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Human acute myeloid leukemia (AML) cells adhere to bone marrow fibroblasts (BMF) and extracellular matrix proteins including fibronectin. Adhesion is increased when fibroblast monolayers are exposed to tumor necrosis factor-alpha (TNF) alone and in combination with interferon-gamma (IFN) or interleukin-4 (IL-4). The combination of TNF and IFN caused enhanced AML cell adhesion to treated BMFs, from a mean of 25.0 +/- 4.1% to 36.3 +/- 5.4% (p = 0.0007). Enhanced binding was partially a result of upregulated vascular cell adhesion molecule-1 expression on BMFs. Intercellular adhesion molecule-1 was also upregulated, but did not appear to play a role in the increased binding to cytokine-stimulated BMFs. In contrast to observed adhesion to resting BMFs, AML cells binding to TNF/IFN-stimulated BMFs rely more heavily on the VLA-4 alpha chain (CD49d). In some cases, alpha4 integrin chain antibody was more effective than beta1 antibody in blocking binding, suggesting that a non-beta1 alpha4 integrin, possibly alpha4 beta7, on AML cells may act as a stromal ligand. The addition of alpha4 antibody to beta1 and beta2 antibodies significantly increased the inhibition of AML cells to stimulated BMFs. The myeloid cytokines granulocyte colony stimulating factor, granulocyte-monocyte colony stimulating factor, interleukin-3 and stem cell factor enhanced the adhesion of AML blast cells to BMFs in some cases. The phorbol ester PMA, however, consistently upregulated AML cell-binding to BMFs, the increase being mediated entirely via beta1 and beta2 integrins without altering AML cell integrin expression. Binding of AML cells to marrow stroma can be enhanced by influences on leukemic cell or stroma. Enhanced binding under these conditions occurs via different pathways, illustrating the heterogeneity of mechanisms underlying leukemic cell retention within the bone marrow stroma.

In this study we have demonstrated that natural killer (NK) cells adhere to elements of the bone marrow stroma (BMS) including fibroblasts, fibronectin and laminin but not to collagen type I, vitronectin and hyaluronic acid. NK cells bind to fibronectin and laminin using the beta 1 integrins VLA-4 and VLA-5, and VLA-6 respectively. The mechanism of adhesion to bone marrow fibroblasts is more complicated with beta 1 and beta 2 integrins being partially responsible but the majority of adhesion remaining unexplained. IL-2 stimulation of NK cells resulted in an increase in the expression of adhesion molecules involved in binding of NK cells to bone marrow fibroblasts (BMF) and extracellular matrix (ECM) proteins including the beta 1 chain CD29, alpha chains of VLA-4 and 5, beta 2 chain CD18 and alpha L chain CD11a. A marked increase in expression of beta 7 was also observed. There was a significant increase in the adhesion of NK cells to fibronectin in response to IL-2 treatment. NK cells also bound more strongly to BMF following IL-2 treatment although the development of cytotoxicity appeared to interfere with the adhesion assay. NK cells competitively inhibit the binding of AML blasts but not ALL blasts to BMF. Mechanisms underlying the inhibition of leukemic growth by NK and LAK cells may include direct and cytokine mediated cytotoxicity and perturbation of the interaction of leukemic blasts with the bone marrow stroma which is essential for blast cell survival.

Acute myeloid leukemia (AML) cells express the surface adhesion proteins intercellular adhesion molecule-1 (ICAM-1, CD54) and lymphocyte function associated molecule-3 (LFA-3, CD58). Exposure to the myeloid growth-promoting cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) upregulates expression of ICAM-1 and LFA-3 on AML cells but does not increase their sensitivity to lysis by interleukin-2-activated natural killer cells (LAK) in 51Cr assays. However when AML cells are exposed to GM-CSF prior to incubation with LAK, their subsequent clonogenic activity is significantly reduced. If a blocking antibody to ICAM-1 is added during the incubation period of AML with LAK, the inhibitory effect is completely ablated. A less pronounced effect is observed with an antibody to LFA-3. ICAM-1 is expressed on a greater proportion of CD34+ than CD34- AML cells and exposure to GM-CSF induces a significantly greater upregulation of ICAM-1 on leukemic CD34+ cells than their CD34- counterparts. These data suggest that the inhibitory effect of IL-2-activated natural killer cells on clonogenic AML cells is mediated principally via the lymphocyte function associated molecule-1 (LFA-1)/ICAM-1 interaction. Interleukin-2 upregulates LFA-1 expression on natural killer cells. Simultaneous administration of effector cell activators such as IL-2 and target cell modulators such as GM-CSF may have a therapeutic benefit in patients with minimal residual myeloid leukemia.

Human acute myeloid leukemia (AML) cells, like normal hematopoietic progenitors, die rapidly by apoptosis when cultured under serum-free conditions. Apoptosis was demonstrated by electron microscopy and agarose gel electrophoresis and quantified by flow cytometry. Culturing AML blasts in the presence of a bone marrow fibroblast (BMF) monolayer reduced the percentage of AML blasts undergoing apoptosis in the majority of cases studied. The effect was more pronounced when AML cells were cultured in the presence of an adherent long-term bone marrow (LTBM) stroma rather than BMF. Overall, the mean percentage of AML cells with fragmented DNA fell from 85 +/- 8% in control cultures to 20 +/- 9% in cultures with adherent stroma (p = 0.0004, n = 7). Supplementation of serum-free medium with recombinant cytokines, including stem cell factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-alpha or with human placenta-conditioned medium (HPCM) matched the degree of inhibition of apoptosis induced by BMF in only 50% of cases. Granulocyte colony-stimulating factor (G-CSF), interleukin-1 beta (IL-1 beta), and IL-6 were completely ineffective. Consistent with this observation, direct contact between leukemic cells and adherent layers was essential for maximum inhibition of leukemic-cell DNA fragmentation. Separation by a porous membrane allowing passage of soluble growth factors, but interrupting direct cell contact, was associated with significantly greater DNA fragmentation and cell death. Inhibition of leukemic-cell apoptosis correlated with improved survival and growth of malignant clonogenic cells. Colonies grown in cultures were identified as leukemic by morphology and by fluorescence in in situ hybridization to demonstrate numerical chromosomal abnormalities identified at diagnosis. Close contact between leukemic cells and bone marrow inhibits blast cell apoptosis and directly promotes survival of clonogenic AML cells.

Adhesion protein expression by acute myeloid leukaemia (AML) cells may affect bone marrow stromal localization and determine exposure of leukaemic cells to stromal derived myeloid growth factors. We have analysed the surface expression by myeloid leukaemic cells of proteins with known adhesive function and the ability of AML cells to adhere to bone marrow fibroblasts and the extracellular matrix proteins fibronectin and laminin. Cells from all six patients tested adhered to bone marrow fibroblast monolayers (mean binding 28.8 +/- 12.8%) and to purified fibronectin in five cases studied (mean binding 33.8 +/- 15.3%). Cells from four patients with AML also adhered to laminin (mean binding 20.9 +/- 4.0%). AML cells from the majority of patients with leukaemia at diagnosis or relapse expressed the ligand pair LFA-1 and ICAM-1, the CD2 ligand LFA-3, alpha and beta chains of the integrins VLA-4, VLA-5 and VLA-6, and the hyaluronate receptor CD44. Antibodies to CD11a, CD18, VLA-4 alpha, and VLA-5 alpha failed to inhibit binding of AML cells to bone marrow fibroblasts but anti-VLA-5 alpha antibodies inhibited AML cell binding to fibronectin by approximately 50%. The ability of AML cells to adhere to bone marrow fibroblasts and extracellular matrix proteins such as fibronectin and laminin may to help explain the capacity of AML cells to persist in the marrow during periods of apparent complete remission and to subsequently proliferate under the influence of locally secreted myeloid growth factors.