Characterization of the 4C8 antigen involved in transendothelial migration of CD26(hi) T cells after tight adhesion to human umbilical vein endothelial cell monolayers.
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In extravasation of T cells, little is known about the mechanisms of transendothelial migration subsequent to the T cells' tight adhesion to endothelium. To investigate these mechanisms, we developed a monoclonal antibody (mAb), termed anti-4C8, that blocks transmigration but not adhesion in a culture system in which high CD26-expressing (CD26(hi)) T cells preferentially migrate through human umbilical vein endothelial cell (HUVEC) monolayers cultured on collagen gels. Anti-4C8 reacted with all CD3(+) T cells and monocytes but not neutrophils or HUVECs. The structure defined by this antibody was an 80-kD molecule. The mAb at 1 mug/ml inhibited 80-90% of migration of CD3(+) T cells through unstimulated and interferon gamma-stimulated HUVEC monolayers without interfering with adhesion and cell motility. When added to the cultures after the adhesion, anti-4C8 completely blocked subsequent transmigration of adherent T cells. Phase-contrast and electron microscopy revealed that T cells are arrested at the intercellular junctions of HUVECs in the presence of anti-4C8. Anti-4C8 exhibited agonistic effects on resting T cells without other stimuli under culture conditions in which anti-4C8 can stimulate T cells. First, in the checkerboard assay using collagen gels, the antibody promoted chemokinetic migration of the cells in a dose-dependent manner from 0.1 to 10 mug/ml. The predominant population of T cells that migrated into collagen gels with impregnated anti-4C8 were CD26(hi). Second, solid-phase-immobilized anti-4C8 induced adhesion of T cells to the substrate, often with polarizations in cell shape and large pseudopods rich in filamentous (F-) actin. Third, soluble anti-4C8 augmented F-actin content preferentially in CD26(hi) T cells when added to T cells at a high dose of 10 mug/ml. Finally, both anti-4C8-induced chemokinetic migration and transendothelial migration were inhibited by pretreatment of T cells with pertussis toxin. These findings suggest that stimulation via the 4C8 antigen increases cell motility of CD26(hi) cells with profound cytoskeletal changes through signaling pathways including G proteins. The 4C8 antigen may be involved in preferential transmigration of CD26(hi) cells adherent to HUVECs. (+info)
Isolation and expression of novel human glutamate carboxypeptidases with N-acetylated alpha-linked acidic dipeptidase and dipeptidyl peptidase IV activity.
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Hydrolysis of the neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) by N-acetylated alpha-linked acidic dipeptidase (NAALADase) to release glutamate may be important in a number of neurodegenerative disorders in which excitotoxic mechanisms are implicated. The gene coding for human prostate-specific membrane antigen, a marker of prostatic carcinomas, and its rat homologue glutamate carboxypeptidase II have recently been shown to possess such NAALADase activity. In contrast, a closely related member of this gene family, rat ileal 100-kDa protein, possesses a dipeptidyl peptidase IV activity. Here, we describe the cloning of human ileal 100-kDa protein, which we have called a NAALADase- "like" (NAALADase L) peptidase based on its sequence similarity to other members of this gene family, and its inability to hydrolyze NAAG in transient transfection experiments. Furthermore, we describe the cloning of a third novel member of this gene family, NAALADase II, which codes for a type II integral membrane protein and which we have localized to chromosome 11 by fluorescent in situ hybridization analysis. Transient transfection of NAALADase II cDNA confers both NAALADase and dipeptidyl peptidase IV activity to COS cells. Expression studies using reverse transcription-polymerase chain reaction and Northern blot hybridization show that NAALADase II is highly expressed in ovary and testis as well as within discrete brain areas. (+info)
Phenotypic analysis of lymphocytes and monocytes/macrophages in peripheral blood and bronchoalveolar lavage fluid from patients with pulmonary sarcoidosis.
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BACKGROUND: The granulomatous inflammation in sarcoidosis is driven by the interplay between T cells and macrophages. To gain a better understanding of this process the expression by these cells of cell surface activation markers, co-stimulatory molecules, and adhesion molecules was analysed. METHODS: CD4+ and CD8+ T lymphocytes from peripheral blood (PBL) or bronchoalveolar lavage (BAL) fluid, as well as paired peripheral blood monocytes and alveolar macrophages from 27 patients with sarcoidosis were analysed by flow cytometry. RESULTS: CD26, CD54, CD69, CD95, and gp240 were all overexpressed in T cells from BAL fluid compared with those from PBL in both the CD4+ and CD8+ subsets, while CD57 was overexpressed only in BAL CD4+ cells. In contrast, CD28 tended to be underexpressed in the BAL T cells. Monocyte/macrophage markers included CD11a, CD11b, CD11c, CD14, CD16, CD54, CD71, CD80 and CD86 and HLA class II. CD11a expression in alveolar macrophages (and peripheral blood monocytes) was increased in patients with active disease and correlated positively with the percentage of BAL lymphocytes. Expression of CD80 in macrophages correlated with the BAL CD4/CD8 ratio. CONCLUSIONS: Our data indicate substantial activation of both CD4+ and CD8+ lung T cells in sarcoidosis. There were also increased numbers of BAL lymphocytes whose phenotypic characteristics have earlier been associated with clonally expanded, replicatively senescent cells of the Th1 type. (+info)
Purification of a new intestinal anti-proliferative factor from normal human small intestine.
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Previous studies suggest that intestinal cell proliferation may be controlled by endogenous mitosis inhibitors. We describe here the isolation of a protein named intestinal anti-proliferative factor (IAF) from human small intestine. Successive DEAE anion exchange, isoelectric focusing and gel filtration chromatographies led to a purified anti-proliferative protein fraction used to produce antibodies. Using these antibodies as affinity chromatography ligand, IAF was purified from human small intestine cytosolic fraction. IAF was a potent inhibitor of adenocarcinoma colon cells (HT-29 D4 line) DNA synthesis and proliferation with 50% inhibition observed at picomolar concentrations. Analyzed on SDS/PAGE under reducing conditions, this protein migrates with an apparent molecular mass of 120 kDa and amino acid sequence of two internal peptides displays no homology with another listed protein. Cell cycle studies showed that the growth inhibitory effect was maximal between mid G1 and early S phases. Moreover, flow cytometry studies demonstrated that IAF inhibited the progression of HT-29 D4 cells from G1 to S phase. Northern blot analysis using a dipeptidyl peptidase i.v. probe revealed that the growth arrest mediated by IAF was not linked to differentiation processes. By Western blotting with polyclonal antibodies against IAF, we found that this protein was not detected in differentiated colonic carcinoma. Our results suggest that IAF might regulate intestinal cell proliferation. (+info)
Hydrolysis of alphas1- and beta-casein-derived peptides with a broad specificity aminopeptidase and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2.
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Aminopeptidase hydrolysis of alpha(s)1 - and beta-casein-derived synthetic peptides containing non-consecutive and consecutive proline residues was characterised. Aminopeptidase P (Pep P) (EC 3.4.11.9) or post-proline dipeptidyl aminopeptidase (PPDA) (EC 3.4.14.5) along with lysine-paranitroanilide hydrolase (KpNA-H) (EC 3.4.11.1) activities are required in the degradation of peptides containing non-consecutive proline residues. However, both Pep P and PPDA along with KpNA-H are required for hydrolysis of peptides containing consecutive proline residues. The results demonstrate the mechanism by which combinations of purified general and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2 hydrolyse peptides containing proline residues. (+info)
Constitutive expression of CD26/dipeptidylpeptidase IV on peripheral blood B lymphocytes of patients with B chronic lymphocytic leukaemia.
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We have investigated the expression of the ectoenzyme dipeptidylpeptidase IV (DPP IV)/CD26 on lymphocytes obtained from patients with B chronic lymphocytic leukaemia (B-CLL) and compared it with healthy subjects. Using two-colour immunofluorescence analysis with CD26 and CD20 or CD23 monoclonal antibodies, CD26 was found undetectable on peripheral resting B-cells (CD20+ CD23-) from normal donors whereas it was expressed on B-cells activated in vitro with interleukin (IL)-4 and Staphylococcus aureus strain cowan I (CD20+ CD23+). The expression of CD26 on leukaemic B-cells (CD20+ CD23+) was clearly induced in 22 out of 25 patients examined. Consequently, induced levels of CD26 cell surface expression on either normal activated and malignant B-cells coincided with the enhancement of DPP IV activity detected on the surface of these cells. Reverse transcription polymerase chain reaction analyses showed that the transcript levels of the CD26 gene was higher in normal activated B-cells and B-CLL cells than in resting B-cells, suggesting that CD26 was expressed at the level of transcriptional activation. These observations provide evidence of the abnormal expression of DPPIV/CD26 in B-CLL which, therefore, may be considered as a novel marker for B-CLL. Further investigation in relation to CD26 expression and other B malignancies needs to be defined. (+info)
Gastric inhibitory polypeptide (GIP) is an important insulin-releasing hormone of the enteroinsular axis that, like glucagon-like peptide 1(7-36) amide (tGLP-1), has a functional profile of possible therapeutic value for type 2 diabetes. Both incretin hormones are rapidly inactivated in plasma by the exopeptidase dipeptidyl peptidase (DPP) IV. The present study examined the ability of NH2-terminal modification of human GIP to protect from plasma degradation and enhance insulin-releasing and antihyperglycemic activity. Degradation of GIP by incubation at 37 degrees C with purified DPP IV was clearly evident after 4 h (54% intact). After 12 h, >60% of GIP was converted to GIP(3-42), whereas >99% of NH2-terminally modified Tyr1-glucitol GIP remained intact. Tyr1-glucitol GIP was similarly resistant to serum degradation. The formation of GIP(3-42) was almost completely abolished by inhibition of plasma DPP IV with diprotin A. Effects of GIP and Tyr1-glucitol GIP were examined in Wistar rats after intraperitoneal injection of either peptide (10 nmol/kg) together with glucose (18 mmol/kg). Plasma glucose concentrations were significantly lower and insulin concentrations higher after both peptides compared with glucose alone. More importantly, individual glucose values at 15 and 30 min together with the areas under the curve (AUCs) for glucose were significantly lower after administration of Tyr1-glucitol GIP compared with GIP (AUC 255 +/- 33 vs. 368 +/- 8 mmol x l(-1) x min(-1), respectively; P < 0.01). This was associated with a significantly greater and more protracted insulin response after Tyr1-glucitol GIP than GIP (AUC 773 +/- 41 vs. 639 +/- 39 ng x ml(-1) x min(-1); P < 0.05). These data demonstrate that Tyr1-glucitol GIP displays resistance to plasma DPP IV degradation and exhibits enhanced antihyperglycemic activity and insulin-releasing action in vivo. (+info)
Microdomain-dependent regulation of Lck and Fyn protein-tyrosine kinases in T lymphocyte plasma membranes.
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Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins. (+info)