP-EPC into ischemic tissues in vivo. Furthermore, the pre-incubation of HPP-EPCs with the combination of CD11a and CD49d antibodies led to synergistically negative effects on adhesion and transmigration of HPP-EPCs in vitro, and on the homing of HPP-EPCs to ischemic tissue and on neovascularization capacity in vivo. These results indicate that LFA-1 andVLA-4 are involved in HPP-EPC homing to ischemic tissues.

Evidence for cell adhesion-mediated drug resistance of multiple myeloma cells in vivo.

BACKGROUND/AIMS: Multiple myeloma is an incurable disease and patients eventually die of disease progression due to drug resistance. VLA-4 (very late antigen 4), VCAM (vascular adhesion molecule), LFA-1 (leukocyte function-associated antigen 1), and ICAM-1 (intercellular adhesion molecule 1)-mediated adhesion of myeloma cells to bone marrow stromal cells induces primary multidrug resistance in vitro. Based on these preclinical data we hypothesized that myeloma cells with strong adhesion - due to strong expression of adhesion molecules on the cell surface - are selected by chemotherapy in patients. To prove this hypothesis we determined the expression levels of adhesion molecules in 31 multiple myeloma patients by flow cytometry. METHODS: A 3-color stain with CD38, CD138 and antibodies against VLA-4, ICAM-1, LFA-1, and VCAM was performed. The patients were either at diagnosis (chemo-naive; n=17) or at relapse (pre-treated; n=15). Furthermore, the response to the next chemotherapy of chemo-naive patients was correlated with the expression levels of adhesion molecules. RESULTS: ICAM-1, VLA-4, and VCAM expression was higher in pre-treated patients than in chemo-naive patients and the expression levels increased with the number of chemotherapy regimens. Primarily multidrug-resistant patients had significantly higher expression levels of VLA-4 and ICAM-1 than responders. CONCLUSION: This study suggests that multiple myeloma cells expressing high levels of VLA-4 and ICAM-1 are drug resistant and that such a subpopulation of cells is selected by chemotherapy.

Serum monocyte chemoattractant protein-1 and monocyte adhesion molecules in type 1 diabetic patients with nephropathy.

BACKGROUND: Monocyte chemoattractant protein (MCP)-1 is suggested to be implicated in the pathogenesis of diabetic nephropathy by activating and recruiting monocytes to the glomerulus via regulation of adhesion molecule expressions. The aim of this study was to test potential associations between serum concentrations of MCP-1, monocyte expression of Mac-1 and LFA-1 and nephropathy in type 1 diabetes mellitus. METHODS: Serum MCP-1 levels and expression of monocyte adhesion molecules in 51 type 1 diabetic patients with and without diabetic nephropathy were compared with matched 15 healthy control subjects. Concentrations of serum MCP-1 were determined by enzyme-linked immunosorbent assays whereas monocyte expression of adhesion molecules Mac-1 and LFA-1 was measured by flow cytometry. RESULTS: Serum MCP-1 levels and expression of Mac-1, but not LFA-1, were significantly higher in diabetic patients compared with controls. The mean serum MCP-1 level was 137.2 +/- 71.4 pg/mL in control patients, whereas it was 246.2 +/- 114.9 pg/ml in diabetic patients (p = 0.002). Serum MCP-1 levels were positively correlated with HbA1c and plasma fasting glucose levels. There was no difference in serum levels of MCP-1 and expression of monocyte adhesion molecules between type 1 diabetic patients with and without diabetic nephropathy. CONCLUSIONS: In type 1 diabetic patients, the levels of circulating MCP-1 concentration and expression of Mac-1 is mostly influenced by glycemic control rather than the existence of diabetic nephropathy.

Selective recruitment of src family kinase Hck by leukocyte integrin alphaMbeta2 but not alphaLbeta2 or alphaXbeta2.

Integrins are type I heterodimeric (alpha/beta) cell adhesion molecules. They trigger cell-signaling by recruiting cytosolic molecules to their cytoplasmic tails. Integrin alpha cytoplasmic tail contributes towards integrin function specificity, an important feature of integrins having different alpha subunits but sharing the same beta subunit. Herein, we show that the src family kinase Hck co-capped selectively with leukocyte integrin alpha(M)beta(2) but not alpha(L)beta(2) or alpha(X)beta(2). This was disrupted when the alpha(M) cytoplasmic tail was substituted with that of alpha(L) or alpha(X). Co-capping was recovered by alpha(L) or alpha(X) cytoplasmic tail truncation or forced separation of the alpha and beta cytoplasmic tails via salt-bridge disruption.

Post-transplant reactivation of hepatitis C virus: lymphocyte infiltration and the expression of adhesion molecules and their ligands in liver allografts.

Hepatitis C virus (HCV) recurrence after liver transplantation has been associated with chronic rejection. Biopsies from 10 patients with post-transplant HCV were examined for expression of adhesion molecules ICAM-1, VCAM-1, and ELAM-1, number of lymphocytes positive for their ligands LFA-1, VLA-4, and SLeX, and activation markers MHC class II antigens and IL2-R by immunohistochemistry. The phenotypes of the graft-infiltrating lymphocytes were determined. Results were compared to those for patients with normal graft function or rejection. Five recipients with HCV reactivation and one with de novo HCV had a biopsy available showing induction of ICAM-1 in sinusoidal endothelium (p<0.05) and hepatocytes (p<0.01), and Class II antigens in hepatocytes (p<0.01), compared to normal controls. Lymphocytes in the graft infiltrate expressed LFA-1, VLA-4, and Class II antigens, but IL2-R was not significantly expressed. CD3+, CD4+, and CD8+ cells were observed. In our study, HCV recurrence was not associated with acute or chronic rejection, and the inflammation was due to the viral infection.

Expression of HIV receptors, alternate receptors and co-receptors on tonsillar epithelium: implications for HIV binding and primary oral infection.

BACKGROUND: Primary HIV infection can develop from exposure to HIV in the oral cavity. In previous studies, we have documented rapid and extensive binding of HIV virions in seminal plasma to intact mucosal surfaces of the palatine tonsil and also found that virions readily penetrated beneath the tissue surfaces. As one approach to understand the molecular interactions that support HIV virion binding to human mucosal surfaces, we have examined the distribution of the primary HIV receptor CD4, the alternate HIV receptors heparan sulfate proteoglycan (HS) and galactosyl ceramide (GalCer) and the co-receptors CXCR4 and CCR5 in palatine tonsil. RESULTS: Only HS was widely expressed on the surface of stratified squamous epithelium. In contrast, HS, GalCer, CXCR4 and CCR5 were all expressed on the reticulated epithelium lining the tonsillar crypts. We have observed extensive variability, both across tissue sections from any tonsil and between tonsils, in the distribution of epithelial cells expressing either CXCR4 or CCR5 in the basal and suprabasal layers of stratified epithelium. The general expression patterns of CXCR4, CCR5 and HS were similar in palatine tonsil from children and adults (age range 3-20). We have also noted the presence of small clusters of lymphocytes, including CD4+ T cells within stratified epithelium and located precisely at the mucosal surfaces. CD4+ T cells in these locations would be immediately accessible to HIV virions. CONCLUSION: In total, the likelihood of oral HIV transmission will be determined by macro and micro tissue architecture, cell surface expression patterns of key molecules that may bind HIV and the specific properties of the infectious inoculum.

Immunohistochemical characterization of glomerular inflammatory cells and expression of adhesion molecules in anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in WKY rats with monoclonal anti-GBM antibodies of different subclasses.

According to previous report, adhesion of CD8-positive cells and macrophages to glomerular endotherial cells through the lymphocyte function-associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) pathway is crucial for the initiation and subsequent progression of anti-glomerular basement membrane (anti-GBM) antibody-induced glomerulonephritis (anti-GBM nephritis) in WKY rats. In the present study glomerular inflammatory cell infiltration and LFA-1/ICAM-1 expression were examined in anti-GBM nephritis induced in WKY rats with monoclonal anti-GBM antibodies of different subclasses: IgG1, IgG2a, and IgG2b. The IgG2a and IgG2b subclasses induced significant proteinuria from day 3 as compared with the IgG1 subclass. Glomerular infiltration of macrophages and CD8-positive cells after administration of IgG2a and IgG2b subclass antibodies was significantly elevated compared to that for the IgG1 subclass. The intensity of glomerular ICAM-1 immunostaining by the IgG2a and IgG2b subclass antibodies tended to be stronger than that by the IgG1 subclass. Glomerular LFA-1-positive cell infiltration by the IgG2a and IgG2b subclasses was significantly higher than that of the IgG1 subclass. These results demonstrate that monoclonal antibodies belonging to the IgG2a and IgG2b subclasses strongly induce glomerular infiltration of inflammatory cells and expression of adhesion molecules in rat anti-GBM nephritis.

Multifocal structure of the T cell - dendritic cell synapse.

The structure of immunological synapses formed between murine naive T cells and mature dendritic cells has been subjected to a quantitative analysis. Immunofluorescence images of synapses formed in the absence of antigen show a diffuse synaptic accumulation of CD3 and LFA-1. In electron microscopy, these antigen-free synapses present a number of tight appositions (cleft size approximately 15 nm), all along the synapse. These tight appositions cover a significantly larger surface fraction of antigen-dependent synapses. In immunofluorescence, antigen-dependent synapses show multiple patches of CD3 and LFA-1 with a variable overlap. A similar distribution is observed for PKCtheta and talin. A concentric organization characteristic of prototypical synapses is rarely observed, even when dendritic cells are paralyzed by cytoskeletal poisons. In T-DC synapses, the interaction surface is composed of several tens of submicronic contact spots, with no large-scale segregation of CD3 and LFA-1. As a comparison, in T-B synapses, a central cluster of CD3 is frequently observed by immunofluorescence, and electron microscopy reveals a central tight apposition. Our data show that it is inappropriate to consider the concentric structure as a "mature synapse" and multifocal structures as immature.

CD226 is expressed on the megakaryocytic lineage from hematopoietic stem cells/progenitor cells and involved in its polyploidization.

OBJECTIVES: In this study, we examined the expression of CD226 on megakaryocytic, granulocytic and erythroid lineage from hematopoietic stem cells/progenitor cells in adult and fetus and its potential role in megakaryocytic maturation. METHODS: CD34(+) cells from adult and fetus were induced to differentiate toward the megakaryocytic lineage by thrombopoietin (TPO) and the granulocytic lineage by granulocyte colony-stimulating factor (G-CSF), respectively. Mononuclear cells from fetal liver and CD34(+) cells from adult were induced to differentiate toward erythroid-lineage by erythropoiesis (EPO). We investigated the expression of CD226 and lymphocyte function associated antigen-1 (LFA-1) (CD11a) during hemopoiesis. We also studied the effect of CD226 monoclonal antibody (MoAb) and LFA-1 MoAb on megakaryocyte with antibody cross-liking technique. RESULTS: CD34(+) cells from adult and fetus and TPO-induced CD41(+) cells all expressed CD226 molecule. CD226 was not expressed on erythroid progenitor cells and erythroblasts and most cells of granulocytic lineage although G-CSF induced a significant increase of the expression of CD226 on CD34(+) cells in early period of time. CD226 MoAb acts on megakaryocytes by inducing intracellular calcium mobilization. The expression of LFA-1 decreased significantly at late stage of differentiation and maturation of fetal megakaryocytes whereas the expression of LFA-1 on adult megakaryocytes retained at a high level. CD226 MoAb in combination with LFA-1 MoAb shifted the ploidy of generated megakaryocytes from adult-derived CD34(+) cells to higher classes significantly although CD226 and LFA-1 MoAb slightly increased the ploidy of the generated megakaryocytes individually. CD226 MoAb or LFA-1 MoAb or CD226 MoAb plus LFA-1 MoAbs did not increase the ploidy of the generated megakaryocytes from fetus-derived CD34(+) cells. CONCLUSION: CD226 molecules play an important role in maturation of the megakaryocytes in combination with LFA-1.