Andrew W. Boyd

The role of the LFA-1/ICAM-1 interaction in human leukocyte homing and adhesion.

During differentiation leukocytes exhibit a complex, changing pattern of adhesive interactions with other cells and with extracellular matrix components. Early in lineage commitment, within bone marrow or thymus, these interactions achor the developing cell within these organs until it is sufficiently mature to migrate into the circulation. Down-regulation of these adhesive bonds must occur to prepare the cell for migration; however, this remains to be studied in detail. Mature (resting) lymphoid cells continuously recirculate between the vascular and lymphatic systems. This pattem of recirculation is broken if the cell is altered, for example by encounter with antigen. The cell then re-expresses adhesive properties which fix it within the tissue. The analysis of some of the mechanisms which mediate leukocyte binding to vascular endothehum and of the subsequent events within the tissues will be the major focus of this review. Under normal conditions of re-circulation the trigger which initiates the movement of lymphoid cells out of the circulation is binding to the specialized postcapillary high endothelial venules (HEV) in secondary lymphoid organs (Gowans &. Knight 1964). Moreover, it has been suggested that within the recirculating pool of lymphocytes there are distinct subsets of lymphocytes which show organ-specific pathways of migration (Jalkanen et al. 1986a). This was demonstrated in vitro with normal murine lymphocytes isolated from peripheral lymph nodes or Peyers patches. In each case these cells showed preferential adherence to HEV from the donor tissue (Butcher et al. 1980). A similar differential pattern of adherence was shown for particular murine lymphomas. By screen-

Ligand for EPH-related Kinase (LERK) 7 Is the Preferred High Affinity Ligand for the HEK Receptor

HEK is a member of the EPH-like receptor tyrosine kinase family, which appear to have roles in development and oncogenesis. Recently, we purified a soluble HEK ligand which is also a ligand (AL1) for the HEK-related receptor EHK1. Promiscuity appears to be a characteristic feature of interactions between the EPH-like receptors and their ligands, termed ligands for EPH-related kinases (LERKs). This prompted us to analyze the interactions between the HEK exodomain and fusion proteins comprising candidate LERKs and the Fc portion of human IgG1 (Fc) or a FLAG™-peptide tag by surface plasmon resonance, size exclusion high performance liquid chromatography, sedimentation equilibrium, and transphosphorylation. Our results indicate that AL1/LERK7 is the preferred high-affinity ligand for HEK, forming a stable 1:1 complex with a dissociation constant of 12 nm. As expected the apparent affinities of bivalent fusion proteins of LERKs and the Fc portion of human IgG1 had significantly reduced dissociation rates compared with their monovalent, FLAG™-tagged derivatives. High-avidity binding of monovalent ligands can be achieved by antibody-mediated cross-linking of monovalent ligands and with LERK7 results in specific phosphorylation of the receptor. By extrapolation, our findings indicate that some of the reported LERK-receptor interactions are a consequence of the use of bivalent ligand or receptor constructs and may be functionally irrelevant.

Low dose cytosine arabinoside: partial remission of acute myeloid leukaemia without evidence of differentiation induction

Summary. Thirteen patients with acute myeloid leukaemia aged from 19 to 81 were treated with low dose cytosine arabinoside (ARA‐C) in a dose of 10–15 mg/m2 twice daily subcutaneously. Three complete remissions were obtained. Partial responses were observed in a further two patients. To analyse the action of low dose ARA‐C freshly isolated leukaemic cells and cells from the cloned promyelocytic leukaemia cell line (HL60) were cultured in vitro in the presence of cytosine arabinoside. Minimal evidence of differentiation induction was observed when compared with the cytotoxic effects of the drug. These results suggest that ARA‐C does not exert its anti‐leukaemic effects by halting proliferation through differentiation induction. Rather, it appeared that the capacity of this agent to kill cells in S‐phase produced a progressive depletion of the cycling leukaemic cells. This resulted in a corresponding steady decline in the total leukaemic cell population.

Tissue factor expression in human leukemic cells

Patients with acute leukemia are at increased risk for thrombotic and hemorrhagic complications, particularly those patients with acute promyelocytic leukemia (APL) undergoing induction chemotherapy. These serious complications have been attributed by some authors to the release of tissue factor (TF) procoagulant activity (PCA), particularly during cytotoxic chemotherapy. In previous studies of normal peripheral blood cells, only cells of the monocyte lineage have been found to express TF PCA. Therefore, several questions remain regarding the origin and characterization of the PCA in malignant leukemic cells, particularly those thought to be derived from granulocyte progenitor cells. We utilized a full-length cDNA probe, several monoclonal antibodies (MAbs) and a sensitive one-stage PCA assay to study the expression of TF in the human cell line, HL-60, in human peripheral blood monocytes/macrophages (Mo/Mø) and in highly purified populations of human polymorphonuclear leukocytes (PMN). In the HL-60 cells we detected low but significant levels of TF mRNA and TF antigen (TF:Ag). In unstimulated cells, coordinate increased levels of TF mRNA, TF:Ag and TF PCA expression were noted following phorbol-ester-induced macrophage differentiation of the cells, but a decreased level of TF mRNA with no change in the basal level of TF:Ag expression occurred following retinoic acid-induced granulocyte differentiation of this cell line. Long-term cultures of stimulated mature Mo/Mø demonstrated initial coordinate expression of TF mRNA, TF:Ag and TF PCA, but TF:Ag expression persisted even after 7 days (when TF PCA was undetectable). No TF PCA, TF:Ag or TF mRNA was demonstrated in highly purified populations of human PMN, regardless of culture conditions. Discordant expression of TF mRNA, TF:Ag and TF PCA in HL-60 cells suggests the possibility of novel, post-synthetic mechanisms for the regulation of TF PCA expression, which might be dependent on the phenotypic differentiation level of the cell. Such mechanisms (yet to be defined) might account for the ability of some leukemic cells, which frequently express characteristics of more than one cell line (e.g. monocytes and granulocytes), to express a TF gene product capable of activating blood coagulation.

Inhibition of intercellular adhesion molecule 1-dependent biological activities by a synthetic peptide analog.

We have used a combination of hydropathy analysis of the intercellular adhesion molecule 1 (ICAM-1) sequence and dot-matrix comparison of the sequence with the homologous, but functionally distinct, protein myelin-associated glycoprotein to identify a putative functional binding region. One polar, and presumably surface-exposed, region of ICAM-1 showed no significant identity with myelin-associated glycoprotein. A synthetic peptide analog based on the sequence of this region (JF9) mimicked the inhibitory effects of the anti-ICAM-1 monoclonal antibody WEHI-CAM-1. These included inhibition of ICAM-1-dependent homotypic aggregation of Raji Burkitt lymphoma and phorbol-ester treated U937 cells at concentrations as low as 80 micrograms/ml (24 microM). In addition, at a concentration of 100 micrograms/ml, the peptide analog effectively inhibited cytotoxic cell activity, an ICAM-1-dependent effector function of the immune response. This simple method of sequence analysis may have general applicability to the identification of functional domains in homologous, but functionally distinct, proteins such as the translated products of gene families.

Human leukocyte antigens: an update on structure, function and nomenclature.

&NA; The study of human leukocyte antigens, predominantly by monoclonal antibody techniques, is a rapidly changing area of basic research and clinical investigation. This review outlines some of the results and trends of research in this field. Of particular importance is the updating of the current nomenclature. The CD classification of these antigens has become the standard form in published literature and provides a basis for standardization of clinical reporting. The current CD classification is presented in the form of a list, with a brief summary of each antigen beside each entry. The results reviewed range from the section on CD1 antigen in which the data presented are primarily concerned with the underlying biology of the antigens to the section on clinical application which has little biological content.

The Effect of Cytokines on the Expression of Mhc Antigens and Icam-1 by Normal and Transformed Synoviocytes

We report the expression on synovial cells of cell surface molecules known to be involved in T cell activation by antigen presenting cells. Normal human synovial fibroblasts and a human synovial cell line transformed with the SV40 large T antigen were used for in vitro stimulation studies with recombinant cytokines. We demonstrate an increase in MHC-A, B, C expression in normal synovial cells in response to recombinant interferon gamma (r gamma IFN), tumour necrosis factor alpha and beta (rTNF alpha and beta) and interleukin-1 (rIL-1 alpha). Intercellular adhesion molecular-1 (ICAM-1) expression was increased in parallel with MHC Class I. The combination of r gamma IFN and rTNF alpha was additive in its effect on ICAM-1 expression. Northern blot analysis suggests that ICAM-1 expression in synovial cells is controlled at the level of transcription. In contrast, MHC Class II (HLA-DR) was only significantly induced by r gamma IFN. Other stimuli including interleukin-4 (IL-4), interleukin 6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF) and prostaglandin E2 (PGE2) did not affect the expression of ICAM-1 or MHC Class I and II. Leucocyte function antigen 3 (LFA-3) expression was not affected by any of the stimuli tested. Immunoperoxidase staining of rheumatoid synovial tissue confirmed enhanced in vivo expression of ICAM-1 in rheumatoid arthritis. These changes are discussed in the context of T cell activation in inflammatory arthritis.

Characterization of CD3 4+HLA-DR−CD38+ and CD34+HLA-DR−CD38−progenitor cells from human umbilical cord blood

In this study we show that depletion of cells expressing mature cell markers, including HLA-DR, followed by positive cell sorting for cells expressing CD34 and CD38, can be used to define functionally distinct hematopoietic cells from human umbilical cord blood (HUCB). The CD34+HLA-DR-CD38+ population contained the majority of directly clonogenic cells, while the optimal ability to maintain long term co-culture with bone marrow stromal cells was present within the CD34+HLA-DR-CD38- population. 1.2 +/- 0.4% of the CD34+HLA-DR-CD38- cells plated at 1 cell/well and grown in the presence of hematopoietic growth factors (HGF) formed hemopoietic colonies. Mesenchymal elements were observed in 20% of these cultures. No cell growth, however, was observed when the CD34+HLA-DR-CD38- cells were cultured in the absence of HGF. This is in contrast with the findings in fetal bone marrow which demonstrated the presence of stem cells that were independent of HGF. Thus, while it is possible to isolate very immature hemopoietic progenitor cells from HUCB defined by the phenotype Lin-CD34+HLA-DR-CD38-, these cells do not appear to exhibit the pluripotentiality of the analogous population reported in fetal bone marrow. We conclude that these cells are absent or at a very small frequency in HUCB.

Characterization of two novel pre-B-cell lines (LK63 and LiLa-1): Potential models of pre-B-cell differentiation

In this report we describe two newly isolated pre-B acute lymphoblastic leukaemia cell lines. Both cell lines lack EBV as detected by the EBNA-1 gene probed Southern-blots. Neither cell line expressed the B-cell-specific CD20 antigen on the cell membrane. However surface expression of CD20 was induced by phorbol ester (TPA) on both LiLa-1 and LK63 cell lines. Other pre-B and B-cell lines, such as Reh, Nalm-1, and BALL-1 did not exhibit these changes in phenotype. Previous immunoprecipitation studies have noted that a broad 50-55 kD band co-precipitates with the characteristic 33-37 kD CD20 protein. We demonstrate that, while the 33-37 kD CD20 species was undetectable on resting LiLa-1 and LK63 cells, in each case a 50-55 kD protein was immunoprecipitated by the CD20 antibody. However, the failure to detect any cell surface CD20-associated antigen on the control cells by immunophenotyping indicated that the CD20 epitope of the 50-55 kD molecule was not expressed on the cell surface. Following exposure to TPA the 50-55 kD species was reduced over 48-72 h while the level of the p33-37 CD20 protein was increased. Northern-blot analysis showed that the 50-55 kD protein was not a cryptic form of CD20 as the uninduced cells contained no detectable CD20 mRNA. The decrease of the 50-55 kD protein and the acquisition of the mature CD20 molecule were paralleled by a decline in proliferative activity in both cell lines. As expression of CD20 by normal pre-B cells also coincides with the cessation of cell division and maturation towards a mature B-cell phenotype, these cell lines appear to represent models for a discrete stage of B-cell differentiation which may be valuable in defining the signals regulating pre-B-cell proliferation.

Induction of the insulin receptor and other differentiation markers by sodium butyrate in the Burkitt lymphoma cell, Raji.

The very low expression of insulin receptors in the Burkitt lymphoma cell Raji was increased 2-fold, 6-fold and 10-fold after 1, 2 and 3 days, respectively, by incubation with the differentiation inducer sodium butyrate. Insulin receptor number was increased without a change in receptor affinity, in association with an increase in the receptor alpha and beta subunits detected after cell-surface labelling and immunoprecipitation. Expression of cell-surface class I and II human leukocyte antigens, the intercellular adhesion molecule-1 and the CD38 leukocyte antigen was also increased, consistent with B cell differentiation. Butyrate effects were not unspecific, as the binding of tumour necrosis factor and growth hormone and the expression of the B cell markers CD20, B5 and CD21 was not increased. The low expression of insulin receptors on Raji cells is therefore a reflection of the less differentiated state of these cells compared to lymphoblastoid cells.