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PubMed1993

Intracellular multiplication of Legionella pneumophila in HL-60 cells differentiated by 1,25-dihydroxyvitamin D3 and the effect of interferon gamma.

We examined leukemic cells, HL-60, an acute promyelocytic leukemia cell line, after differentiation induced by 1,25-dihydroxyvitamin D3 (D3) and retinoic acid (A) for infection of Legionella pneumophila, the etiologic agent of Legionnaires' disease. We investigated the effect of interferon gamma (IFN-gamma) on the differentiated cells and on the intracellular growth of the bacteria. An examination of morphological and antigenic changes in the cells was also included in the study. After 4-day incubation with 10(-6)M D3 or A, the HL-60 cells differentiated into monocyte-like (D3-HL-60) or mature granulocyte-like (A-HL-60) cells, respectively. They were then infected with L. pneumophila. Intracellular multiplication of the bacteria was evident in D3-HL-60 cells but not in HL-60 or A-HL-60 cells. D3-HL-60 cells required a 24-h infection time for the intracellular growth of L. pneumophila. D3-HL-60 cells activated with human recombinant IFN-gamma for 1-24 h (gamma-IFN-D3-HL-60 cells) before infection markedly inhibited L. pneumophila multiplication, the effect of IFN-gamma being dose dependent. Surface marker analysis was carried out in HL-60, D3-HL-60, and gamma-IFN-D3-HL-60 cells. On D3-HL-60 cells, CD11b, CD11c, CD14, and CD35 antigen increased, whereas CD71 and HLA-DR antigen decreased. This finding suggested that HL-60 cells differentiated into monocyte-like cells; the acquisition of the complement receptors, CD11b(CR3) and CD35(CR1), seemed to be important for phagocytosis and for the subsequent intracellular multiplication of L. pneumophila. The gamma-IFN-D3-HL-60 cells showed an increase of CD16, CD36, CD71, and HLA-DR antigen, suggesting that they were in an activated state. Our study indicated, first, that D3 can induce human leukemic cells to differentiate into functional monocyte-macrophage-like cells that can support the intracellular multiplication of L. pneumophila and, second, that these differentiated leukemic cells can be activated by IFN-gamma to markedly inhibit bacterial growth.

Animals↗
PubMed1993

Luminol-enhanced chemiluminescence induced in peripheral blood-derived human phagocytes: obligatory requirement of myeloperoxidase exocytosis by monocytes.

Luminol-enhanced chemiluminescence (LCL) of phagocytes is often used to monitor for the generation of reactive oxygen mediators. A strong LCL reaction, as observed in triggered peripheral blood monocytes and neutrophils, depends on both the activation of an NAD(P)H-dependent oxidase and a functional myeloperoxidase (MPO). The aim of this work was to compare the LCL response induced by soluble and particulate stimuli in monocytes with that of polymorphonuclear leukocytes (PMNs). In agreement with earlier results, neutrophils showed a first peak of LCL activity within 1 min and a second peak around 3 min when stimulated with soluble stimuli. The first peak is sensitive to oxygen scavengers and requires the presence of extracellular luminol, whereas the second peak is much less sensitive to oxygen scavengers and does not require the presence of extracellular luminol. The first peak of LCL is therefore thought to represent extracellular and the second peak intracellular LCL. Monocytes, in contrast, showed only the first peak of LCL activity. This peak was sensitive to oxygen scavengers, required the extracellular presence of luminol, and could be suppressed by a rapid pH shift to a pH not allowing LCL (i.e., to pH 5). These results suggest that the stimulus-dependent extracellular release of oxygen metabolites and of MPO is an obligatory requirement for LCL induced in monocytes. An exclusively extracellular LCL was noted upon stimulation with aggregated immunoglobulin G, f-Met-Leu-Phe, phorbol myristate acetate, A23187, anti-CD36 antibodies, opsonized zymosan, and opsonized E. coli. With the addition of H2O2 in excess (1 microM), the LCL response of monocytes therefore reflected the stimulus-dependent degranulation of MPO, and LCL-based determination of MPO release closely correlated with enzymatic MPO determination. The question of why LCL induced in monocytes, but not in PMNs, is restricted to the extracellular compartment was addressed. Although flow cytometric experiments were consistent with the hypothesis that extracellular H2O2 entered the cytoplasm of monocytes less efficiently than that of PMNs, other more important factors are assumed to contribute. Collectively, these results point to previously unrecognized differences in monocyte and PMN LCL which may reflect cellular differences of potential pathophysiological significance.

Azides↗
PubMed1999

Dexamethasone inhibits dendritic cell maturation by redirecting differentiation of a subset of cells.

To investigate how corticosteroids affect differentiation of human dendritic cells (DC) in a defined inflammatory environment, we incubated immature DC with dexamethasone in the presence of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and prostaglandin E2. Dexamethasone inhibited differentiation into mature DC, as indicated by the reduced expression of antigen-presenting molecules, costimulatory and adhesion molecules, a marker of mature DC, and IL-12. Dexamethasone increased expression of CD14, CD36, and CD68, molecules characteristic of monocytes/macrophages and induced CD14+CD83- cells, a subset distinct both from immature DC and mature DC. The effects were concentration-dependent, with ID50 values between 2 and 30 nM dexamethasone. Unlike T and B cells, in DC dexamethasone induced no apoptosis, although it suppressed activated nuclear transcription factor NF-kappaB. Dexamethasone reduced the ability of DC to stimulate proliferation of allogeneic T cells in proportion to the level of CD14+CD83- cells in the population. CD83+ cells, isolated from dexamethasone-treated populations, retained the synthesis of IL-12 and the ability to stimulate proliferation of allogeneic T cells. Our data demonstrate that the dominant effect of the drug was redirecting differentiation of a subset of cells despite the presence of inflammatory cytokines. The observed ID50 values indicate that inhibition of DC differentiation might contribute significantly to in vivo immunosuppression by chronic administration of corticosteroids.

Antigen Presentation↗
PubMed1992

Expression of myeloid differentiation antigens in acute nonlymphocytic leukemia: increased concentration of CD33 antigen predicts poor outcome--a report from the Childrens Cancer Study Group.

Ninety-eight cryopreserved specimens of acute nonlymphocytic leukemia (ANLL) cells obtained at initial diagnosis of children enrolled on the Childrens Cancer Study Group 251 protocol (CCG 251) were examined by indirect immunofluorescence using four monoclonal antibodies to myeloid differentiation antigens. The relationship between the level of differentiation of ANLL cells as determined by their antigen phenotype and the clinical outcome of treatment, including complete remission (CR) rate, survival, and event-free survival, was evaluated. Most leukemic specimens were determined to express the CD33 antigen (L4F3), a 67-kD protein. Because the level of differentiation of normal myeloid cells is reflected by the concentration of the CD33 antigen expressed, samples were categorized as CD33-bright (immature) versus CD33-dull (mature). Patients with CD33-bright leukemic blasts had a marginally inferior CR rate to those with CD33-dull blasts (P = 0.08). With respect to survival and event-free survival, there was a significantly inferior outcome in the CD33-bright patients (P = 0.04 and P = 0.06, respectively). Reactions of ANLL with anti-CD15 antibody (1G10), anti-CD36 antibody (5F1), or anti-CD17 antibody (T5A7) did not predict clinical outcome. This study indicates that patients whose ANLL blasts displayed the CD33 antigen in an amount associated with immature myeloid cells experienced a worse outcome than patients with ANLL blasts that expressed a phenotype associated with more mature cells.

Antibodies, Monoclonal↗
PubMed2026

Successful Live Birth Following Treatment of Persistent Endometrial Dysbiosis and Recurrent Chronic Endometritis: A Case Report.

CASE: To study the cause of recurrent endometritis, which recurred after standard antibiotic therapy, we report the case of a 40-year-old woman with a history of recurrent pregnancy, preterm birth, and CE. The endometritis recurred following a standard antibiotic regimen. OUTCOME: Microbiome analysis via 16S rRNA gene sequencing revealed persistent dysbiosis in both vaginal and endometrial samples despite antibiotic regimens. Whole-exome sequencing (WES) identified rare variants in TRPV3 and CD36, potentially associated with epithelial barrier dysfunction. Following an extended course of antibiotic therapy, the woman gave birth to a healthy baby at GA 32 weeks. CONCLUSIONS: This case highlights the possibility that barrier gene variants may be associated with persistent endometrial dysbiosis and recurrence of CE. An intensive antibiotic regimen may help achieve a viable pregnancy in patients with recurrent CE following standard antibiotic therapy.

antibiotics↗
PubMed1995

Differential expression of cell adhesion molecules by human hematopoietic progenitor cells from bone marrow and mobilized adult peripheral blood.

The mechanisms responsible for mobilization of hematopoietic progenitor cells (HPC) from the bone marrow into the circulation are unknown. One possibility is that HPC downregulate cell adhesion molecule expression. We studied normal human bone marrow and adult peripheral blood following 4 g/m2 cyclophosphamide and recombinant human granulocyte colony-stimulating factor (rHuG-CSF). Each sample was studied for the coexpression of CD34 and a panel of cell adhesion molecules by dual immunocytometry. Bone marrow HPC express the immunoglobulin gene superfamily members of ICAM-1 (CD54), PECAM-1 (CD31) and LFA-3 (CD58), the integrins VLA-4 (CD49d/CD29), VLA-5 (CD49e/CD29) and LFA-1 (CD11a/CD18), L-Selectin (CD62L), HCAM (CD44) and CD36. Mobilized peripheral blood HPC display less expression of LFA-3 (CD54) and VLA-5 (CD49e). Significant differences in cell adhesion molecule expression do exist between sessile and circulating HPC, but the biological relevance of these observations is currently unclear.

Adult↗
PubMed1995

Putative membrane fatty acid translocase and cytoplasmic fatty acid-binding protein are co-expressed in rat heart and skeletal muscles.

A membrane protein (FAT) homologous to CD36 has recently been implicated in the binding and transport of long-chain fatty acids (FA). Expression of this protein in rat heart, skeletal muscles and in isolated cardiac cells was studied. Changes in expression during development of the heart were also examined. Expression of FAT was compared to that of the cytoplasmic fatty acid-binding protein (H-FABP) to determine whether coexpression, indicative of related biological functions, could be demonstrated. FAT and H-FABP mRNAs showed a similar muscle tissue distribution and similar cellular localization in the heart. During development, heart mRNA levels for both proteins were upregulated in the same way. In conclusion, expression of FAT and H-FABP in muscle tissues and cell-types with high FA metabolism and the upregulation of mRNA levels associated with heart development, when FA utilization increases, support the suggested role of both proteins in FA metabolism.

Adipose Tissue↗
PubMed1997

Angiotensin II injection into mice increases the uptake of oxidized LDL by their macrophages via a proteoglycan-mediated pathway.

Angiotensin II (Ang-II) has been shown to possess several atherogenic properties including its ability to induce macrophage-mediated oxidation of LDL and to form Ang-II-modified LDL which is taken up by macrophages at enhanced rate. Oxidized-LDL (Ox-LDL) is also taken up by macrophages at enhanced rate via several scavenger receptors, leading to macrophage cholesterol accumulation. In the present study we examined the effect of Ang-II on the uptake of Ox-LDL by peritoneal macrophages derived from Balb/c mice (MPM). Intraperitoneal injection of Ang-II (10(-7) M, once daily for a period of 2 days) to the mice resulted in an increased Ox-LDL uptake up to 60%, in comparison to macrophages from placebo-treated mice. Similar results were obtained when Ang-II (10(-7) M) was injected to the mice twice a week for a period of three months. This Ox-LDL uptake was Ang-II dose-dependent. The cellular uptake of acetylated-LDL (Ac-LDL), another ligand for scavenger receptors, however, was not affected by Ang-II injection to the mice. Furthermore, preincubation of the MPM with the monoclonal antibody, anti CD36, reduced macrophage uptake of Ox-LDL in Ang-II-treated mice by only 11%. Ang-II administration to mice resulted in a 60% increase in the macrophage cellular proteoglycan content. Chondroitinase treatment of MPM decreased Ox-LDL cellular uptake by 20% and by 38% in placebo-treated and Ang-II-treated cells, respectively. We thus conclude that Ang-II administration to mice enhances their macrophage Ox-LDL uptake via its stimulating effect on cellular proteoglycan content and this process can lead to foam cell formation and atherosclerosis.

Angiotensin II↗
PubMed2000

Transforming growth factor-beta(1) increases the expression of lectin-like oxidized low-density lipoprotein receptor-1.

Lectin-like oxidized low-density lipoprotein (Ox-LDL) receptor-1 (LOX-1) is a novel cell-surface receptor for Ox-LDL, which can be expressed by vascular endothelial cells, smooth muscle cells, and macrophages. On the other hand, transforming growth factor (TGF)-beta(1), which plays crucial roles in vascular remodeling and the pathogenesis of atherosclerosis, has been shown to inhibit expression of class A scavenger receptors and CD36 in macrophages. Here we provide the evidence that TGF-beta(1) (0.1-10 ng/mL) induces LOX-1 protein and mRNA expression in both bovine aortic endothelial cells and smooth muscle cells in a dose- and time-dependent fashion, probably at the transcriptional level. TGF-beta(1) also upregulates LOX-1 mRNA expression in murine peritoneal macrophages. Thus TGF-beta(1) can highly induce LOX-1 expression in vascular endothelial cells, smooth muscle cells, and macrophages, suggesting that TGF-beta(1) appears one of the key regulators that modulates expression of scavenger receptors.

Animals↗
PubMedDate not supplied

Phenotypic characterization of immunomagnetically purified umbilical cord blood CD34+ cells.

This study describes the multilineage differentiation pattern of purified CD34+ stem cells obtained from human umbilical cord blood. CD34+ cells were collected from 49 umbilical cord blood samples. Following immunomagnetic purification, cells were double stained with anti CD34 and CD71, CD61, CD7, CD19, CD33, CD36 and triple stained with anti CD34, CD38 and HLA-DR. Analysis were performed using a FACScan flow cytometer. After purification, the mean CD34+ cells' purity was 85.49 +/- 7.08%. Several subpopulations of umbilical cord blood CD34+ cells were identified indicating different lineage commitment. The majority of CD34+ cells expressed both CD38 and HLA-DR (91.74 +/- 3.76%), while those lacking CD38 were 3.43 +/- 2.12% (CD38-DR+) and 1.81 +/- 1.54% (CD38-DR-). These data were compared to the expression of lineage commitment markers on purified CD34+ cells from 5 mobilized peripheral blood samples. The percentage of peripheral blood CD34+CD38-DR+) and CD34+CD38-DR- cells was significantly lower than umbilical cord blood, 0.24 +/- 0.18% and 0.04 +/- 0.03% respectively. The knowledge and standardized of umbilical cord blood CD34+ cells phenotype is critical since umbilical cord blood volume is limited. The homogeneity of CD34+ subpopulation phenotype suggests that monitoring of lineage differentiation antigens may not be relevant for clinical use of umbilical cord blood samples. However, the observed higher percentage of pluripotent CD34+38- stem cells in umbilical cord blood compared to peripheral blood, that might explain the successful clinical use of umbilical cord blood even when low number of cells are used, candidates these antigens as the predictive parameter for clinical use of umbilical cord blood samples.

Antigens, CD↗
PubMed1996

Characterization of human leucocytes bearing the IL-3 receptor.

Human leucocytes from peripheral blood and tonsil were examined for the presence of the IL-3 receptor using monoclonal antibodies directed to epitopes of the alpha and beta chains of the receptor. We found that the beta chain, common to IL-3, IL-5, and GM-CSF, was either present at low levels or not detected on the majority of peripheral blood and tonsil B lymphocytes, while the alpha chain showed a distinct but restricted distribution. In peripheral blood the IL-3R alpha chain was limited to a subpopulation of peripheral B lymphocytes and a population of cells which lack lineage-specific markers. Dimly staining cells were identified as B lymphocytes as they coexpressed CD19, CD20, CD22, CD24, and HLA-DR. A brightly staining population lacks T and B lymphocyte, NK specific, and macrophage lineage markers but expresses CD9, CD45RO, CD26, and, in a proportion of cells, CD36 and CD60. This population remains unclassified. In tonsil tissue IL-3R alpha chain expression was strongest on B lymphocytes present in the T cell rich areas of tonsillar tissue. The IL-3R alpha bearing B tonsil cells included cells in both CD23 and IgD positive and negative populations. The phenotype of the IL-3R alpha positive B cells defines them as a population of B lymphocytes distinct from previously characterized cells in the lymphoid architecture. Lymphoblastoid cell lines with a corresponding phenotype were also identified.

Antigens, CD↗
PubMed2000

Extensive surface phenotyping of alveolar macrophages in interstitial lung disease.

There is increasing evidence implicating activated macrophages in the pathogenesis of interstitial and other lung diseases. We investigated whether there was a unique pattern of cell surface expression that constituted a disease-specific phenotype on alveolar macrophages from patients with interstitial lung disease (ILD). Macrophage cell surface receptor expression of 19 selected markers was assessed by indirect immunofluorescence and flow cytometry in bronchoalveolar lavage (BAL) fluids from patients with idiopathic pulmonary fibrosis (IPF, n = 4), scleroderma (SCL-ILD, n = 14), mild asthma (n = 7), allergy without asthma (n = 2), and normal subjects (n = 9). There was increased expression of adhesion receptors (CD11c, CD29, CD36, CD44, CD49e, CD54), receptors involved in signal transduction and/or inflammation (CD13, CD45, CD53), and other markers (CD9, CD52, CD71, CD98, HLA Class I) on macrophages from ILD patients compared to the non-ILD group. Most markers upregulated on macrophages in ILD were significantly inversely correlated with clinical parameters of disease activity such as FEV(1), FVC, and DL(CO) and positively correlated with numbers of BAL neutrophils and eosinophils. Increased expression of several cell surface markers suggests that activated alveolar macrophages may contribute to the pathophysiology of IPF and SCL-ILD.

Adult↗
PubMed1997

A novel technique for culture of human dermal microvascular endothelial cells under either serum-free or serum-supplemented conditions: isolation by panning and stimulation with vascular endothelial growth factor.

Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microvasculature require homogenous primary cultures of microvascular endothelial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contamination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonatal human foreskin by panning the cells using EN4, an anti-endothelial cell monoclonal antibody. Panned cells uniformly expressed von Willebrand factor and CD36, confirming their microvascular endothelial characteristics, whereas cells cultured without panning showed a significant degree of contamination with fibroblasts. In the presence of vascular endothelial growth factor (VEGF), HDMEC could be cultured under serum-free conditions. VEGF stimulated the growth of HDMEC in a dose-dependent manner in serum-free medium or in media supplemented with either human serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we compared the response to VEGF stimulation of HDMEC with human umbilical vein endothelial cells (HUVEC). The dose response of the two cell types to VEGF was different. This effect of VEGF on endothelial cells may be mediated by the VEGF receptor kdr, since mRNA for kdr was detected using RT-PCR in both HDMEC and HUVEC. The procedure described in this study will make possible the culture of highly enriched HDMEC without contamination with fibroblasts and facilitate studies with these cells under defined assay conditions in a serum-free environment.

Animals↗
PubMed2000

Plasmodium falciparum: cloned and expressed CIDR domains of PfEMP1 bind to chondroitin sulfate A.

Adherence of erythrocytes infected with mature asexual Plasmodium falciparum parasites (iRBC) to microvascular endothelial cells contributes to the pathology of P. falciparum malaria. It has been shown that the variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) confers adhesion to a wide range of cell surface receptors. Previously, the cysteine-rich interdomain region (CIDR) of PfEMP1 has been identified as binding site to CD36. We provide evidence that the same region can also mediate binding to chondroitin sulfate A (CSA). CIDR domains of two different parasite strains were expressed in Escherichia coli as a 6xHis-tagged protein. Purified recombinant protein bound to Chinese hamster ovary (CHO) cells which naturally express chondroitin sulfate A. Treatment of wild-type CHO cells with chondroitinase ABC reduced binding up to 94.4%. Competitive binding using soluble CSA inhibited binding to CHO cells by up to 100% at 2 mg/ml and by 62.4% at 0.5 mg/ml, whereas 1 mg/ml heparan sulfate had only a little effect (18.1%). In contrast, a recombinant 6xHis-tagged DBL1 domain showed no binding to wild-type CHO cells. Such an approach of analyzing various domains of PfEMP1 as recombinant proteins may elucidate their functions and may lead to novel anti-adherence therapeutics, especially for maternal malaria infections.

Amino Acid Sequence↗
PubMed2001

Activation of natural interferon-alpha producing cells by apoptotic U937 cells combined with lupus IgG and its regulation by cytokines.

We recently demonstrated that IgG from patients with systemic lupus erythematosus (SLE) in combination with U937 cells made apoptotic by UV-irradiation, can induce interferon-alpha (IFN-alpha) production in normal peripheral blood mononuclear cells (PBMC). In the present study we show by flow cytometry that the actual IFN-alpha producing cells (IPC) among PBMC had the same phenotype (HLA-DR+, CD4+, CD11b-, CD11c-, CD14-, CD19-, CD32-, CD36+, CD40+, CD45RA+, CD68+, CD83+, CD86-, IL-3R+ and IL-10R-) and low frequency (approximately 2/10(4)PBMC) as the IPC activated by Herpes simplex virus type I. Consequently, these cells correspond to the natural IPC, also described as type 2 precursor dendritic cells. We also demonstrated that cytokines of possible importance in the pathogenesis in SLE had effects on the IFN-alpha production. Specifically, the IFN-alpha production was strongly increased by the type I IFNs, IFN-alpha and -beta, but markedly inhibited by IL-10 and also to some extent by TFN-alpha. In contrast, the cytokines IFN-gamma, IL-6, TGF-beta and GM-CSF had no clear effects. No production of IL-10 was detected in PBMC stimulated by apoptotic U937 cells and SLE IgG. These results may explain the cause of the ongoing IFN-alpha production in SLE patients and its relation to the autoimmune process.

Adolescent↗
PubMed2001

Endothelin-1 synthesis and endothelin B receptor expression in human coronary artery smooth muscle cells and monocyte-derived macrophages is up-regulated by low density lipoproteins.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide exerting its effects predominantly by paracrine and autocrine mechanisms. ET-1 acts as a mitogen and co-mitogen on vascular smooth muscle cells, and accumulating evidence suggests that ET-1 is involved in the pathogenesis of atherosclerosis. Deposition of low density lipoproteins (LDL) in the vessel wall is known to play a crucial role in the development of atherosclerotic lesions. In the present study, we have investigated the effect of native LDL (nLDL) and oxidatively modified LDL (oxLDL) on ET-1 synthesis and endothelin receptor expression in cultured human coronary artery smooth muscle cells and human monocyte-derived macrophages. Native LDL and oxLDL induced a significant stimulation of ET-1 release and ET-1 mRNA expression in human coronary artery smooth muscle cells and monocyte-derived macrophages. Antibodies against the scavenger receptor CD36 significantly reduced the oxLDL-induced stimulation of ET-1 synthesis. The antioxidants trolox and probucol did not significantly inhibit the LDL-induced rise of ET-1 release. Endothelin B receptor expression was up-regulated in both cell types after incubation with nLDL and oxLDL. In coronary smooth muscle cells, endothelin A receptor expression was slightly increased by LDL, whereas endothelin A receptor was not detectable in monocyte-derived macrophages. Coronary artery smooth muscle cells secreted a more than 150-fold higher amount of immunoreactive ET-1 into the cell culture medium than monocyte-derived macrophages. In summary, the present data, demonstrating a LDL-induced up-regulation of the endothelin system in coronary smooth muscle cells and in monocyte-derived macrophages, provide further support for a pathophysiological role of endothelin in coronary atherosclerosis and suggest that ET-1 might be involved in mediating the atherogenic effects of LDL.

Cells, Cultured↗
PubMed1995

Expression of adhesion molecules in cultured human pulmonary microvascular endothelial cells.

HPMECs were successfully isolated by differential trypsinization from peripheral lung lobes. The cells proliferated rapidly in EGM-MV with 10% FBS and were serially cultivated for more than 20 passages (1:4 split ratio) in vitro. Cells were characterized as endothelial based upon their cobblestone morphology, the presence of factor VIII-related antigen, incorporation of DiI-Ac-LDL, tubule-like structure formation in Matrigel, and positive staining for ACE. Adhesion molecules were tested at passage 3 and passage 12. Cells demonstrated intense staining for PECAM-1 both unstimulated and stimulated with TNF-alpha (20 ng/ml). The adhesion molecules ICAM-1, VCAM-1, ELAM-1, and P-selectin differed in expression on unstimulated cells. ICAM-1 was constitutively expressed on unstimulated cells and the expression was increased by TNF-alpha stimulation (20 hr). In contrast, VCAM-1, ELAM-1, and P-selectin were not detected on unstimulated cells but were detected after stimulation with TNF-alpha. The inducibility of adhesion molecules was different. VCAM-1 (10 hr) and ELAM-1 (4 hr) were expressed more strongly than P-selectin (minutes to 4 hr). The adhesion molecule profile found on passage 12 was the same as on passage 3. CD36 was not detected on both unstimulated and stimulated (4 and 8 hr) cells. The peak of adhesion of HL-60 cells to TNF-alpha activated HPMEC monolayers was around 8 hr. The results indicate that HPMEC can be continuously grown in vitro for many passages without losing their adhesion molecule expression. This expression of adhesion molecules confirms that HPMECs might be a good in vitro model in the understanding of various aspects of pulmonary microvascular endothelial cell function and may be useful as the basis for studies of adhesion molecule targeted therapies of pulmonary inflammatory diseases.

Cell Adhesion Molecules↗
PubMed1996

BMEC-1: a human bone marrow microvascular endothelial cell line with primary cell characteristics.

Bone marrow microvascular endothelial cells (BMEC) are a functional component of the bone marrow stroma and have been shown to release hematopoietic regulatory factors as well as to selectively adhere and support the proliferation and differentiation of CD34+ hematopoietic progenitors. An early passage of these cells was immortalized by transfection with a vector (pSVT) encoding the large T antigen of SV40. The transformed cell line (CDC/CU.BMEC-1) expresses the SV40 transcript, retains the primary cell expression of Ulex europeaus and vWF/ FVIII, and incorporates acetylated low-density lipoprotein. In addition, BMEC-1 mirrors the phenotype of the primary cells with only a few exceptions. Both cell populations express the cellular adhesion molecules ICAM-1 and PECAM and also VCAM-1 and ELAM-1 after upregulation by tumor necrosis factor-alpha. The fibronectin receptor, hyaluronate receptor, collagen receptor, integrins VLA-alpha 3, VLA-alpha 4, and beta 4, endoglin, collagen IV, CD58, and CD61 are also expressed. The only differences are that BMEC-1 expresses higher levels of ICAM-1, CD58, CD34, CD36, and c-kit than the primary cells. The supernatants of primary cell and BMEC-1 contain stem cell factor, interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1 alpha, IL-11, and G-CSF. The functional significance of these hematopoietic cytokines was demonstrated in transwell cultures. Both cell populations supported the expansion of progeny from CD34+ cell-enriched cord blood mononuclear cells suspended in the upper chamber. These characteristics, plus the fact that BMEC-1 can be maintained independently of exogenous growth factors and exhibit contact inhibition, indicate that this cell line can be used to further define the role of BMEC in hematopoiesis.

Bone Marrow↗