Coordinate expression of OPN and associated receptors during monocyte/macrophage differentiation of HL-60 cells

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits α_v, β₃, β₁, were examined. Results were as follows: (1) PMA induced OPN mRNA and OPN secretion into media; (2) untreated cells expressed β₁ and CD44 mRNA, and PMA induced α_v and β₃ mRNA and increased β₁ and CD44 mRNA expression; (3) PMA increased levels of α_v, β₃, β₁ and CD44 protein on the cell surface; and (4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, α_v, β₃, β₁, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells. J. Cell. Physiol. 175:229–237, 1998. © 1998 Wiley-Liss, Inc.     

Antisense CD11b integrin inhibits the development of a differentiated monocyte/macrophage phenotype in human leukemia cells

Macrophage-like development of myeloid leukemia cells which can be induced by agents such as phorbol esters (TPA) is accompanied by integrin expression and cell adhesion. Thus, in differentiating myeloid leukemia cells CD11b is predominantly expressed which can associate with CD18 to form the functional heterodimeric integrin Mac-1. To elucidate the role of cell adhesion during macrophage-like differentiation, we transfected human U937 myeloid leukemia cells with a vector containing the CD11b gene in antisense orientation. Expression of the CD11b antisense gene in stably transfected U937 cells (as-CD11b cells) resulted in an attenuated response to TPA. As-CD11b cells demonstrated poor adhesion to solid substrate upon TPA treatment in contrast to U937 control cells. Constitutive expression of c-myc in as-CD11b transfectants was higher than in control cells and failed to be repressed by TPA treatment. Moreover, unlike control cells, antisense transfectants failed to induce expression of early response genes such as c-jun and the redox factor ref-1 upon TPA stimulation. Consequently, the induction of monocytic differentiation markers such as the activity of alpha-naphthyl acetate esterase, the capacity to reduce nitroblue tetrazolium and the expression of the vimentin gene was much lower in antisense transfectants than in control U937 cells. According to the failure to undergo a monocytic differentiation program, TPA treatment of as-CD11b cells resulted in a progressively increasing amount of apoptotic cells whereas the differentiated population of U937 control cells remained alive. Taken together, these data suggest that the integrin-mediated (particularly CD11b-mediated) adhesion of myeloid leukemia cells in the course of induced monocytic differentiation is crucial for cell attachment, development of a monocytic phenotype and subsequent survival.     

The role of integrin alpha D beta2 (CD11d/CD18) in monocyte/macrophage migration

Integrin α_Dβ₂ (CD11d/CD18) is a multiligand macrophage receptor with recognition specificity identical to that of the major myeloid cell-specific integrin α_Mβ₂ (CD11b/CD18, Mac-1). Despite its prominent upregulation on inflammatory macrophages, the role of α_Dβ₂ in monocyte and macrophage migration is unknown. In this study, we have generated model and natural cell lines expressing different densities of α_Dβ₂ and examined their migration to various extracellular matrix proteins. When expressed at a low density, α_Dβ₂ on the surface of recombinant HEK293 cells and murine IC-21 macrophages cooperates with β₁/β₃ integrins to support cell migration. However, its increased expression on the α_Dβ₂-expressing HEK293 cells and its upregulation by PMA on the IC-21 macrophages result in increased cell adhesiveness and inhibition of cell migration. Furthermore, ligation of α_Dβ₂ with anti-α_D blocking antibodies restores β₁/β₃-driven cell migration by removing the excess α_Dβ₂-mediated adhesive bonds. Consistent with in vitro data, increased numbers of inflammatory macrophages were recovered from the inflamed peritoneum of mice after the administration of anti-α_D antibody. These results demonstrate that the density of α_Dβ₂ is critically involved in modulating macrophage adhesiveness and their migration, and suggest that low levels of α_Dβ₂ contribute to monocyte migration while α_Dβ₂ upregulation on differentiated macrophages may facilitate their retention at sites of inflammation.     

Effect of integrin beta 2 subunit truncations on LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) assembly, surface expression, and function

LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are members of the beta2 integrins involved in leukocyte function during immune and inflammatory responses. We aimed to determine a minimized beta2 subunit that forms functional LFA-1 and Mac-1. Using a series of truncated beta2 variants, we showed that the subregion Q23-D300 of the beta2 subunit is sufficient to combine with the alphaL and alphaM subunits intracellularly. However, only the beta2 variants terminating after Q444 promote cell surface expression of LFA-1 and Mac-1. Thus, the major cysteine-rich region and the three highly conserved cysteine residues at positions 445, 447, and 449 of the beta2 subunit are not required for LFA-1 and Mac-1 surface expression. The surface-expressed LFA-1 variants are constitutively active with respect to ICAM-1 adhesion and these variants express the activation reporter epitope of the mAb 24. In contrast, surface-expressed Mac-1, both the wild type and variants, require 0. 5 mM MnCl2 for adhesion to denatured BSA. These results suggest that the role of the beta2 subunit in LFA-1- and Mac-1-mediated adhesion may be different.     

Human Thy-1 (CD90) on activated endothelial cells is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18)

Leukocyte recruitment in response to inflammatory signals is in part governed by interactions between endothelial cell receptors belonging to the Ig superfamily and leukocyte integrins. In our previous work, the human Ig superfamily glycoprotein Thy-1 (CD90) was identified as an activation-associated cell adhesion molecule on human dermal microvascular endothelial cells. Furthermore, the interaction of Thy-1 with a corresponding ligand on monocytes and polymorphonuclear cells was shown to be involved in the adhesion of these leukocytes to activated Thy-1-expressing endothelial cells. In this study, we have identified the specific interaction between human Thy-1 and the leukocyte integrin Mac-1 (CD11b/CD18; alphaMbeta2) both in cellular systems and in purified form. Monocytes and polymorphonuclear cells were shown to adhere to transfectants expressing human Thy-1 as well as to primary Thy-1-expressing human dermal microvascular endothelial cells. Furthermore, leukocyte adhesion to activated endothelium as well as the subsequent transendothelial migration was mediated by the interaction between Thy-1 and Mac-1. This additional pathway in leukocyte-endothelium interaction may play an important role in the regulation of leukocyte recruitment to sites of inflammation.     

Regulation of myeloperoxidase gene expression during differentiation of human myeloid leukemia HL-60 cells

When human myeloid leukemia HL-60 cells were induced to differentiate into mature cells by dimethyl sulfoxide or retinoic acid, the amount of myeloperoxidase activity per cell decreased to 20 to 30% of that of uninduced cells, and the rate of myeloperoxidase biosynthesis decreased to an undetectable level in 19 h after induction of differentiation. After 19-h exposure to an inducer, the cells could not resume myeloperoxidase synthesis on further incubation in inducer-free medium. When polysomes and mRNAs prepared from untreated and treated cells were translated in rabbit reticulocyte lysates, the former showed myeloperoxidase polypeptide synthesis, and the latter did not. These results indicate that the inability of induced cells to synthesize myeloperoxidase is due to the absence of myeloperoxidase mRNA.     

CD40 signaling activates CD11a/CD18 (LFA-1)-mediated adhesion in B cells.

Cell-cell adhesion events play critical roles in the sequential migrations and multiple specific cell-cell interactions which B cells undergo during normal development and function. We have observed that mAb to several B cell-associated molecules, including mAb to CD19, CD37, and CD40, induce homotypic aggregation of freshly isolated human B cells. The aggregation of B cells induced by CD40 mAb was due to activation of a cell-cell adhesion system, and not due to agglutination by mAb, because 1) in addition to being energy dependent and cation dependent, the aggregation was blocked by inhibitors of messenger RNA and protein synthesis; and 2) a mouse B cell line transformed with intact human CD40 aggregated in response to CD40 mAb, whereas a line expressing surface CD40, but lacking the cytoplasmic tail and previously shown incapable of transmitting a signal from the cell surface, did not aggregate. The aggregation, although of slow onset, was persistent and of high avidity. In addition, CD40 mAb induced increased surface expression of intercellular adhesion molecule-1 (CD54), a ligand for CD11a/CD18 (LFA-1), and CD18 mAb blocked aggregation. CD40 mAb also augmented the ability of dense B cells to stimulate the proliferation of allogeneic T cells via a CD18-dependent process. We conclude that signaling through CD40, elicited by cross-linking the CD40 protein on the cell surface, activates the CD18/intercellular adhesion molecule adhesion system; in addition, CD40 cross-linking may activate a second adhesion system since CD40 mAb induced aggregation of the B cell line Ramos, which does not express surface CD18. B cell adhesion may be triggered by signaling through multiple surface proteins, thereby lending specificity of activation to adhesion systems which are broadly expressed.     

Heparin is an adhesive ligand for the leukocyte integrin Mac-1 (CD11b/CD1)

Previous studies have demonstrated that the leukocyte integrin Mac-1 adheres to several cell surface and soluble ligands including intercellular adhesion molecule-1, fibrinogen, iC3b, and factor X. However, experiments with Mac-1-expressing transfectants, purified Mac- 1, and mAbs to Mac-1 indicate the existence of additional ligands. In this paper, we demonstrate a direct interaction between Mac-1 and heparan sulfate glycans. Heparin affinity resins immunoprecipitate Mac- 1, and neutrophils and transfectant cells that express Mac-1 bind to heparin and heparan sulfate, but not to other sulfated glycosaminoglycans. Inhibition studies with mAbs and chemically modified forms of heparin suggest the I domain as a recognition site on Mac-1 for heparin, and suggest that either N- or O-sulfation is sufficient for heparin to bind efficiently to Mac-1. Under conditions of continuous flow in which heparins and E-selectin are cosubstrates, neutrophils tether to E-selectin and form firm adhesions through a Mac- 1-heparin interaction.     

Expression of leukocyte adherence-related glycoproteins during differentiation of HL-60 promyelocytic leukemia cells

We used the HL-60 human promyelocytic leukemia cell line to analyze the surface expression of a family of adherence-related leukocyte surface antigens during myeloid differentiation. These antigens are composed of discrete alpha subunits, designated alpha L, alpha M, and alpha X, that are each noncovalently associated with a common beta subunit. Monoclonal antibodies directed against the individual subunits served as markers in both indirect immunofluorescence studies and immunoprecipitations from HL-60 cells differentiated preferentially towards mature granulocytes (DMSO, retinoic acid) or monocyte/macrophages (PMA, vitamin D3). In undifferentiated HL-60 cells, the alpha L and alpha X subunits were constitutively expressed, whereas the alpha M subunit was not. Differentiation of HL-60 cells along the granulocytic pathway with DMSO resulted in a marked increase in alpha M and minimal increases in alpha L and alpha X. The phenotypic expression of these antigens on DMSO-treated HL-60 cells closely resembled that on normal circulating PMN. Differentiation along the monocyte/macrophage pathway when using PMA or vitamin D3 resulted in major increases in alpha L and alpha X expression, as well as alpha M. These changes resulted in a surface phenotype characteristic of that present on human monocyte-derived macrophages. Triggering of undifferentiated HL-60 cells with PMA caused no increase in subunit expression, whereas stimulation of DMSO-differentiated HL-60 cells with PMA produced more than a 1.5-fold enhancement of both the alpha M and alpha X subunits, and stimulation of human PMN with PMA increased the surface expression of alpha M more than fourfold and alpha X subunit twofold. Stimulation with PMA produced no change in expression of the alpha L subunit in any of the three cell populations. These results indicate that the alpha subunits of this glycoprotein family can be selectively regulated during in vitro differentiation of a human promyelocytic leukemia cell line. Second, DMSO-differentiated HL-60 cells and human PMN possessed an intracellular pool of alpha M and alpha X, but not alpha L, that could be translocated to the surface. Thus, despite structural and functional relationships among the alpha subunits in this glycoprotein family, they undergo disparate surface expression and intracellular regulation during differentiation.     

Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.     

Human endothelial-cell specific molecule-1 binds directly to the integrin CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1.

ICAMs are ligands for LFA-1, a major integrin of mononuclear cells involved in the immune and inflammatory processes. We previously showed that endothelial cell specific molecule-1 (ESM-1) is a proteoglycan secreted by endothelial cells under the control of inflammatory cytokines. Here, we demonstrate that ESM-1 binds directly to LFA-1 onto the cell surface of human blood lymphocytes, monocytes, and Jurkat cells. The binding of ESM-1 was equally dependent on Ca(2+), Mg(2+), or Mn(2+) divalent ions, which are specific, saturable, and sensitive to temperature. An anti-CD11a mAb or PMA induced a transient increase in binding, peaking 5 min after activation. Direct binding of ESM-1 to LFA-1 integrin was demonstrated by specific coimmunoprecipitation by CD11a and CD18 mAbs. A cell-free system using a Biacore biosensor confirmed that ESM-1 and LFA-1 dynamically interacted in real time with high affinity (K(d) = 18.7 nM). ESM-1 consistently inhibited the specific binding of soluble ICAM-1 to Jurkat cells in a dose-dependent manner. These results suggest that ESM-1 and ICAM-1 interact with LFA-1 on binding sites very close to but distinct from the I domain of CD11a. Through this mechanism, ESM-1 could be implicated in the regulation of the LFA-1/ICAM-1 pathway and may therefore influence both the recruitment of circulating lymphocytes to inflammatory sites and LFA-1-dependent leukocyte adhesion and activation.     

Pertussis toxin treatment in vivo reduces surface expression of the adhesion integrin leukocyte function antigen-1 (LFA-1)

Pertussis toxin treatment in macaques inhibits lymphocyte extravasation from the blood and leads to transient lymphocytosis and leukocytosis. We examined lymphocyte adhesion molecules known to be involved in the extravasation process to find possible mechanisms for the effects of pertussis toxin treatment. The two subunits of LFA-1, CD11a and CD18, showed decreased surface expression on lymphocytes from pertussis toxin treated animals compared to untreated animals. The adhesion molecule CD44, and the alpha subunit of the integrin VLA-4 (CD49d) were not decreased by pertussis toxin treatment. Lower surface expression of CD11a and CD18 was observed on all lymphocyte subsets and was correlated inversely with the extent of lymphocytosis. The magnitude of lymphocytosis after pertussis toxin treatment was higher in SIV-infected macaques than in uninfected animals. However, changes in LFA-1 levels were similar in both groups. These data show that LFA-1 surface levels are affected by pertussis toxin in vivo and this change may account in part, for the ability of pertussis toxin to induce lymphocytosis.     

Altered expression profiles of microRNAs during TPA-induced differentiation of HL-60 cells

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that regulate gene expression through translational repression by base-pairing with partially complementary mRNAs. The expression of a set of miRNAs is known to be regulated developmentally and spatially, and is involved in differentiation or cell proliferation in several organisms. However, the expression profiles of human miRNAs during cell differentiation remain largely unknown. In an effort to expand our knowledge of human miRNAs, we investigated miRNAs during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of human leukemia cells (HL-60) into monocyte/macrophage-like cells. Several hundred RNAs ranging from 18 to 26 nucleotides were isolated from HL-60 cells with or without TPA-induction, and subsequently characterized by sequencing, database searching, and expression profiling. By removing non-miRNA sequences, we found three novel and 38 known miRNAs expressed in HL-60 cells. These miRNAs could be further classified into subsets of miRNAs that responded differently following TPA induction, either being up-regulated or down-regulated, suggesting the importance of regulated gene expression via miRNAs in the differentiation of HL-60 cells.     

Pertussis toxin treatment In vivo reduces surface expression of the adhesion integrin leukocyte function antigen-1 (LFA-1)

Pertussis toxin treatment in macaques inhibits lymphocyte extravasation from the blood and leads to transient lymphocytosis and leukocytosis. We examined lymphocyte adhesion molecules known to be involved in the extravasation process to find possible mechanisms for the effects of pertussis toxin treatment. The two subunits of LEA-1, CD11a and CD18, showed decreased surface expression on lymphocytes from pertussis toxin treated animals compared to untreated animals. The adhesion molecule CD44, and the alpha subunit of the integrin VLA-4 (CD49d) were not decreased by pertussis toxin treatment. Lower surface expression of CD 11 a and CD 18 was observed on all lymphocyte subsets and was correlated inversely with the extent of lymphocytosis. The magnitude of lymphocytosis after pertussis toxin treatment was higher in SIV-infected macaques than in uninfected animals. However, changes in LFA-1 levels were similar in both groups. These data show that LEA-1 surface levels are affected by pertussis toxin in vivo and this change may account in part, for the ability of pertussis toxin to induce lymphocytosis.     

Heat shock protein is a unique marker of growth arrest during macrophage differentiation of HL-60 cells

Prior to morphologic and functional maturation, terminally differentiating hematopoietic cells first exit the cell cycle and undergo growth arrest. Relatively little is known about which molecules regulate differentiation-induced growth arrest. In the present report, we sought to determine whether the mammalian low molecular weight heat shock protein (hsp28) was a candidate growth-regulatory molecule during human hematopoiesis. To this end, hsp28 protein expression was examined during phorbol ester (PMA)-induced macrophage differentiation of the human HL-60 promyelocytic leukemic cell line. Whereas hsp28 was constitutively expressed at relatively low levels in an unphosphorylated state, hsp28 was rapidly phosphorylated within 4 hr following PMA-induced differentiation, preceding increased hsp28 protein levels at 24–48 h. In contrast to other differentiative agents, hsp28 steady state mRNA and protein were regulated concordantly in response to macrophage differentiation. More importantly, these changes were transient, and occurred concomitant with the down-regulation of cellular proliferation and the onset of G₁ phase cell cycle arrest. In total, these observations implicate hsp28 as an intermediary in the myelomonocytic differentiative pathway of promyelocytic leukemic cells, and will shed light on the events regulating this process. © 1993 Wiley-Liss, Inc.