Expression and function of adhesion molecules on human hematopoietic stem cells: CD34+ LFA-1- cells are more primitive than CD34+ LFA-1+ cells.

Advances in fluorescence-activated cell sorter technology have brought about multicolor analysis of cell phenotypes. To clarify the phenotypes of human hematopoietic stem cells (HSCs), we initially prepared novel antibodies against CD34 and labeled one of them (4A1) with allophycocyanin (APC). With this, we analyzed the phenotypes of CD34+ HSCs and showed that primitive HSCs or CD34+CD33- cells expressed adhesion molecules such as CD43, CD44, CD11a, CD11c, CD18, and leukocyte adhesion molecule (LAM-1). The more primitive hematopoietic cells or CD34+CD38- cells also expressed CD11a and CD18 with an incidence of 20% to 30%. To clarify the role of adhesion molecules in HSCs, we examined the colony forming capacity after long-term culture with allogeneic irradiated stromal layers. Among CD34+CD33- cells, CD18+ cells gave rise to colony-forming cells (CFCs) on stromal layers, but reached a maximum at week 2, after which the number of generated CFCs decreased. On the other hand, CD18- cells generated less CFCs than CD18+ cells at 2 to 3 weeks, but increased after 4 weeks of culture. When CD18 or CD11a antibody was added to a coculture system of CD34+CD33- cells with stromal layers, the number of generated CFCs decreased significantly compared with the no antibody control. Leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) was expressed on some populations of hematopoietic cells and contributed to the proliferation by interacting with stromal cells. However, more primitive cells capable of reconstituting hematopoiesis did not express LFA-1. These data provide a rationale for the administration of anti-LFA-1 antibody after bone marrow transplantation for reducing the graft failure.     

Expression of adhesion molecules on acute leukemic blast cells and sensitivity to normal LAK activity

Summary Antigenic expression of CD54 and CD58 adhesion molecules was investigated on leukemic blast samples from ten patients with acute lymphoblastic (ALL) and 14 with acute myeloblastic (AML) leukemia. The mean intensity of fluorescence (MIF) was calculated and correlated with sensitivity of blast cells to the lytic activity of allogeneic lymphokine-activated killer (LAK) cells. CD54 antigen was expressed in all AML cases with an MIF of 11.2 while in ALL, though present in the majority of cases, it was absent in one case and expressed in less than 30% of blasts in two others, with an overall MIF of 3.0. CD58 expression was similar in both groups of patients, with an MIF of 7.6 in ALL and 7.0 in AML. In addition, in six of the ten ALL cases and in two of the 14 AML cases, leukemic blasts proved to be resistant to the cytotoxic activity of normal allogeneic LAK effectors. In these LAK-resistant patients, the CD54 antigenic expression was lower (p=0.03) than in LAK-sensitive patients with an MIF of 1.7 vs 4.9 in ALL and 1.35 vs 12.9 in AML cases. Finally, blocking of CD54 and/or CD58 receptors on leukemic blasts resulted in a slight reduction of⁵¹Cr release. Findings suggest that CD54 is differently expressed on myeloid and lymphoid blasts and that there is a correlation between CD54 MIF and susceptibility of blasts to the LAK activity.     

Expression of junctional adhesion molecules on the human lymphatic endothelium

Human lymphatic vessels express several leukocyte adhesion molecules. The study here investigated the expression of three junctional adhesion molecules (JAM) which are a newly reported glycoprotein family of adhesion molecules on human lymphatic endothelium. In this study, JAM-1 and JAM-3 but not JAM-2 were detected in cultured human neonatal dermal lymphatic endothelial cells (LEC) at the gene and protein levels by microarray, RT-PCR, real-time PCR, and immunohistochemical analysis. The JAM-1 and JAM-3 expression was not altered in the TNF-alpha-treated LEC or in the untreated cells. In human tissue, the expression of JAM-1, and the expression of JAM-1, JAM-2, and JAM-3 were observed in collecting lymphatic vessels of uninflamed small intestine, and in initial lymphatics of inflamed tongue and uninflamed gingival tissue. It is thought that JAM-2 mRNA could be produced in mature vascular endothelium but not in cultured cells, and that human intestinal and oral lymphatic vessels usually express JAM-1, JAM-2, and JAM-3. There were initial lymphatics simultaneously expressing JAM-1, JAM-2, and JAM-3 in the mucosal connective tissue papillae of gingival tissue. The three JAM expressions on the lymphatic endothelium may contribute to both seal the cell-cell contact at interendothelial junctions and also allow lymphocytes to transmigrate into lymphatic vessels from tissue, independent of inflammatory cytokines.     

Surface antigen expression of human neoplastic plasma cells includes molecules associated with lymphocyte recirculation and adhesion

Adherent lymphokine activated killer (ALAK) cells are a subpopulation of activated natural killer (NK) cells with MHC unrestricted antitumour activity distinguished by their propensity to adhere to plastic in the presence of interleukin-2 (IL-2). We generated ALAK cells from seven patients with chronic myeloid leukaemia (CML) following Campath-1-depleted bone marrow transplantation (BMT). Five had relapsed and were in chronic phase, one had cytogenetic evidence of relapse and one had prior evidence of cytogenetic relapse but was in complete remission at time of study. Phenotypically the ALAK cells included both CD56+/CD3- NK cells and CD56-/CD3+ T cells. The CD3- subpopulation were studied cytogenetically and their functional activity tested in a 4 h 51Cr release cytotoxicity assay using the pretransplant leukaemia cells as targets. Cytogenetic studies showed that the ALAK cells from six patients were Ph negative, and where donor and recipient were sex mismatched, ALAK cells were exclusively of donor origin. In one patient ALAK cells were Ph positive and of recipient origin in eight of nine metaphases. In the 51Cr release assay the ALAK cells showed significant lysis of the pretransplant leukaemia in five of the seven patients tested. These data indicate that in CML patients who relapse post-BMT the NK cells are usually of donor origin but may be recipient-derived. In most patients these ALAK cells have antileukaemic activity in vitro.     

FLT3 receptor expression on the surface of normal and malignant human hematopoietic cells

FLT3 ligand is a hematopoietic growth factor that plays a key role in growth of primitive hematopoietic cells. FLT3 receptor mRNA is found in early hematopoietic progenitors and in human myeloid leukemia blasts. Much less is known about the surface expression of FLT3 receptor on human hematopoietic cells. Using human 125I-FLT3 ligand, we have identified and characterized surface FLT3 receptors on normal and malignant human hematopoietic cells and cell lines. Our results showed that surface display of FLT3 receptor was greatest in fresh myeloid leukemia blast cells and myeloid leukemia cell lines. Erythroleukemic and megakaryocytic leukemia cell lines (n = 5) bound little to no 125I- FLT3 ligand. Scatchard analysis of 125I-FLT3 ligand binding data shows that three myeloid leukemia cell lines, ML-1, AML-193, and HL-60, as well as normal human marrow mononuclear cells, exhibit high affinity FLT3 receptors. Crosslinking of 125I-FLT3 ligand to FLT3 receptors on the surface of ML-1 myeloid leukemia cells indicates that the FLT3 ligand. The rates of FLT3 ligand internalization and degradation were determined by binding 125I-FLT3 ligand to ML-1 cells and acid stripping to distinguish surface bound from internalized ligand. Internalized 125I-FLT3 ligand was detected within 5 minutes after binding to ML-1 cells. In addition, we evaluated the effect of FLT3 ligand on megakaryocytic colony growth and nuclear endoreduplication, alone or in the presence of thrombopoietin. FLT3 ligand did not promote colony forming unit megakaryocyte (CFU-Meg) colony growth or megakaryocyte nuclear maturation, nor did FLT3 ligand augment the effects of thrombopoietin on these measures of megakaryopoiesis. These data indicate that the FLT3 receptor shares several characteristics with the c-kit receptor including dimerization and rapid internalization. However, the more restricted cellular distribution of the FLT3 receptor may target the effects of FLT3 ligand to primitive hematopoietic cells and to myeloid and lymphoid progenitor cells, in contrast to the pleiotropic effects of the c-kit receptor ligand, stem cell factor.     

Homotypic cell aggregations of human myeloma cells with ICAM-1 and LFA-1 molecules

Some myeloma cells freshly isolated from bone marrow aspirates in human myelomas and some myeloma cell lines formed spontaneous cell aggregations in vitro (homotypic cell aggregations). In order to clarify the surface molecules involved in homotypic cell aggregations and physiological roles of these cell aggregations, we investigated the expressions of intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function-associated antigen 1 (LFA-1) on 20 samples of freshly isolated myeloma cells and three myeloma cell lines and the effect of anti-ICAM-1 and anti-LFA-1 alpha antibodies on myeloma cell proliferation in vitro. All myeloma cells that we tested expressed ICAM-1 on their surface. Among them, myeloma cells that strongly coexpressed LFA-1 alpha, formed homotypic cell aggregates in vitro. These spontaneous cell aggregations were completely released by adding either anti-ICAM-1 or anti-LFA-1 alpha antibody. During short-term culture, spontaneous proliferation of myeloma cells in vitro and their proliferative responses to recombinant interleukin-6 (rIL-6) were not affected by pretreatment of myeloma cells with anti-ICAM-1 or anti-LFA-1 alpha antibody. Therefore these data suggest that homotypic cell aggregation of myeloma cells is mediated by ICAM-1 and LFA-1 molecules, but myeloma cell proliferation may not be modulated by these adhesion molecules during short-term cultures.     

Expression of adhesion molecules on CD34 cells does not correlate with property to adhere to normal marrow-derived stroma

The differences of engraftment kinetics after umbilical cord blood (CB) and mobilized peripheral blood (MPB) transplantation are not yet fully understood. Since homing into bone marrow microenvironment would certainly play crucial role during engraftment, we have investigated adhesion capacities of CB and MPB CD34⁺ cells to bone marrow stromal cells (SC) and expression of several molecules known to be important during homing process. Cells, at day 0 and after 48 hour culture with SCF+ IL-3, are plated for 2 hours on BM confluent stromal layer. The non adherent (na) and adherent (a) fractions are then recovered. Cells are counted and evaluated for the coexpression of CD34 and VLA-4 (CD49d), VLA-5 (CD49e), L-selectin (CD62L) or CXCR4. The adhesion molecule expression is expressed in term of antigen density (Mean Equivalent Soluble Fluorescence (MESF)x 10³).Our results show that a) cell adhesion capacity is significantly increased after culture in CB (78.6±4.0% vs 58.2±7.4%) as well in MPB (72.5±5.9% vs 51.5±5.4%) and is not different between the two sources of progenitors; b) VLA-4, VLA-5, CD62L and CXCR4 expression increased significantly after culture, in CB and in MPB; c) no specific expression on adherent cells could be observed except for L-selectin after 48h culture; d) the only significant difference between CB and MPB concerns the CXCR4 expression,lower on PB cells but independent of cell adhesion capacities. In conclusions, it seems that neither adhesion capacities nor expression of evaluated adhesion molecules could explain differences between CB and MPB engraftment.     

Proliferation of normal and malignant human immature lymphoid cells

In this study, the proliferative activity of human B and T cell precursors in central lymphoid organs, acute lymphoblastic leukemia (ALL) cells, and permanent cell lines was investigated with double- and triple-color-labeling methods for the analysis of cell cycle-associated features such as 5-bromo-2'-deoxyuridine (BrdU) incorporation and the expression of a nuclear proliferation-associated antigen, Ki67, together with the phenotypic profile of the cells. In infant and regenerating bone marrow (BM), 41.5% +/- 4.0% of terminal deoxynucleotidyl transferase (TdT+) cells were Ki67+, and 30.0% +/- 4.0% incorporated BrdU. A similar proportion of TdT+ dividing cells was observed in adult BM. The proliferative activity of the B cell progenitors reached the peak at the pre-B stage: 80.8% +/- 7.6% and 35.3% +/- 6.1% of c mu +, RFB7- cells were Ki67+ and BrdU+, respectively. In contrast, greater than 95% of surface immunoglobulin- positive BM lymphocytes were resting cells. In infant thymus the highest dividing capacity (95% Ki67+, 60% to 90% BrdU+) was observed in large cortical thymocytes (TdT+, CD1-, cCD3+), and TdT+, CD1+ cortical thymocytes also showed a high proliferative activity (74.3% +/- 2.3% Ki67+, 22.0% +/- 1.0% BrdU+), but TdT-, mCD3+ thymic lymphocytes were mainly resting cells (less than 5% Ki67+, less than 1% BrdU+). The proliferative activity of null and common ALL blasts was significantly lower than that of normal BM TdT+ cells (15.5% +/- 4.2% Ki67+, 6.2% +/- 2.1% BrdU+; P less than .001). Dividing ALL blasts were TdT+ and expressed surface antigens detected by CD10 and/or CD19 antibodies. In T cell-ALL, the percentages of Ki67+ and BrdU+ blasts were also lower than those found in the corresponding normal immature thymocytes (13.0% +/- 3.1% and 2.4% +/- 1.3%, respectively; P less than .001). Thus, ALLs derive from actively proliferating lymphoid precursors but have a lower dividing capacity than the corresponding normal cell types. In ALL cases with heterogeneous expression of markers such as cmu and CD1, dividing blasts were distributed among both negative and positive populations, thus indicating that blasts with signs of differentiation also remain within the dividing pool.     

Distribution of complement receptors on human normal and malignant mononuclear cells

Mononuclear cells from normal human subjects and patients with chronic lymphocytic leukemia (CLL), chronic lymphosarcoma cell leukemia (LCL), and hairy cell leukemia (HCL) were labeled with fluoresceinated, purified human C3b (FI-C3b) and analyzed using the fluorescence- activated cell sorter (FACS). FI-C3b labeled 17.6% +/- 6.0% of peripheral blood mononuclear cells (PBM) from 20 normal subjects, which, when separated by the FACS, consisted of B lymphocytes and approximately 5% monocytes. Analyses in which either monocytes or B lymphcoytes were excluded from consideration demonstrated that both these cell types were labeled by the FI-C3b with a heterogeneous distribution of fluorescence intensity, indicating either heterogeneity of CR density or variable avidity of individual CR for the FI-C3b. FACS profiles of PBM ( < 5% monocytes) from 14 of 15 patients with CLL showed a homogeneous distribution of very low fluorescence intensity, with > 60% of the cells being slightly more fluorescent than unlabeled controls. This low, homogeneous distribution of fluorescence is strikingly similar to profiles of CLL cells labeled with anti-Ig reagents and suggests homogeneity of low CR density and/or avidity. Similarly, CR+ mononuclear cells from five patients with HCL and three patients with LCL displayed more homogeneous FI-C3b labeling than normal CR+ PBM. Homogeneity of FI-C3b binding to CLL, LCL, and HCL cells further supports the concept for a clonal origin for these disorders.     

IgG plasma cells display a unique spectrum of leukocyte adhesion and homing molecules

Long-lived antibody-secreting plasma cells are formed in the secondary lymphoid organs and subsequently home to the bone marrow, although the mechanisms that control this migration remain primarily unknown. In this study, we show that IgG plasma cells constitute a significant fraction of cervical lymph node cells from older mice deficient in both E- and P-selectin (E/P(-/-)), and that these cells can be prospectively isolated by phenotype. These IgG plasma cells were polyclonal, cytoplasmic Ig(+), spontaneously secreted antibody, were in the G(0)/G(1) phase of the cell cycle, and failed to express multiple B-cell surface markers. The plasma cells exhibited up-regulated cell surface expression of multiple adhesion molecules, including alpha(4) and leukocyte function-associated antigen 1 (LFA-1) integrins, CD44, and P-selectin glycoprotein ligand 1 (PSGL-1). IgG plasma cells bound to vascular cell adhesion molecule 1 (VCAM-1) significantly better than IgM(+) B cells, indicating that the alpha(4) integrins were constitutively active. A subset of IgG plasma cells also bound hyaluronic acid, the ligand for CD44. In addition, the IgG plasma cells interacted strongly with E-selectin, but poorly with P-selectin, despite elevated levels of PSGL-1 protein. The preferential interaction of plasma cells with E-selectin, but not P-selectin, correlated with elevated alpha1,3-fucosyltransferase-VII messenger RNA levels, but selective down-regulation of core 2 beta1-6-N-glucosaminyltransferase levels, compared to B cells. These results demonstrate a unique adhesion profile for murine IgG plasma cells. Furthermore, the E/P(-/-) mice represent a novel system to isolate and purify significant numbers of primary IgG plasma cells.     

Apolipoprotein E and colon cancer. Expression in normal and malignant human intestine and effect on cultured human colonic adenocarcinoma cells

Background: Apolipoprotein E (apo E) is a key regulatory protein in lipoprotein metabolism and it is also a potent inhibitor of cell proliferation. Although genetic alterations of apo E affect enterohepatic cholesterol transport and, presumably, the risk of colon carcinoma, the expression and potential functions of apo E in the human intestine are poorly known. Methods: The localization of apo E in normal and malignant gastrointestinal tract was studied using immunohistochemistry and in situ hybridization. The effect of apo E3 on cell polarity and the distribution of beta-catenin was examined in HT29 human colon adenocarcinoma cell lines. Results: Both apo E protein and mRNA were present throughout human intestine. The macrophages in the superficial lamina propria of normal colon were more strongly positive for apo E than those in the small intestine, where the most positively stained cells were dendritic cells and macrophages in the germinal centers of lymphoid follicles. In carcinomas, intensely positive macrophages surrounded the tumor area. In cultured undifferentiated HT29 cells, treatment with apo E improved cell polarity and translocated beta-catenin from the cytoplasm to cell--cell adhesion sites. Conclusions: Mononuclear phagocytes and endocrine cells are the main source of apo E in the gastrointestinal tract. We hypothesize that macrophage-derived apo E may modulate epithelial integrity and thus contribute to cell growth.     

Expression of adhesion molecules in endothelial cells during allogeneic bone marrow transplantation

Abstract: Endothelial cell activation during allogeneic bone marrow transplantation, mainly in acute graft-versus-host disease (aGvHD) was studied in 23 recipients and 5 controls using anti-von Willebrand factor (vWF) antibody, antibodies to endothelial leukocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and anti-HLA-DQ antibody, by immunohistological staining of skin. vWF extravasation, ELAM-1 and VCAM-1 expression were present in most recipients with a cutaneous rash which was confirmed as an aGvHD by histological examination (documented aGvHD) (p = 0.005 for vWF extravasation and ELAM-1 expression and p = 0.03 for VCAM-1 expression in comparison with the controls). In recipients with a rash, the cases displaying vWF extravasation and ELAM-1 expression were significantly more numerous in those with a documented aGvHD than in those without histological features of aGvHD (p = 0.01). vWF extravasation and ELAM-1 occurred concomitantly (p<0.01). This study demonstrates that, during the course of skin aGvHD following bone marrow transplantation, there is transient expression of ELAM-1 and VCAM-1 by endothelial cells and simultaneous vWF extravasation, indicating an intense inflammation with endothelial cell participation.     

Effect of an LH-RH analogue on adhesion molecules in human prostate cancer cells

BACKGROUND. The reduction in or the loss of the cell-cell adhesion often characterizes epithelial tumor initiation and progression. In the present study, we investigated the effects of the LH-RH analogue Leuprorelin acetate (LA), alone or associated with Dihydrotestosterone (DHT), on the expression of the adhesion proteins E-cadherin, α-, β- and γ-catenin in androgen-sensitive (LNCaP) and -insensitive (PC-3 and DU-145) prostate cancer cells. MATERIALS AND METHODS. Protein expression was evaluated by Western blotting on cells treated for 48 h with LA (10-11 or 10-6 M) and 10-9 M DHT, alone or combined. RESULTS. In LNCaP cells, all the above mentioned molecules are expressed. PC-3 cells lack α-catenin, while DU-145 cells only express β- and γ-catenin. In both LNCaP and PC-3 cells two truncated forms (97 and 35 kDa) of E-cadherin are present other than the functional protein (120 kDa). In LNCaP cells, no significant changes in E-cadherin (120 and 97 kDa) level were produced by DHT, while the 35 kDa fragment was reduced by 34%. LA increased the full length E-cadherin (26-30%) as well as the two fragments (30-49%). The addition of DHT to LA significantly reduced the analogue-induced E-cadherin raising. In LNCaP cells β- and γ-catenin were up-regulated either by DHT (24% and 20%, respectively) or LA (up to 18% and up to 40%, respectively), while the expression of α-catenin was not modified. The combined DHT/LA treatment results in a less marked increase in β- and γ-catenin levels. In PC-3 cells no changes in adhesion molecule expression were produced by LA treatment, while in DU-145 cells the analogue determined an appreciable reduction in β- (20%) and γ-catenin (up to 35%) levels. CONCLUSIONS: The up-regulation of E-cadherin, β- and γ-catenin in LNCaP cells by LA may be considered as another feature of the direct antitumor LH-RH analogue activity, as it may contribute to the maintenance/restoration of the normal architecture of prostate epithelium. The LA-induced modifications of catenins in DU-145 cells are worth some further investigations.     

Expression sequences of cell adhesion molecules.

A reexamination of the expression of cell adhesion molecules (CAMs) during the development of the chicken embryo was carried out using more sensitive immunocytochemical techniques than had been used previously. While the previously determined sequence of CAM expression was confirmed, neural CAM (N-CAM) was also detected on endodermal structures such as the lung epithelium, gut epithelium, and pancreas and on budding structures such as the pancreatic duct and gall bladder. It was also found on ectodermal derivatives of the skin. In most of these sites, N-CAM expression was transient, but in the chicken embryo lung, the epithelium remained positive for N-CAM and liver CAM (L-CAM) into adult life. Thus, at one time or another, both of these primary CAMs can be expressed on derivatives of all three germ layers. At sites of embryonic induction, epithelial cells expressing both L-CAM and N-CAM, or L-CAM only, were apposed to mesenchymal cells expressing N-CAM. Examples included epiblast (NL) and notochord (N); endodermal epithelium (NL) and lung mesenchyme (N); Wolffian duct (NL) and mesonephric mesenchyme (N); apical ectodermal ridge (NL) and limb mesenchyme (N); and feather placode (L) and dermal condensation (N). The cumulative observations indicate that cell surface modulation of the primary CAMs at induction sites can be classified into two modes. In mode I, expression of N-CAM (or both CAMs) in mesenchyme decreases to low amounts at the cell surface, and then N-CAM is reexpressed. In mode II, one or the other CAM disappears from epithelia expressing both CAMs. As a result of the primary processes of development, collectives of cells linked by N-CAM and undergoing modulation mode I are brought into the proximity of collectives of cells linked by L-CAM plus N-CAM or by L-CAM undergoing modulation mode II. Such adjoining cell collectives or CAM couples were found at all sites of embryonic induction examined.