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Androgenesis represents one of the most fascinating examples of cell differentiation in plants. In barley, the conversion of stressed uninucleate microspores into embryo-like structures is highly efficient. One of the bottlenecks in this process is the successful release of embryo-like structures out of the exine wall of microspores. In the present work, morphological and biochemical studies were performed during the transition from multicellular structures to globular embryos. Exine wall rupture and subsequent globular embryo formation were observed only in microspores that divided asymmetrically. Independent divisions of the generative and the vegetative nuclei gave rise to heterogeneous multicellular structures, which were composed of two different cellular domains: small cells with condensed chromatin structure and large cells with normal chromatin structure. During exine wall rupture, the small cells died and their death marked the site of exine wall rupture. Cell death in the small cell domain showed typical features of plant programmed cell death. Chromatin condensation and DNA degradation preceded cell detachment and cytoplasm dismantling, a process that was characterized by the formation of vesicles and vacuoles that contained cytoplasmic material. This morphotype of programmed cell death was accompanied by an increase in the activity of caspase-3-like proteases. The orchestration of such a death program culminated in the elimination of the small generative domain, and further embryogenesis was carried out by the large vegetative domain. To date, this is the first report to show evidence that programmed cell death takes part in the development of microspore-derived embryos. © Springer-Verlag 2005.

The aim was to investigate mechanisms contributing to quercetin's previously described effects on cell-proliferation and -differentiation, which contradicted its proposed anticarcinogenic potency. In a 10-day experiment, 40 μM quercetin stabilized by 1 mM ascorbate reduced Caco-2 differentiation up to 50% (p < 0.001). Caco-2 RNA from days 5 and 10, hybridized on HG-U133A2.0 Affymetrix GeneChips®, showed 1743 affected genes on both days (p < 0.01). All 14 Caco-2 differentiation-associated genes showed decreased expression (p < 0.01), including intestinal alkaline phosphatase, that was confirmed technically (qRT-PCR) and functionally (enzyme-activity). The 1743 genes contributed to 27 pathways (p < 0.05) categorized under six gene ontology (GO) processes, including apoptosis and cell-cycle. Genes within these GO-processes showed fold changes that suggest increased cell-survival and -proliferation. Furthermore, quercetin down-regulated expression of genes involved in tumor-suppression and phase II metabolism, and up-regulated oncogenes. Gene expression changes mediated by ascorbate-stabilized quercetin were concordant with those occurring in human colorectal carcinogenesis (≈80-90%), but were opposite to those previously described for Caco-2 cells exposed to quercetin without ascorbate (≈75-90%). In conclusion, gene expression among Caco-2 cells exposed to ascorbate-stabilized quercetin showed mechanisms contrary to what is expected for a cancer-preventive agent. Whether this unexpected in vitro effect is relevant in vivo, remains to be elucidated. © 2007 WILEY-VCH Verlag GmbH & Co. KGaA.

The effect of therapeutic doses of ultrasound was tested on endochondral ossification of in vitro developing metatarsal long bone rudiments of 16- and 17-day-old fetal mice. Bone growth, calcification and resorption following exposure to several doses of pulse-wave (PW) or continuous-wave (CW) ultrasound were examined. PW was applied at intensities between 0.1 W cm-2 and 0.77 W cm-2 (I(satp)) and CW intensities were 0.1 W cm-2 or 0.5 W cm-2 (I(sata)). After 1 week of culture, the metatarsal long bone rudiments were fixed and paraffin sections were prepared for histological evaluation and for measurement of the relative contribution of the various cartilage zones to the total bone length. In contrast to treatment with CW ultrasound, treatment of 16-day-old metatarsal long bone rudiments with PW ultrasound resulted after 4 days of culture in significantly increased longitudinal growth. Histology revealed a significant increased length of the proliferative zone, whereas the length of the hypertrophic cartilage zone was unaltered. This might indicate that proliferation of the cartilage cells is stimulated without influence on cell differentiation.

Epidermal cell proliferation and differentiation were investigated in vitro after exposure to the anionic surfactant sodium dodecyl sulfate (SDS). Human skin organ cultures were exposed topically to various concentrations of SDS for 22 h, after which the irritant was removed. Cell proliferation was measured immunohistochemically by incorporation of bromodeoxyuridine (BrdU) into the DNA of cells during S-phase, while the expression of transglutaminase and involucrin were used as markers of differentiation. Cell proliferation was moderately increased at concentrations of SDS that did not affect the histomorphology (0.1% and 0.2% SDS). A marked increase of cell proliferation was observed 22 to 44 h after removal of SDS at a concentration (0.4%) that induced slight cellular damage. Exposure of human skin organ cultures to a toxic concentration of SDS (1.0%) led to decreased cell proliferation. Transglutaminase and involucrin were expressed in the more basal layers of the epidermis after exposure to 0.4% or 1.0% SDS. Moreover, intra-epidermal sweat gland duets were positive for transglutaminase at these irritant concentrations. These in vitro data demonstrate that SDS-induced alterations of epidermal cell kinetics, as described in vivo are at least partly due to local mechanisms and do not require the influx of infiltrate cells. However, we were unable to relate the altered cell kinetics to the release of interleukin-1α or interleukin-6. Furthermore, supplementation of the culture medium with 12-hydroxyeicosantetraenoic acid did not affect epidermal cell proliferation. Rabbit skin cultures appeared more sensitive to SDS than human skin. At nontoxic doses, the irritant induced an increase of epidermal cell proliferation, similar to that observed in human skin discs.

In the present review the results obtained so far with thymic epithelial culture supernatants (TES) or cocultivation of potential precursor T cells with TE cells will be discussed and compared with the effects reported for other thymic factors in studies on T cell differentiation in vitro. These studies can be divided into those involved with T cell markers, T cell proliferative capacity and effector T cell functions. In sections 4, 5 and 6 the effects of thymic factors, TES and TE cells with regard to their capacity to modify these three T cell parameters in vitro will be summarized. Since it has not yet been conclusively demonstrated which are the target cells for the action of thymic factors, the putative target cells will be dealt with separately in each section. Additionally, I will present some of our recent data with TES which support the suggestion that factors present in TES can induce maturation of the subpopulation of immature thymocytes, the peanut-agglutinin (PNA)-agglutinating, corticosteroid-sensitive cells with a high density of Thy 1-antigen. The TES used routinely in our studies is derived from rats TE cultures prepared as described in detail previously. Similar results have been obtained with human TES (A.M. Kruisbeek and G.C.B. Astaldi, unpubl. obs.) and with TES from mouse TE cultures derived from mice syngeneic to the mice used as target cell donors for the experiments shown here (A.M. Kruisbeek, unpubl. obs.). However, mouse TES is more difficult to obtain because cultures tend to be rapidly overgrown by fibroblasts, reason why for routine studies (once it was established that no differences between xeno- and syngeneic TES were observed) the rat TES is preferred. Chemicals/CAS: Antigens; Concanavalin A, 11028-71-0; Cortisone, 53-06-5; Phytohemagglutinins; Thymic Factor, Circulating, 78922-62-0; Thymus Extracts; Thymus Hormones

Tissue microenvironment plays a critical role in guiding local stem cell differentiation. Within the intervertebral disc, collagen type II and nucleus pulposus (NP) cells are two major components. This study aimed to investigate how collagen type II and NP cells affect adipose tissue-derived stem cells (ASCs) in a 3D environment. ASCs were cultured in collagen type I or type II hydrogels alone, or co-cultured in transwells with micromass NP cells for 4 and 14 days. ASCs seeded in collagen type II gels acquired dentritic cell shapes, and orchestrated cell density-dependent gel contraction rates. Up-regulation of collagen type X, but not of other chondrogenic markers was observed at day 4, irrespective of the hydrogel type. Strikingly, in co-cultures with NP cells, more pronounced differentiation of ASCs along the cartilaginous lineage was observed (up-regulation of collagen IIA, IIB and aggrecan gene expression, as well as stronger alcian blue staining), when ASCs were embedded in collagen type II in comparison with type I hydrogels. Interestingly, strong cellular condensations/aggregations were observed in ASC-seeded type II, but not type I gels, and this aggregation was markedly delayed when the same gels were co-cultured with NP cells. The NP cell-mediated inhibition of ASC aggregation in collagen type II gels coincided with down-regulation of integrin subunit α2 gene expression. We conclude that soluble factors released by NP cells can direct chondrogenic differentiation of ASCs in collagen hydrogels, and that combination with a nucleus-mimicking collagen type II microenvironment enhances differentiation towards a more pronounced cartilage/NP lineage relative to collagen type I hydrogels. © 2008 The Authors. Chemicals/CAS: Collagen Type I; Collagen Type II; Gels; Integrin alpha2; Protein Subunits

The effect of the dietary flavonoid quercetin was investigated on proliferation and differentiation of the human colon cancer cell line Caco-2. Confluent Caco-2 monolayers exposed to quercetin showed a biphasic effect on cell proliferation and a decrease in cell differentiation (0.001<P<0.05). During differentiation Caco-2 cells formed 5 phase II metabolites, of which the amount of 4′-O-methyl-quercetin-3′-O-glucuronide correlated with the differentiation grade (r=0.99, P<0.003). The increment of cell proliferation at low quercetin concentrations and the decrease in cell differentiation are effects opposite to what would be expected for a functional food ingredient with anti-carcinogenic potential. © 2005 Elsevier Ireland Ltd. All rights reserved.

The members of the 14-3-3 isoform family have been shown to be developmentally regulated during animal embryogenesis, where they take part in cell differentiation processes. 14-3-3 isoform-specific expression patterns were studied in plant embryogenic processes, using barley (Hordeum vulgare L.) microspore embryogenesis as a model system. After embryogenesis induction by stress, microspores with enlarged morphology showed higher viability than non-enlarged ones. Following microspore culture, cell division was only observed among the enlarged microspores. Western blot and immunolocalization of three barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C were carried out using isoform-specific antibodies. The level of 14-3-3C protein was higher in enlarged microspores than in non-enlarged ones. A processed form of 14-3-3A was associated with the death pathway of the non-enlarged microspores. In the early embryogenesis stage, 14-3-3 subcellular localization differed among dividing and non-dividing microspores and the microspore-derived multicellular structures showed a polarized expression pattern of 14-3-3C and a higher 14-3-3A signal in epidermis primordia. In the late embryogenesis stage, 14-3-3C was specifically expressed underneath the L₁ layer of the shoot apical meristem and in the scutellum of embryo-like structures (ELSs). 14-3-3C was also expressed in the scutellum and underneath the L₁ layer of the shoot apical meristem of 21 d after pollination (DAP) zygotic embryos. These results reveal that 14-3-3A processing and 14-3-3C isoform tissue-specific expression are closely related to cell fate and initiation of specific cell type differentiation, providing a new insight into the study of 14-3-3 proteins in plant embryogenesis. Chemicals/CAS: protein 14 3 3, 136047-16-0; tyrosine 3 monooxygenase, 9036-22-0; 14-3-3 Proteins; Protein Isoforms; Tyrosine 3-Monooxygenase, EC 1.14.16.2

Activated microglia are found in a variety of neuroinflammatory disorders where they have attributed roles as effector as well as antigen-presenting cells (APC). Critical determinants for the multifaceted role of microglia are the differentiation potential of microglia and their mode of activation. In this study, we have investigated the effects of M-CSF and GM-CSF-mediated differentiation of adult primate microglia on their cellular phenotype, antigen presentation, and phagocytic function as well as on Toll-like receptor (TLR)-mediated responses. We show that although cell morphology and expression levels of activation markers were markedly different, differentiation with either factor yielded microglia that phenotypically and functionally resemble macrophages. Both M-CSF and GM-CSF-differentiated microglia were responsive to TLR1/2, 2, 3, 4, 5, 6/2, and 8-mediated activation, but not to TLR7 or 9-mediated activation. Intriguingly, M-CSF-differentiated microglia expressed higher levels of TLR8-encoding mRNA and protein, and produced larger amounts of proinflammatory cytokines in response to TLR8-mediated activation as compared to GM-CSF-differentiated microglia. While differentiation of adult microglia by growth factors that can be produced endogenously in the central nervous system is thus unlikely to change their APC function, it can alter their innate responses to infectious stimuli such as ssRNA viruses. Resident primate microglia may thereby help shape rather than initiate adaptive immune responses. © 2007 Wiley-Liss, Inc.

The monocyte-like human histiocytic lymphoma cell line U937 can be induced by phorbol 12-myristate 13-acetate (PMA) to undergo differentiation into a macrophage-like phenotype. We have used two-dimensional gel electrophoresis (2-DE), oligonucleotide microarrays and principal component analysis (PCA) to characterize the U937 cell line as a model system for the differentiation of monocytes into macrophages. A total of 226 differentially expressed proteins were found, of which 41 were selected by PCA for identification using matrix-assisted laser desorption/ionization tandem mass spectrometry. Based on the PCA results, three marker proteins were selected for confirmation of differential expression using Western blot and quantitative real time-PCR. The selected marker proteins were: gamma interferon inducible lysosomal thiol reductase, cathepsin D and adipocyte-fatty acid binding protein. All three proved to be good differentiation markers for macrophage maturation of U937 cells as well as peripheral blood-derived macrophages. The transcriptomics data revealed a large number of additional putative differentiation markers in U937 macrophages, many of which are known to be expressed in peripheral blood-derived macrophages. These include osteospontin, matrix metalloproteinase 9, and HC-gp39. Our results show that the characteristics of U937 macrophages resemble those of inflammatory (exudate) macrophages, exemplified by the down-regulation of 5′ nucleotidase and the up-regulation of leucine aminopeptidase mRNAs. In conclusion, using the powerful combination of transcriptomics, 2-DE and PCA, our results show that U937 cells differentiated by PMA treatment are an excellent model system for monocyte derived macrophage generation from blood.

Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pH_i) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pH_i in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pH_i of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na⁺/H⁺-antiporter, corroborating an important role of the pH_i in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH_i may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers. © 2010 Elsevier Inc.

The embryonic stem cell test (EST) has been proposed as an in vitro assay that might reduce animal experimentation in regulatory developmental toxicology. So far, evaluation of the EST was not performed using compounds within distinct chemical classes. Evaluation within a distinct class of chemically related compounds can define the usefulness of the assay for the chemical class tested. The aim of the present study was to evaluate the relative sensitivity of the EST for a selected series of homologous compounds and to compare the data to the relative developmental toxicity of the compounds in vivo. To this end a series of proximate developmentally toxic glycol ether alkoxy acid metabolites was tested in the EST. All glycol ether alkoxy acid metabolites tested showed a concentration-dependent inhibition of cardiomyocyte differentiation at noncytotoxic concentrations, with methoxyacetic acid as the most potent compound followed by ethoxyacetic acid, butoxyacetic acid, and phenoxyacetic acid, respectively. The potency ranking of the compounds in the EST corresponds with the available in vivo data. The relative differences between the potencies of the compounds appeared more pronounced in the in vivo studies than in the EST. A possible explanation for this discrepancy could be the difference in the kinetics of the compounds in vivo as compared with their in vitro kinetics. This study illustrates that the EST can be used to set priorities for developmental toxicity testing within classes of related compounds. © The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

The Embryonic Stem cell Test (EST) is an in vitro alternative test designed for the prediction of embryotoxicity. The endpoint of the test is the interference with mesoderm-derived cardiac muscle differentiation observed under the microscope as beating muscle foci. The relative subjectivity of this endpoint, as well as the applicability domain and related predictivity need further to be defined to facilitate implementation of the EST into regulatory strategies. The use of transcriptomics techniques to monitor differentiation-related gene expression changes in the EST might improve the EST in each of these aspects. Therefore, we studied the gene expression profile in embryonic stem cells (ESC) in the early phase of differentiation and its modulation by exposure to the well known embryotoxicant monobutyl phthalate (MBP). Cells were exposed from the early embryoid body stage onwards and RNA was collected after 6, 12 and 24 h of exposure. Samples were hybridized to spotted microarrays, containing 21,997-mer oligonucleotides. Differential gene expression patterns were analyzed. A total number of 43 genes that were found to be upregulated in this study as a consequence of induction of cardiomyocyte differentiation were combined in a gene set, named 'VAN_DARTEL_HEARTDIFF_24H'. Gene Set Enrichment Analysis (GSEA) comparative analysis using multiple gene set collections clearly showed that temporal changes in gene expression were functionally related to cardiomyocyte differentiation. Furthermore, exposure of embryoid bodies (EB) to MBP increased expression of pluripotency-, proliferation- and nonmesodermal differentiation-related gene sets, which indicates inhibition of mesodermal differentiation. The inhibition of mesoderm-derived cardiomyocyte differentiation by MBP exposure was most obvious through the downregulation of our novel gene set identified in this study, 'VAN_DARTEL_HEARTDIFF_24H', which specifically describes the niche of early cardiomyocyte differentiation. The gene set defined in this study might serve as a starting point for defining a dedicated gene set for early detection of embryotoxicity in the EST. Such a gene set may serve as an improved endpoint in the EST as compared to morphology, and will allow a more detailed definition of the applicability domain and predictivity of EST. © 2008 Elsevier Inc. All rights reserved. Chemicals / CAS: RNA, 63231-63-0; phthalic acid, 88-99-3; Phthalic Acids; monobutyl phthalate, 131-70-4

Arteriogenesis, the enlargement of collateral vessels, seems a promising new target to improve blood flow to ischemic regions in patients suffering from cardiovascular conditions. With the growing knowledge of the mechanisms involved in arteriogenesis and the factors that influence the process, an increasing number of clinical trials are being performed to stimulate arteriogenesis, providing more insight in therapeutic opportunities for arteriogenesis. The expression of growth factors and the cooperation of surrounding and infiltrating cells seem to be essential in orchestrating the complex processes during arteriogenesis. In this review, we will discuss the regulating mechanisms of arteriogenesis, including the role of growth factors and different cell types and their implementation in a clinical setting. Furthermore, individual differences in the arteriogenic response will be considered, in light of the effect this will have on the success of therapeutic strategies to improve blood flow to ischemic tissue. © Society for Leukocyte Biology.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) in the SJL mouse is a Th1-mediated autoimmune demyelinating disease model for human multiple sclerosis and is characterized by infiltration of the central nervous system (CNS) by Th1 cells and macrophages. Disease relapses are mediated by T cells specific for endogenous myelin epitopes released during acute disease, reflecting a critical role for epitope spreading in the perpetuation of chronic central CNS pathology. We asked whether blockade of the CD40-CD154 (CD40L) costimulatory pathway could suppress relapses in mice with established R-EAE. Anti-CD154 antibody treatment at either the peak of acute disease or during remission effectively blocked clinical disease progression and CNS inflammation. This treatment blocked Th1 differentiation and effector function rather than expansion of myelin-specific T cells. Although T-cell proliferation and production of interleukin (IL)-2, IL-4, IL- 5, and IL-10 were normal, antibody treatment severely inhibited interferon- γ production, myelin peptide-specific delayed-type hypersensitivity responses, and induction of encephalitogenic effector cells. Anti-CD154 antibody treatment also impaired the expression of clinical disease in adoptive recipients of encephalitogenic T cells, suggesting that CD40-CD154 interactions may be involved in directing the CNS migration of these cells and/or in their effector ability to activate CNS macrophages/microglia. Thus, blockade of CD154-CD40 interactions is a promising immunotherapeutic strategy for treatment of ongoing T cell-mediated autoimmune diseases. Chemicals/CAS: Antibodies; CD40 Ligand, 147205-72-9; Interferon Type II, 82115-62-6; Interleukins; Membrane Glycoproteins; Myelin Proteolipid Protein; Peptide Fragments; proteolipid protein 139-151; proteolipid protein 178-191, 172228-98-7

Tracheal organ cultures and isolated tracheal epithelial cells are frequently used to study effects of carcinogens and retinoids on both proliferation and differentiation of respiratory tract epithelial cells. For each of these in vitro models, optimal culture conditions have been established, varying in type of culture medium and composition of growth factor and hormone supplementation, which by themselves may influence cellular proliferation and differentiation. In this study, we investigated the influence of medium composition and growth factor supplementation on the effect of benzo[a]pyrene (B[a]P) and vitamin A on cellular proliferation and differentiation in hamster tracheal epithelium in organ culture. In tracheae cultured in Ham's F12 medium, cell proliferation was decreased by B[a]P relative to untreated controls, whereas vitamin A in combination with B[a]P increased cell proliferation compared with that in tracheae treated with B[a]P alone. The effects in tracheae cultured in CMRL-1066 medium were just the opposite: B[a]P increased cell proliferation and vitamin A decreased B[a]P-induced proliferation. To explain this difference in cell proliferation, the effects of various growth factors (epidermal growth factor and transferrin) and medium components (nucleotides, NAD+/NADP and CaCl2.2H2O) on B[a]P and vitamin A-induced cell proliferation were investigated. The main factor responsible for the different effects on cell proliferation appeared to be the concentration of Ca2+ in the culture medium; addition of CaCl2.2H2O to Ham's F12 medium resulted in effects of B[a]P and vitamin A on cell proliferation comparable with those observed in tracheae cultured in CMRL-1066 medium. These results clearly show that the composition of the culture medium, and particularly the concentration of Ca2+, strongly influences the effect of vitamin A and B[a]P on cell proliferation in hamster tracheal epithelium in organ culture.

IL-10 and IL-4 were studied with respect to their capacity to inhibit experimental allergic encephalomyelitis (EAE) induced in SJL/J mice by immunization with the proteolipid protein peptide PLP139-151. Treatment with 2 μg IL-10/day from day 0 until day 12 delayed onset of disease and inhibited the severity of EAE. By contrast, a daily dose of 0.5 μg IL-4 was ineffective. Instead of acting in a synergistic fashion, IL-4 even abrogated the inhibitory effect of IL-10. The effects of IL-10 and IL-4 treatment were largely consistent with the (lack of) ability of these cytokines to down-regulate the inflammatory response in brain tissue. Although IL-4 was ineffective in the inhibition of EAE, lymph node cells from IL-4-treated mice displayed a strongly inhibited peptide-specific IFN-γ production. By contrast, IL-10, which was effective in inhibiting EAE, showed no significant inhibition of IFN-γ at this level. Neither cytokine treatment resulted in detectable levels of peptide-specific IL-4. Indirect evidence for the activity of T(h)2 cells in vivo came from the observation that IL-10 inhibited the primary PLP139-151-specific IgG2a and IgG3 response in favor of IgG1, whereas IL-4 inhibited the primary antibody response to the peptide, regardless of subclass. The combination of IL-4 and IL-10 did not affect the subclass composition. The observation that IL-10-treated mice remained sensitive to re-induction of EAE is not in support of an important role of T(h)2 cells in regulating disease activity in this model of actively induced EAE.

The balance of tissue-type plasminogen activator (t-PA) production and degradation determines its concentration in blood and tissues. Disturbance of this balance may result in either increased or decreased proteolysis. In the present study, we identified the receptor systems involved in the degradation of t-PA by human monocytes/macrophages in culture. Monocytes were cultured and became macrophages within 2 days. At 4°C, 125I-t-PA bound to macrophages with high (apparent dissociation constant [kd], 1 to 5 nmol/L) and low affinity (kd > 350 nmol/L). At 37°C, the cells internalized and degraded t-PA via the high affinity binding sites, which were partially inhibited by mannan. The low affinity binding sites were 6-aminohexanoic acid-inhibitable and not involved in t-PA degradation. Degradation of t-PA was upregulated during differentiation of monocytes to macrophages. Dexamethasone further upregulated the mannan-inhibitable t-PA degradation. Lipopolysaccharide downregulated both mannan-inhibitable and non-mannan- inhibitable t-PA degradation. Non-mannan-inhibitable degradation was completely blocked by recombinant 39-kD receptor-associated protein (RAP, inhibitor of lipoprotein receptor-related protein [LRP]), whereas mannan- inhibitable degradation was blocked by the addition of a monoclonal antibody against the mannose receptor. No differences between the degradation of t-PA and functionally inactivated t-PA were observed. We conclude that human monocyte-derived macrophages are able to bind, internalize, and degrade t- PA. Degradation of t-PA does not require complex formation with plasminogen activator inhibitors. The macrophages use two independently regulated receptors, namely, the mannose receptor and LRP, for the uptake and degradation of t-PA. Chemicals/CAS: aminocaproic acid, 1319-82-0, 60-32-2; tissue plasminogen activator, 105913-11-9; Dexamethasone, 50-02-2; LDL-Receptor Related Protein 1; Lipopolysaccharides; mannose receptor; Receptors, Cell Surface; Receptors, Immunologic; Tissue Plasminogen Activator, EC 3.4.21.68

Recently we developed mouse monocloual antibodies (mAb) against the isolated human 175-kDa mannose receptor. In the present study we tested whether these mAb are suitable for the detection of the mannose receptor on cultured macrophages using flow cytometry and on cells in human tissues using immunohistochemistry. Human monocytes did not react with the mAb in flow cytometry. Mannose receptor expression became detectable on monocytes cultured for 3 days (macrophages), and was maximal from 4 days onward. The mannose receptor was up-regulated on dexamethasone-treated (immunosuppressed) macrophages, and down-regulated on lipopolysaccharide-treated (activated) macrophages. Immunohistochemically the staining pattern of our mAb was compared with the marker of monocytes/macrophages KP1. In a bone marrow smear, only macrophages were stained with our mAb, whereas all myeloid cells were stained with KP1. In the thymus and lymph node, mannose receptor-positive branched cells (macrophages and dendritic cells) were detected in connective tissue, thymus cortex (not medulla), and in the T cell area (not the B cell area) of lymph nodes, whereas KP1 stained branched cells in all areas. It was concluded that the mAb are useful tools in flow cytometry and immunohistochemistry for the specific detection of cells expressing mannose receptor. Chemicals/CAS: Antibodies, Monoclonal; Biological Markers; Dexamethasone, 50-02-2; Glucocorticoids; Lectins, C-Type; Lipopolysaccharides; mannose receptor; Mannose-Binding Lectins; Receptors, Cell Surface