Biochimica et Biophysica Acta, 1092 (1991) 165-168 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100137Z

165

BBAMCR 12912

Induction of LFA-l-mediated homotypic adhesions in promonocytic U-937 cells occurs independently

of cell differentiation

Carlos Cabahas 1, Pedro Lastres 1, Teresa Bell6n 1, Patricio Aller 1, Carl G. Figdor 2,

Angel Corbi 3 and Carmelo Bernabeu 1 s Centro de lnoestigaciones Biol6gicas, C.S.I.C., Madrid (Spain). 2 The Netherlands Cancer Institute, Amsterdam (The Netherlands)

and ~ Seroicio de lnmunologla, Hospital de La Princesa-UAM, Madrid (Spain)

(Received 11 September 1990)

Key words: LFA-I; Homotypic adhesion; Monocyte differentiation

The differentiation of monocytes into macrophages occurs along with a marked increase in LFA-l-dependent intercellular adhesions. Similarly, the phorboi ester-induced differentiation of U-937 promonocytic cells into macro- phage-like cells is morphologically characterized by an important increase in LFA-I/ICAM-l-dependent intercellular homotypic adhesions. Since an important functional role in activation of human T ceils has been demonstrated for LFA-I-dependent adherence, we have analyzed whether the induction of LFA-l-dependent intercellular adhesion of human monocytic cells is necessarily accompanied by differentiation of these cells. We found that treatment of the promonocytic U-937 cells with the anti-LFA-I mAb NKI-LI6 induces formation of intercellular clusters, but does not induce cell differentiation as determined by several differentiation markers. These markers include the arrest of cell proliferation, production of reacti~ oxygen species, changes in the cell surface expression of differentiation-associated antigens such as the transferrin receptor, CDI |h and CDIIc and chango~ in the levels of several specific gene transcripts such as CDI8 antigen, c-myc, ornithine decarboxylase and vimentin. Th, ~e findings suggest that LFA-I-de- pendent adhesion and differentiation of monocytic cells are independent processes.

Introduction

In mononuclear phagocytes, the regulation of cell-cell adhesion is an important aspect of their function and development. Thus, monocytes initiate diapedesis by adherence to the endothelium, extravasate and maturate into tissue macrophages. In addition, upon antigen chal- lenge macrophages infiltrate into the site of inflamma- tion and display homotypic (macrophage-macrophage) and heterotypic (macrophage-T cell) interactions. These cellular associations can lead to granuloma formation in certain diseases (lepromatous leprosy, tuberculosis, sarcoidosis, etc.) [1]. When monocytes are exposed in vitro to immunologic or inflammatory stimuli such as interferon gamma, LPS or TNF, an induction of homo-

Abbreviations: DCFH-DA, 2',7'-dichlnrofluorescein diacetate; OrnDC, ornithine decarboxylase.

Correspondence: Carmelo Bernabeu, Centro de lnvestigaciones, Binlbgicas, CSIC Velazquez 144, 28006 Madrid, Spain.

typic adhesions occurs concomitantly with activation and differentiation of these cells [2-4]. Similarly, phor- bol esters induce cellular differentiation as well as an increase in intercellular adhesions, as evidenced by cell cluster formation, in the human promonocytic U-937 cells and in the myelomonocytic cell line HL-60 [5-10]. This adhesion of monocytic cells is mediated by the LFA-1 and ICAM-1 molecules [10,11]. Since binding of ligand to LFA-1 is required for T cell function [12] and the existence of regulatory signals conveyed by LFA-1 has been suggested for T lymphocytes [13,14], we have analyzed in this report the possible occurrence of cell differentiation in the human promonocytic U-937 cells when these cells are induced to agregate via LFA- 1/ICAM-1 by a mAb specific for LFA-1.

Materials and Methods

Antibodies The mAb NKI-L16 (anti-LFA-1 a or CDl l a ) in-

duces LFA-1/ICAM-1 mediated homotypic interac-

166

tions [15]. The mAb FG1/6 (anti-transferrin receptor or CD71) [16], Bearl (anti-CDllb) [17] and HC1/1 (anti- CDllc) [18] were generated in our laboratories. Anti- bodies were of the lgG1 (FG1/6, HC1/1 and Bearl) and IgG2a (NKI-L16) subclasses. Antibody NKI-L16 was added to cell cultures as 1:I000 dilution of ascites fluid.

Cells The promonocytic U-937 and the myelomonocytic

HL-60 cells were cultured in RPMI 1640 medium supp- lemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, penicillin (100 U/ml) and streptomycin (100 ng/ml) (Flow Laboratories, Rockwell, MD) in a 5% CO,. atmosphere at 37°C. When necessary, cells were incubated with either NKI-L16 mAb (1:1000 dilu- tion of ascites fluid) or 10 ng/mi of PMA for 24-48 h to induce intercellular cluster formation or cell differ- entiation.

Markers of monocytic differentiation Cellular proliferation was measured as previously

described [19]. Flow cytometry analysis was performed with an EPICS-CS flow cytometer (Coulter Cientifica, Mostoles, Spain) using logarithmic amplifiers. For anti- gen expression analysis, cells were incubated with the corresponding mAb for 30 min at 4°C. After two washes with NaCI/P i (140 mM NaCI, 3 mM KCI, 8 mM N v, ttPO4 and 8 mM KH2PO 4, pH 7.4), FITC-labeled rablf, anti-mouse IgG1 (Nordic Immunology, Tilburg, Denmark) were added and incubation followed by an additional period of 30 rain at 4°C. Finally, cells were washed twice with NaCI/P i and their fluorescence estimated.

Determination of generation of hydrogen peroxide was carried out as reported by Bass et al. [20]. Briefly, U-937 calls were loaded with the nonfluorescent com- pound 2',7'-dichlorofluorescein diacetate (DCFH-DA), which is deacetylated by cellular esterases into DCFH. Then, the oxidation of DCFH by the stimulus-induced generation of hydrogen peroxide results in the fluo-

rescent compound DCF which is detected by flow cy- tometry.

RNA blot analyses were performed basically as de- scribed [19]. The probes used were: the 1.5 kb Clal- EcoR! fragment of pMC413rC plasmid [21], which con- tams the 3 rd e x o n of human c-myc; the 1.1 kb human vimentin-specific XhoI fragment of L3ATA plasmid [22]; the 1.5 kb OrnDC-specific Xhol fragment of OD- 821 plasmid (a generous gift from Dr. L. Kaczmareck, Varsow, Poland); and the 1 kb EcoRl fragment from the eDNA of the human CD18 antigen [231.

Results

The addition of the anti-LFA-1 mAb NKI-L16 to U-937 cultures results in an important induction of intercellular homotypic adhesions evidenced by forma- tion of large cell clusters which remain in suspension in the culture medium (Fig. 1). These aggregates are in- duced as soon as 2 h after the addition of the mAb and the cells remain clustered for up to 3 days. A similar effect on cluster formation is induced by treatment of U-937 cells with the phorbol ester PMA. However, this PMA-induced aggregation of U-937 cells is somehow different from that induced by NKI-L16, since it is not observable before 15-20 h of treatment with PMA and is accompanied by an important increase in cellular adhesiveness to the plastic substrate. Both, NKI-L16 and PMA induced cell aggregates are LFA-1/ICAM-1- mediated, since mAb to either of these ligands are able to inhibit cluster formation.

To address the question of whether this LFA-l-medi- ated aggregation of U-937 cells induced by the mAb NKI-L16 is also accompanied by induction of cell dif- ferentiation, as is the case for PMA, we have analyzed the changes in several myeloid differentiation markers. Proliferation of U-937 cells completely ceases upon induction of differentiation into macrophage-like cells with PMA (Table I). On the contrary, the addition of the mAb NKI-L16 had no effect on U-937 prolifera- tion.

C o n t r o l + N K I - L 16 + P M A

Fig. 1. Photomicrographs of LFA-I-mediated homotypic adhesions induced by the mAb NKI-LI6. U937 cells were incubated in the absence (control) or in the presence of either the mAb NKI-L]6 (anti-LFA-]) or PMA as described in Materials and Methods,

TABLE 1

Effect of homotypic adhesions induced by the NKI-LI6 mAb on the proliferation of U937 cells

U937 cells were cultured in the absence or in the presence of either PMA or NKI-LI6 mAb. The [3H]-thymidine uptake was measured as described in Materials and Methods. The mAb 8F3 (anti-CDl3) was included as a control.

Treatment [ 3HlThymidine uptake (cpm)

None 72682 PMA 1102 NKI-LI6 64453 8F3 65 571

Fig. 2 shows the changes in the cell surface expres- sion of CD11b, CD11c and CD71 antigens during the PMA-induced differentiation of U-937 cells. An im- portant increase in CD11b and CDl l c expression and the loss of CD71 characterize the differentiation of U-937 into macrophage-like cells [18]. However, the induction of intercellular homotypic adhesion induced by NKI-L16 is not accompanied by any of the afore- mentioned antigenic changes.

Also, the differentiation of U-937 cells induced with PMA is characterized by an important decrease in the levels of c-myc and OrnDC as well as the marked increase of the levels of CD18 and vimentin transcripts [19,24]. In contrast, the treatment of U-937 cells with NKI-L16 has no effect on the levels of any of these gene transcripts (Fig. 3).

Finally, the differentiation of U-937 into macro- phage-like cells is functionally characterized by the acquisition of capacity to generate reactive oxygen de- rivatives. Fig. 4 shows that PMA-differentiated U-937 cells but not NKI-L16-treated cells are able to generate

CDllb

CDIIc

CDTI

Control i i 5 t

t 4 i

9 5

A

+PMA I e0 I

I

33

IZ

+NKI-LI6

I

I 9 2

I I

.Q

E

Z

t j

Fluorescence Intensity

Fig. 2. Analysis of the expression of CDI Ib , C D t l c and CD71 antigens on U937 cells treated with the mAb NKI-L16. Cells treated in the absence or in the presence of either PMA or NKI -L16 mAb were analyzed by f low cytometry using H C ] / ] (ant i -CDl lc ) , Bear 1 (an t i -CDl lb ) or FG1 /6 (anti-CD71) mAb. Numbers at the upper

right corner indicate the percentage of positive cells.

28S - -

w

- - -J -.I O

O Z CL O Z Q. tO ÷ ÷ O + +

C- myc Orn DC

1 6 7

1 8 S - -

2 8 S - -

0'0 Oo ;:17

Vimentin CDt8

1 8 S - -

W

Fig. 3, Effect of NKI-L16 induced homotypic adhesions on the levels of c-myc, OrnDC, vimentin and CD18 transcripts. Total cytoplasmic RNA was extracted from untreated cells (0 h) or cells treated with either NKI-L16 mAb (24 h) or PMA (24 h). RNA blots (15 ~tg per lane) were prepared and hybridized with c-myc, ornithine decarboxy-

lase (OrnDC), CD18 and vimentin probes.

hydrogen peroxide upon stimulation with high doses of PMA.

Taken together, these results suggest that LFA-I-deo pendent homotypic adhesions are not sufficient to in- duce monocytic differentiation of U937 cells.

D i s c u s s i o n

Interactions of monocytes with endothelial cells and with a variety of cell types and matrices occur concom- itanly with their differentiation into tissue macrophages. The importance in this process of the CDl l /CD18-de- pendent interactions is evidenced in CD18 immunodefi- cient patients by the paucity of phagocytes at ti,,; site of inflammation [25]. Using the human promonocytic cell line U937 as an experimental model of differentiation, we show that extensive LFA-1/ICAM-l-dependent in- teractions can occur in the absence of monocytic differ- entiation. This conclusion is supported by the findings that (a) PMA is able to differentiate U937 cells into macrophage-like cells in the absence of LFA-1/ICAM- 1-dependent cellular interactions [19]; and (b) in CD18 deficient patients, LFA-1 adhesion independent func- tions of phagocytes are all normal, including shape changes, binding of chemotactic compounds (FMLP) and oxygen radical generation to soluble stimuli [26].

Our results using monocytic cells do not support the existence of LFA-l-mediated transductive signals as

168

$ 4

e o

Fluorescence intensity

Fig. 4. Effect of NKI-LI6 induced homotypic adhesions on the generation of reactive oxygen species. U-937 cells were incubated in the absence or in the presence of either PMA or the mAb NKI-L16 for 48 h. Then, cells were loaded with DCFH-DA followed by stimulation with PM~ and the fluorescence of the DCF generated was estimated by flow cytometry using logarithmic amplifiers. Cells treated with hydrogen peroxide (500/tM) were included as a positive control, Numbers at the upper right corner indicate the mean fluores-

cence intensity.

reported for T cells [13,14]. On the contrary, we think that the NKI-L16 mAb affects the conformation of LFA-1, thereby increasing the affinity for ICAM-1 without the need for signal transduction [27]. On the other hand, we can not exclude the requirement for other types of cell-cell and cell-matrix interactions during the monocytic differentiation process. In this sense, the monocytic antigen VLA-4 is able to interact with the endothelial molecule VCAM-1 [28]. Further- more, the endothelial antigens CD62 and ELAM-1 and the homing receptor MEL-14 present in monocytes, are able to interact with the corresponding counterstruc- tures (so far unknown) to allow monocyte-endothelial cell adhesions [29]. The possible implication of these molecules in the differentiation process needs to be elucidated.

Aelmowledgements

We are thankful for the helpful discussiens of Dr. S~nchez-Maddd. This work was supported by grants from Direccibn General de lnvestigacibn Cientifica y T6cnica PB87-0286 and PB87-0351.

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