Eur. J . Biochem. 191, 599-604 (1990) (0 FEBS 1990

Phorbol esters induce differentiation of U-937 human promonocytic cells in the absence of LFA-1/1CAM-1-mediated intercellular adhesion Carlos CABAfiAS Francisco SANCHEZ-MADRID’, Patricio ALLER’, Enriqueta YAGUE’ and Carmelo BERNABEU’

Centro de lnvestigaciones Biologicas, CSIC, Madrid, Spain ’ Servicio de Inmunologia, Hospital de la Princesa, Madrid, Spain

(Received October 25, 1989/April 9, 1990) - EJB 89 1285

Intercellular adhesions which occur during the mononuclear phagocyte differentiation are predominantly mediated by the lymphocyte-function-associated antigen- 1 (LFA- 1) family and the intercellular-adhesion molecule- 1 (ICAM-1) which is a ligand for LFA-1. Thus, differentiation of U-937 promonocytic cells induced by phorbol esters occurs concomitantly with intercellular LFA-l/ICAM-1 -dependent cluster formation. Since these homotypic adhesions can be inhibited by monoclonal antibodies (mAb) directed to either LFA-1 or ICAM-1, we have analyzed whether the lack of cell - cell adhesions impairs the differentiation process. Treatment of U-937 cells with the phorbol ester 12-0-tetradecanoylphorbol 13-acetate in the presence of mAb to LFA-1 or ICAM-1 antigens yielded cells free from homotypic adhesions but differentiated as evidenced by their decreased proliferation and enhanced capacity for generation of superoxide anion. In addition, expression of the CDl lc antigen was increased, whereas the transferrin receptor disappeared from the cell surface. Vimentin gene transcription was also greatly augmented as opposed to a clear diminution in the levels of c-myc and ornithine decarboxylase transcripts. These results clearly demonstrate that phorbol esters can induce differentiation of monocytic cells independently of cell - cell adhesion.

Intercellular adhesions play an important role in many immunological functions. Many of the adhesion phenoma of leucocytes are mediated by the lymphocyte-function-associ- ated antigen 1 (LFA-1) family and the intercellular-adhesion molecule 1 (ICAM-1, CD54) molecule, which is a ligand for LFA-1 [I - 61. These molecules are involved in antigen presen- tation [2, 7, 81, T cell effector functions [9- 121, cooperation between B and T lymphocytes [I31 and chemotaxis of myeloid cells [I, 14, 151. The LFA-1 family of antigens is composed of three structurally related glycoproteins, LFA-1 (CD1 la), Mol (CDllb) and p150,95 (CDllc) [16]. These three antigens are noncovalently linked a : j heterodimers which have distinct a subunits of 170, 165 and 150 kDa, respectively, and share a common j subunit of 95 kDa (CD18) [16,17].

In mononuclear phagocytes, the regulation of cell - cell adhesion is an important aspect of their function and develop- ment. Thus, monocytes initiate diapedesis by adherence to the endothelium. This adhesion to endothelial cells allows the monocytes to exit from blood vessels by passing between adjacent endothelial cells. At the same time that these adhe- sion processes take place, monocytes maturate into tissue macrophages. Also, upon antigen challenge, there is a massive infiltration of macrophages showing homotypic (macro- phage - macrophage) and heterotypic (macrophage - T-cell) interactions. These cellular associations can lead to granuloma

Correspondence to C. Bernabeu, Centro de Investigaciones Bio- logicas, CSIC, Velazquez 144, E-28006 Madrid, Spain

Abbreviutions. mAb, monoclonal antibody(ies); PMA, 12-0- tetradecanoyl-phorbol 13-acetate; FITC, fluorescein isothiocyanate; LFA-1, lymphocyte-function-associated antigen-1 ; ICAM-1, inter- cellular-adhesion molecule-1 ; OrnDC, ornithine decarboxylase.

formation in certain pathological states (lepromatous leprosy, tuberculosis, sarcoidosis, etc.) [18]. When monocytes are ex- posed to immunologic or inflammatory stimuli such as y- interferon, an induction of homotypic adhesions occurs con- comitantly with activation and differentiation of these cells [19 - 211. Similarly, phorbol esters induce cellular differen- tiation as well as an increase in intercellular adhesions, as evidenced by cell cluster formation, in the human promono- cytic U-937 cells and in the myelomonocytic cell line HL-60 [22 - 271. This adhesion of monocytic cells is mediated by the LFA-1 and ICAM-1 molecules, since monoclonal antibodies to these molecules are capable of inhibiting the cell cluster formation [6, 271.

Several markers for monocytic differentiation of myeloid cell lines have been used, such as an arrest in their proliferative capacity [23 - 25, 281, variations in the levels of specific gene transcripts [26, 29 - 331, changes in the expression of specific myeloid surface antigens [23,24,26,34 - 361 and development of the superoxide-generating system [23, 26, 371. Thus, at the antigenic level, expression of the CDI Ic antigen is highly increased in U-937 and HL-60 cells upon differentiation to mature macrophage-like cells induced by 12-0-tetradecanoyl- phorbol 13-acetate (PMA) [34, 351, whereas the expression of the transferrin receptor molecule (CD71) is down-regulated [26,35]. In addition, a decrease in the expression of ornithine- decarboxylase (OrnDC) and c-myc genes as well as an induc- tion of transcription of the vimentin gene take place during the PMA-induced differentiation of U-937 and HL-60 cells [29 - 331. Using these differentiation markers, we have investi- gated in the present report the possible requirement of LFA- l/ICAM-I-dependent intercellular homotypic adhesions for the differentiation of U-937 cells induced by PMA.

600

MATERIALS AND METHODS

Induction of intercellular homotypic adhesions and d(fferentiation in U-937 cells

The promonocytic U-937 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, penicillin (100 Ujml) and strep- tomycin (100 ng/ml; Flow Laboratories, Rockwell, MD) in a 5% C 0 2 atmosphere at 37'C. When necessary, cells were treated with 10 ng PMA/ml for 48 h to induce cellular cluster formation and differentiation.

An t ih odies

The mAb D3/9 (anti-CD45) [38], FG1/6 (anti-transferrin receptor or CD71) [39] and HCl / l (anti-p150,95 or CDllc) 1351 were generated in our laboratory. The mAb TSIjl1 (anti- LFA-1 or CDl la) and TS1/18 (anti-LFA-I or CD18) have been previously described 1161 and the RRl / l mAb (anti- ICAM-1 or CD54) (61 was kindly provided by Dr T. A. Springer (Dana Farber Cancer Institute, Boston, MA).

Antibodies RR1/1, TS1/11, TS1/18 and D3/9 were added to cell cultures either as 1 :200 dilution of ascites fluid or hybridonia culture supernatant previously dialyzed against RPMI 1640 medium.

Inimunqfluorescence and f low cytometry

Flow cytometry analysis was performed with an EPICS- CS flow cytometer (Coulter Cientifica, Mostoles, Spain) using logarithmic amplifiers. For indirect immunofluorescence, cells were incubated with the corresponding mAb for 30 min at 4 T . After two washes with NaCI/P, (140 mM NaC1, 3 mM KCI, 8 mM Na2HP04 and 8 mM KH2P04, pH 7.4), rabbit anti-mouse IgG labeled with fluorescein isothiocyanate (FITC; Dakopatts, Denmark) was added and incubation fol- lowed for an additional period of 30 min at 4°C. Finally, cells were washed twice with NaCI/P, and their fluorescence estimated. Direct immunofluorescence was carried out by in- cubating the cells for 30 min with the corresponding FITC- labeled mAb [40]. After two washes with NaCI/Pi, their fluo- rescence was analyzed.

Measurenient of superoxide anion generation

U-937 cells were differentiated for two days with 10 ng/ml of PMA in the presence of the corresponding mAb, washed twice with a balanced salt solution (10 mM Hepes, 150 inM NaCl, 1.2 mM MgCI2, 1.3 mM CaCI,, 5.5 mM glucose, pH 7.5) and resuspended in this buffered solution. lo6 PMA- differentiated U-937 cells were incubated with 0.90 mg of cytochrome c, 5.0 pg cytochalasin B either in the presence or absence of 0.4 pg superoxide dismutase in a total reaction volume of 1.0 ml for 10 min at 37°C. Subsequently, 100 ng PMA was added and the reaction proceeded for an additional period of 15 min at 37°C. At the end of this period, samples were centrifuged to remove cells and the supernatants were collected. The extent of cytochrome c reduction was measured in a Baush-Lomb (Spectronic 710 model) spectrophotometer at 550 nm. The difference in absorbance between samples treated and untreated with superoxide dismutase was a mea- sure of the amount of reduced cytochrome c.

Measurement of prolijeration

After culture of U-937 cells in the presence of PMA and the corresponding mAb for 48 h, cells were washed, resus- pended at 2.5 x l o5 cells/ml, distributed in 96-well flat-bottom microtiter plates (Costar, Cambridge, MA) and pulsed with 1 pCi/well [3H]thymidine for 12 h. After this time, cells were harvested using a Skatron semiautomatic cell harvester Skatron, Norway) and the radioactivity was determined.

RNA blot analyses

Total cytoplasmic RNA was prepared as described in a previous work 1411. RNA samples (15 pg) were denatured, then fractionated in 1.1 %O (massivol.) agarose/formaldehyde gels 1421 and blotted to nylon membrane. RNA blots were hybridized with excess 32P-labelled probes and washed under highly stringent conditions, as previously described [43] and finally autoradiographed. The probes used were: the 1.5-kb C1aI - EcoRI fragment of pMC413rC plasmid 1441, which con- tains the third exon of human c-myc; the 1.1-kb human vimentin-specific XhoI fragment of L3A7A plasmid 1321 ; the 1.5-kb OrnDC-specific XhoI fragment of OD-821 plasmid (a generous gift of Dr L. Kaczmareck, Varsow, Poland) and the 5.1-kb BamHI fragment of pcD-TR1 plasmid [45], which contains the full-length cDNA of human transferrin receptor. The fragments were isolated by elution after binding to NA45- DEAE membrane (Schleicher & Schiill, FRG) in agarose gels and labelled to 1 .O - 1.5 x lo9 cpm/pg DNA with [cx-~'P]~CTP (3000 Ci/mmol, Amersham, UK) by random hexanucleotide priming [46].

RESULTS

Ejject of anti-LFA-1 or anti-ICAM-1 mAb on the honiot-vpic adhesions and proliferation of PMA-differentiated U-937 cells

Two major changes that the promonocytic U-937 cells undergo during phorbol-ester-induced differentiation to mac- rophage-like cells are an important increase in their adhesive properties, evidenced as cell cluster formation and adhesion to plastic surface, and a marked decrease in proliferation. The PMA-induced intercellular adhesions of U-937 cells are completely inhibited by monoclonal antibodies directed to the cx (CDlla) or (CD18) chains of the LFA-1 heterodimer and also by mAb specific for the ICAM-1 molecule (CD54) [6], whereas mAb directed to other highly expressed antigens, such as CD45, had no effect on the adhesive properties of these cells (Fig. 1).

Also, the mAb to the CDl la , CDI 8 or CD54 antigens did not restore the loss of the proliferative capacity shown by U-937 cells after treatment with PMA, which indicates that differentiation to terminal macrophage-like cells effectively occurs despite the complete inhibition of cell cluster formation (Table 1).

Antigenic changes during PMA-induced differentiation o j U-937 cells in the presence of anti-LFA-1 or anti-ICAM-I mAb

Important changes in the expression of several relatively restricted myeloid antigens, such as the CDllc , have been described during the PMA-induced differentiation of U-937 and HL-60 cells [34, 351. Also, changes in the expression of

60 1

Fig. 1. Effect qfthe addition of unti-CDllu, anti-CD18 or anti-CD54 mAh on the PMA-induced homotypic adhesions of U-937 cells. Photomicro- graphs of untreated U-937 cells (A), PMA-treated (I0 ng/ml, 24 h) U-937 cells (B), PMA-treated U-937 cells in the presence of anti-CDlla (C), anti-CD18 (D), antLCD54 (E) or anti-CD45 (F) mAb. Magnification x 90

602

I 1

I

I 1 -

A. Indirect Immunofluorescence B. Direct Immunofluorescence

I I I I I I I I I I

I I

I I I I I I

I Transferrin I

Receptor I I I

Fig. 2. Changes in the expression of the C D l l c and transferrin receptor (CD71) differentiation antigens on U-937 cells upon treatment with P M A (10 nglml, 24 h ) in the absence or presence of anti-CDlla, anti-CDl8, or anti-CD54 mAb. Flow cytometry analysis of the expression of CD1 lc and transferrin receptor by indirect (A) and direct immunofluorescence (B). The mAb X63 was included as a negative control

Oh 10h 24h - + - + --- Table I . Proliferation of undifferentiated and PMA-differentiated U-

937 cells in the presence or absence of homotypic adhesions U-937 cells were cultured for two days in the presence or absence of PMA and the indicated mAb. Anti-LFA-I ct (CDlla), anti-LFA-1 f i (CD18) and anti-ICAM-I mAb inhibited cell cluster formation. The anti-CD4S mAb allowed cell-cell adhesions and was included as a control. The [3H]thymidine uptake was measured as described in Materials and Methods

TfR

28S-

Treatment Homotypic 10-3 x [3H]- adhesions Thymidine c-myc

uptake 18S-

-

PMA - anti-CDlla (LFA-1) PMA + anti-CD11 a (LFA-1) - anti-CD18 (LFA-ID) PMA + anti-CD18 (LFA-1P) - anti-CD54 (ICAM-1) PMA + anti-CD54 (ICAM-1) - anti-CD45 PMA + anti-CD45

CPm 47 820

8157 46371

3 075 51 406

5 536 48397

6235 44 300

6 402

28S-

OrnDC

18S-

28 S-

broadly distributed antigens, such as the transferrin receptor which is a marker for cell proliferation, occur during this differentiation process [26, 351. We have thus analyzed these antigenic changes to assess whether the treatment with PMA in the presence of mAb to LFA-1 or ICAM-1 induces differen- tiation of the promonocytic U-937 cells, despite the inhibition of intercellular homotypic adhesions. Fig. 2 shows that the important increment in the expression of the CD1 l c antigen and the dramatic decrease of the transferrin receptor observed during PMA-induced differentiation of U-937 cells also occur in the absence of intercellular homotypic adhesions.

Changes in specific gene transcripts levels during the PMA-induced dgferentiution of U-937 cells in the presence of anti-CDllu mAb

The PMA-induced differentiation of U-937 and other my- eloid cell lines is accompanied by drastic changes in the ex-

vim.

18s-

Fig. 3. Changes in specific gene transcript levels. Total cytoplasmic RNA was extracted from untreated cells (0 h) and from cells treated for 10 h and 24 h with 20 ng/ml PMA in the absence (-) and in the presence (+) of anti-CDlla mAb. RNA blots (15 pg/lane) were prepared and hybridized with transferrin receptor (TfR), c-myc, orni- thine decarboxylase (OrnDC) and vimentin (vim.) probes

pression of specific genes. In agreement with previous studies [29-331, we found that PMA treatment provoked a great increase in vimentin RNA levels, while it greatly reduced the levels of c-myc and OrnDC RNA (Fig. 3). The inhibition of intercellular adhesions by anti-CD1 l a mAb did not alter the changes in those transcripts induced by the phorbol ester. Under the conditions used here, the levels of transferrin recep-

603

Table 2. PMA-inducedproduction of superoxide anion by PMA-dij’er- entiated U-937 cells in the presence or absence of LFA-1-mediated homotypic adhesions Undifferentiated and PMA differentiated U-937 cells (10 ng/ml of PMA for two days) were stimulated for generation of superoxide anion with 100 ng/ml of PMA. Values of cytochrome c reduced rep- resent the mean * standard error of three different experiments

Treatment Cytochrome c reduced by 1 O6 cells in 15 min

nmol

None 4.20 k 0.25 PMA 15.87 + 3.36 PMA + anti-CDlla 17.72 * 1.53

tor RNA were not significantly modified by the PMA treat- ment of U-937 cells either in the presence or in the absence of cell cluster formation mediated by the anti-LFA-1 mAb (Fig. 3). Although this finding contrasts with the rapid inter- nalization of the transferrin receptor upon PMA treatment [47], it is concordant with the presence of transferrin receptor transcripts during monocyte differentiation [48].

Effect of anti-LFA-1 mAb on the superoxide-anion-generating capacity of PMA-differentiated U-937 cells

The capacity of generating superoxide anion is developed in the promonocytic U-937 cells and in the myelomonocytic cell line HL-60 during treatment with PMA [23, 251 and rep- resents an important functional parameter to assess the differ- entiation of these cells into macrophage-like cells. Thus, U- 937 cells differentiated for two days with low doses of PMA produce a significant amount of superoxide anion upon stimu- lation with high doses of PMA, whereas undifferentiated U- 937 cells are not capable of generating superoxide anion (Table 2). The presence of mAb to the LFA-1 a chain (CD1 la) during the treatment of U-937 cells for two days with 10 ng/ ml of PMA, although inhibiting completely the intercellular homotypic adhesions, did not affect their capacity to generate superoxide anion upon stimulation with high doses of PMA (Table 2).

DISCUSSION

Cell - cell adhesion phenomena constitute an important aspect of human mononuclear phagocyte differentiation and function. Thus, the LFA-1-dependent interaction of mono- cytes with lymphocytes is a crucial event at the initiation of an immune response [8]. Monocytes also adhere through LFA-1 to endothelial cells as they differentiate into macrophages and migrate into tissues, and to tumour cells to exert their cytotoxic activity [lo, 491. Mononuclear phagocytes parti- cipating in all these adhesion processes show distinct differen- tiation characteristics which are regulated by different agents

The human promonocytic cell line U-937 provides a valu- able experimental model for the study of the differentiation and intercellular adhesion of mononuclear phagocytes. When these cells are induced to differentiate into macrophage-like cells by treatment with the phorbol ester PMA, an important

~501.

increase in cellular adhesiveness occurs, as evidenced by at- tachment to the plastic substrate and cell cluster formation. These intercellular adhesions induced by PMA in U-937 cells are completely inhibited by mAb to the LFA-1 or ICAM-1 surface antigens, which morphologically, could be interpreted as a blockade of cell differentiation by these mAb. However, the development of other phenotypic and functional charac- teristics during the PMA-induced differentiation process of U-937 cells is not inhibited by the mAb directed to the CDlla , CD18 or CD54 antigens. These important modifications as- sociated with the differentiation of U-937 cells include changes in the expression of myeloid-restricted antigens and in the levels of specific gene transcripts, the loss of the proliferative capacity and the acquisition of capacity to generate superoxide anion. Thus, at the antigenic level, the important increment in the expression of the CDl lc antigen and the disappearance of the transferrin receptor from the cell surface, which charac- terize the differentiation of U-937 cells into macrophage-like cells, occur in the presence of the aforementioned mAb. Also, the great increase in vimentin RNA levels and the marked decrease in the levels of ornithine decarboxylase and c-myc RNA that accompany the PMA-induced differentiation of U- 937 cells are observed in the absence of intercellular adhesions mediated by the anti-CDlla mAb. Our results based on all these differentiation markers clearly demonstrate that PMA induces the differentiation of the monocytic U-937 cells inde- pendently of intercellular adhesion. It could be argued that PMA bypasses the physiological signalling pathways triggered by the LFA-l/ICAM-1-mediated adhesion. Nevertheless, the fact that in patients suffering from a CD11/CD18 deficiency, adherence-independent functions of phagocytes are all normal including shape changes, Met-Leu-Phe binding and oxygen radical generation to soluble stimuli [51], supports our con- clusions. Experiments with monocytes similar to those de- scribed here with U-937 cells are hampered by the fact that control monocytes adhere spontaneously and adhesion in- duced by y-interferon or phorbol esters has only a limited enhancing effect on aggregation. In addition, homotypic ad- hesions of monocytes are only partly inhibited by anti-CD18 mAb [27]. A second LFA-1 ligand, ICAM-2 [52], could prob- ably account for the total homotypic binding in monocytes. This contrasts with our aggregation model system with U-937 cells, which is only dependent on ICAM-1.

It has been proposed that the mechanism whereby PMA induces homotypic intercellular adhesions is the induction of a conformational change in the LFA-1 molecule, possibly due to phosphorylation mediated by protein kinase C 1531. This change in the conformation of LFA-1 induced by PMA would make this molecule more accessible for interacting with its ligand ICAM-1, thus facilitating occurrence of cell -cell ad- hesion. According to this hypothesis, the inhibition of the PMA-induced intercellular adhesions mediated by anti- LFA-1 or anti-ICAM-1 mAb, should not affect cell differen- tiation, since PMA, besides inducing the conformational change of LFA-1, phosphorylates via protein kinase C several other specific substrate proteins, which in turn leads to cell differentiation. Therefore, our results are in accord with this explanation for the mechanism by which intercellular ad- hesion and differentiation of mononuclear phagocytes occur.

Finally, our results using monocytic cells do not exclude the existence of LFA-l/ICAM-1-dependent signals across the plasma membrane in other cell types. In fact, recent studies have postulated that binding of ligand to LFA-1 can convey regulatory signals as well as activation of T lymphocytes [54, 551. The different role in activation/differentiation of the

604

LFA-1 molecule depending on the hematopoietic lineage de- serves to be further studied.

We thank Drs de Landazuri, Larraga and Marquet for helpful discussions. The technical assistance of P. Lastres and D. Hernandez is also acknowledged. This work was supported by grants from Direccidn General de Investigacidn Cientifica y Ticnica (PB87-0286 and PB87-0351) and Fondo de Investigaciones Sanitarias (8811726).

REFERENCES 1.

2.

3. 4. 5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23. 24.

Springer, T. A. &Anderson, D. C. (1986) Ciba Found. Svmp. 118,

Springer, T. A., Dustin, M. L., Kishimoto, T. K. & Marlin, S. D. (1987) Annu. Rev. Immunol. 5, 223-252.

Bierer, B. E. & Burakoff, S. J. (1988) FASEB J . 2, 2584-2590. Martz, E. (1987) Hum. Immunol. 18, 3-37. Dustin, M. L., Rothlein, R., Bhan, A. K., Dinarello, C. A. &

Springer, T. A. (1986) J . Immunol. 137, 245-254. Rothlein, R., Dustin, M. L., Marlin, S. D. & Springer, T. A.

(1986) J . Immunol. 137,1270-1274. Dougherty, G. J. & Hogg, N. (1987) Eur. J . Immunol. 17, 943-

947. Dougherty, G. J., Murdoch, S. & Hogg, N. (1988) Eur. J .

Inimunol. 18, 35 - 39. Krensky, A. M., Sanchez-Madrid, F., Robbins, E., Nagy, J. A,,

Springer, T. A. & Burakoff, S. J. (1983) J . Immunol. 131,611 - 616.

Krensky, A. M., Robbins, E., Springer, T. A. & Burakoff, S. J. (1984) J . Immunol. 132,2180-2182.

Davignon, D., Martz, E., Reinolds, T., Kurzinger, K. & Springer, T. A. (1981) Proc. Natl Acad. Sci. USA 78,4535-4539.

Sanchez-Madrid, F., Krensky, A. M., Ware, C. F., Robbins, E., Strominger, J. L., Burakoff, S. J. & Springer, T. A. (1982) Proc. Natl Acad. Sci. U S A 79,7489-1493.

Davignon, D., Martz, E., Reynolds, T., Kurzinger, K. & Springer, T. A. (1981) J. Immunol. 127, 590-595.

Arfors, K. E., Lundberg, C., Lindbom, L., Lundberg, K., Beatty, P. G. & Harlan, J. M. (1987) Blood 63, 338 -340.

Keizer, G. D., Te Velde, A. A,, Schwarting, R., Figdor, C. G. & DeVries, J. E. (1987) Eur. J . Immunol. 17, 1317-1321.

Sanchez-Madrid, F., Nagy, J., Robbins, E., Simon, P. & Springer, T. A. (1983) J . Exp. Med. 158, 1785-1803.

Keizer, G. D., Borst, J., Figdor, C. G., Spits, H., Miedema, F., Tcrhorst, C. & De Vries, J. E. (1985) Eur. J. Immunol. 15,

Boros, D. L. (1989) in Human monocytes, 1st edn (Zembala, M. & Asherson, G. L., eds) pp. 373 -381, Academic Press, London.

Mentzer, S. J., Faller, D. V. & Burakoff, S. J. (1 986) J . Immunol.

Le, J., Prensky, W., Yip, Y. K., Chang, Z., Hoffman, T., Stevenson, H. C., Balacs, I., Sadlik, J. R. & Vicek, J. (1983) J. Immunol. 131, 2821 -2827.

Hamilton, T. A. & Adams, D. 0. (1987) Immunol. Today 8,151 - 158.

Gidlund, M., Orn, A,, Pattengale, P. K., Jansson, M., Wigzell, H. & Nilsson, K. (1981) Nature292, 848-851.

Minta, J . 0. &Pambrun, L. (1985) Am. J. Pathol. 119, I11 -126. Hass, R., Bartels, H., Topley, N., Haldam, M., Kohler, L.,

Goppelt-Strube, M. & Resch, K . (1989) Eur. .I. Cell Bid. 48,

102- 126.

1142- 1147.

137,108-113.

282-293.

25. Rovera, G., Santoli, D. & Damsky, C. (1979) Proc. Natl Acad.

26. Collins, S . J. (1987) Blood 70, 1233- 1244. 27. Patarroyo, M., Clark, E. A,, Prieto, J . , Kantor, C. & Gahmberg,

C. G. (1987) FEBS Lett. 210, 127-131. 28. Yen, A., Brown, D. & Fishbaugh, J. (1987) Exp. Cell Res. 168,

29. Einat, M., Resnitzky, D. & Kimchi, A. (1985) Nature 313, 597-

30. Sariban, E., Mitchell, T. & Kufe, D. (1985) Nature. 316, 64-66. 31. Muller, R. (1986) Trend.y Biochem. Sci. 11, 129-132. 32. Ferrari, S., Baltini, R., Kaczmarek, L., Rittling, S., Calabretta,

B., De Riel, J. K., Philiponis, V., Wei, J. F. & Baserga, R. (1986) Mol. Cell. Biol. 6. 3614-3620.

Sci. USA 76,2779-2783.

247 - 254.

600.

33. Rius, C. & Aller, P. (1989) Cell Defer. Dev. 28, 39-46. 34. Miller, L., Schwarting, R. & Springer, T. A. (1986) J . Immunol.

35. Cabaiias, C., Sanchez-Madrid, F., Acevedo, A., Bellon, T., Fernandez, J. M., Larraga, V. & Bernabeu, C. (1988) Hybrid- oma 7,167-176.

36. Ferrero, D., Pessano, S., Pagliardi, G. L. & Rovera, G. (1983)

37. Newburger, P. E., Speier, C., Borregaard, N., Walsh, C. E., Whitin, J. C. & Simons, E. R. (1984) J . Biol. Chem. 259,3771 - 3776.

38. Bernabeu, C., Carrera, A. C., De Landazuri, M. & Sanchez- Madrid, F. (1987) Eur. J . Immunol. 17, 1461 - 1466.

39. Bernabeu, C., Morago, G., De Landazuri, M. O., Carreira, J. & Sanchez-Madrid, F. (1986) Inmunologia 5, 83 -90.

40. Rindernecht, H. (1962) Nature 193, 167-168. 41. Aller, P. & Baserga, R. (1986) J . Cell Physiol. 128, 362-366. 42. Lehrach, H., Diamond, D.. Wozney, M. & Boedtkc, H. (1977)

Biochemistry 16, 4743 -4749. 43. Hirschhorn, R. R., Aller, P., Yuan, Z. A., Gibson, C. W. &

Baserga, R. (1984) Proc. Natl Acad. Sci. USA 81,6004-6008, 44. Dalla-Favera, R., Gelmann, E. P., Martinotti, S., Franchini, G.,

Papas, T. S., Gallo, R. C. & Wong-Staal, F. (1982) Proc. Natl Acad. Sci. U S A 79, 6497 - 6501.

45. Kuhn, L. C., McClelland, A. & Ruddle, F. H. (1984) Cell 37,

46. Feinberg, B. P. & Vogelstein, B. A. (1984) Anal. Biochem. 137,

47. May, W. S., Jacobs, S. & Cuatrecasas, P. (1984) Proc. Natl Acad.

48. Hirata, T., Bitterman, P. B., Mornex, J . F. &Crystal, R. G. (1986)

49. Te Velde, A. A., Keizer, G. D. & Figdor, C. G. (1 987) Immunology

50. Figdor, C. G., Te Velde, A. A., Leemans, J. M. M. & Bont, W. S. (1986) in Leucocytes and host defense (Oppenheim, J. J. & Jacobs, D. M., eds) pp. 283-288, Alan R. Liss, New York.

51. Anderson, D. C., Schmalstieg, F. C., Arnaout, M. A,, Kohl, S., Tosi, M. F., Dana, N., Buffone, G. J., Hughes, B. J., Brinkley, B. R., Dickey, W. D., Abramson, J. S., Springer, T. A., Boxer, L. A., Hollers, J. M. & Smith, C. W. (1984) J. Clin. Invest. 74,

52. Staunton, D. E., Dustin, M. L. & Springer, T. A. (1989) Nature

53. Keizer, G. D., Visser, W., Vliem, M. & Figdor, C. G. (1988) J. lmmunol140, 1393 - 1400.

54. van Noesel, C., Miedema, F., Brouwer, M., de Rie, M. A., Aarden, L. & Lier, A. W. (1988) Nature 333, 850-852.

55. Wacholtz, M. C., Patel, S. S. & Lipsky, P. E. (1989) 1. Exp. Med.

137,2891 -2900.

Blood61, 171-179.

95-103.

266 - 267.

Sci. USA 81, 2016-2020.

J . Immunol. 136, 1339-1345.

61,261 -267.

536- 551.

339, 61 - 64.

170, 431 -448.