Eur. J. Immunol. 1989.19: 1373-1378 Regulated expression of a novel myeloid antigen 1373

Carlos CabaiiasoA, Francisco Sanchez-Madrido, Teresa Bellon’, Carl G. FigdorA, Anje A. Te VeldeA, Jesus M. Fernandez’, Augustin Acevedo’ and Carmelo Bernabeu”

Centro de Investigaciones Biologicas’, C.S.I.C., Hospital de La Princess', Madrid and The Netherlands Cancer InstituteA, Amsterdam

Characterization of a novel myeloid antigen regulated during differentiation of monocytic cells*

The monoclonal antibody HC1/6 generated against phorbol 12-myristate 13-acetate- treated U-937 cells recognizes a new cell surface antigen with a broad relative molecu- lar mass ranging from 100 to 150 kDa. This antigen is also present on monocytes, platelets and endothelial cells and is weakly expressed by granulocytes. In contrast, it is absent from T, B and erythroblastoid cells. The antigen HC1/6 is also expressed by normal tissue macrophages in tonsil, lung and kidney, as well as in skin biopsies from pathologies such as sarcoidosis and lepromatous leprosy. The expression of the HC1/6 antigen is increased up to 5-fold when U-937 (promonocytic) and HL-60 (myelomonocytic) cell lines are stimulated with phorbol 12-myristate 13-acetate. Con- versely, the expression of the HC1/6 antigen is down-regulated in monocytes upon treatment with interferon-y. These findings are discussed in relation with other myeloid cell surface markers.

1 Introduction

The differentiation of monocytes is a multistep process charac- terized by major changes in their morphological and functional properties [l, 21. These changes can correlate with the reg- ulated expression of different cell surface antigens which are useful for the analysis of discrete stages of differentiation. A variety of agents including interferon (1FN)-y, retinoic acid (RA), phorbol12myristate 13-acetate (PMA) and 1,25-dihyd- roxyvitamin D3 [1,25-(OH)zD3] are able to generate function- ally different subpopulations and to regulate the expression of cell surface proteins of the monocytic lineage [3-71. The exist- ence of these subpopulations can be analyzed by using mono- clonal antibodies (mAb) specific for surface antigens [8]. Thus, CR3 (CD11b or Mol) is mainly present in circulating monocytes and granulocytes, whereas CDllc (p150,95) is highly expressed in tissue macrophages (McP) [9-111. Also, the expression of CR1 (CD35), CR3 (receptor for C3bi), CDllc and FcRI (CD16) is rapidly increased when cells are treated with certain stimuli such as IFN-y, PMA or the N- formyl-Met-Leu-Phe peptide [9-151.

The promonocytic cell line U-937 and the myelomonocytic cell line HL-60 have often been used to analyze the myeloid differ- entiation process [16-181. Here, we have used the M@-like U-937 cells after differentiation in the presence of PMA as a model system to generate mAb against novel cell surface anti- gens of the myeloid family which are up-regulated after differ-

[I 74661 ~~ ~ ~ ~

* This work was supported by grants from “Fondo Nacional para la Investigacih Cientifica” (CSIC-424) and “Fondo de Inves- tigaciones Sanitarias” (FISS 116187 and FISS 1726/88). Recipient of a PFPI (1985) fellowship from “Ministerio de Educa- cion y Ciencia”. Recipient of a short-term fellowship (ASTF, 5607) of the European Molecular Biology Organization.

Correspondence: Carmelo Bernabeu, Centro de Investigaciones Biologicas (CSIC), Velhzquez 144, 28006 Madrid, Spain

Abbreviations: mAb: Monoclonal antibody(ies) 1,25-(OH)2D3 or Vitamin D3: 1,25-Dihydroxycholecalciferol PMA: Phorbol12-myris- tate 13-acetate RA: Retinoic acid IFN: Interferon Mo: Mono- cyte(s) M 8 : Macrophage(s) SF: Specific fluorescence

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1989

entiation. Thus, we have obtained the mAb HC1/6 which rec- ognizes a novel monocytic antigen of 100-150 kDa. The characterization of this antigen is described.

2 Materials and methods

2.1 Cell lines

U-937 (promonocytic), HL-60 (myelomonocytic), K-562 (erythroblastoid), Jurkat (T lymphocytic), JM (T leukemic) and JY (B lymphoblastoid) cell lines were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, penicillin (100 Ulml) and streptomycin (100 ng/ml) (Flow Laboratories, Rockwell, MD), in a 5% C 0 2 atmosphere at 37°C. When necessary, cells were treated with PMA (10 ng/ml), 1,25-(OH)2D3 M), IFN-y (100 U/ml) or RA (100 ng/ml).

2.2 Preparation of leukocytes

Peripheral blood leukocytes were obtained from heparinized whole venous blood. After elimination of most erythrocytes by gravity sedimentation in the presence of 0.4% dextran, mono- cytes (Mo) and lymphocytes were separated from granulocytes by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gra- dient centrifugation. Granulocytes were obtained from the pellet and separated from contaminant erythrocytes by a 20-s hypotonic lysis in distilled water. Mo were isolated by incubat- ing the mononuclear cell fraction in a slightly hypertonic medium followed by a second modified Ficoll-Hypaque density centrifugation [ 191. Alternatively, Mo were purified by centrifugal elutriation as described by Figdor et al. (201.

Mo (3 X 106/ml) were incubated at 37”C, 5% COz and 100% humidity in Teflon bags (Jansen’s MNL, St. Niklaas, Belgium) in a modified Iscove’s medium containing human albumin and supplemented with 2% autologous heat-inactivated serum [21]. After 2 days in culture, Mo were collected from the Tef- lon bags, washed, resuspended in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin, 0.02% sodium a i d e and kept on ice until the detection of surface membrane determinants. Platelets were obtained by density centrifuga- tion [20].

OO14-2980/89/0808-1373$02.50/0

1374 C. Cabahas, F. Sanchez-Madrid, T. Bellon et al. Eur. J . Immunol. 1989.19: 1373-1378

2.3 Reagents

Recombinant IFN-y (a gift from Dr. Adolf, Boehringer- Ingelheim, Vienna, Austria) was solubilized in RPMI 1640 containing 10% FCS and stored at -2O"C, 1,25-(OH)zD3 (a gift of Dr. Uskokovic, Hoffmann-La Roche, Nutley, NJ) and RA (Sigma, St. Louis, MO) were stored at -20°C in ethanol. PMA (Sigma) was dissolved in dimethylsulfoxide to a concentration of 1 mg/ml.

2.4 mAb

The mAb HClll (anti-CDllc) [22], FG116 (specific for the transferrin receptor) [23], HP2/6 (anti-CD4) [24] and 8F3 (anti-CD13) have been generated in our laboratory. The mAb Bear 1 (anti-CDllb) and Bear 2 (anti-CD14) [25, 261 were kindly provided by Dr. Jan De Vries (UNICET, Lyon, France). The mAb Q5/13, which detects a determinant com- mon to HLA-DR, -DQ and -DP molecules (271, was a gift of Dr. S. Ferrone (New York Medical College, New York, NY).

2.5 Production of the hybridoma HC1/6

BALB/c mice were injected i.p. with 15 x lo6 PMA-treated U-937 cells on days 48, 33 and i.v. on day 3 prior to fusion. Spleen cells were fused on day 0 with P3-X63Ag8.653 mouse myeloma cells at a 4 : 1 ratio according to standard techniques, distributed into 96-well plates (Costar, Cambridge, MA) and grown as described [22]. After 2 weeks, hybridoma culture supernatants were harvested and screened for binding to PMA-differentiated U-937 cells using the '251-labeled rat anti- mouse x chain mAb 187.1 as second antibody [28]. The mAb HC1/6 was selected due to its enhanced reactivity for PMA- treated over untreated U-937 cells. This antibody is of the IgGl subclass.

2.6 Radiolabeling, immunoprecipitation and electrophoresis

Cells were iodinated using chloroglycoluril (IODO-GEN, Sigma, [29]) and lysed in PBS, pH 7.4, containing 1% Triton X-100, 1 % hemoglobin and 1 mM phenlymethylsulfonyl fluoride. Immunoprecipitation was carried out with protein A- Sepharose (Pharmacia; [22]) and samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis [30] and autoradiography with enhancing screens [31].

2.7 Flow cytometry

Flow cytometry analysis was performed using either an EPICS-C (Coulter Cientificia, Mostoles, Spain) or a FACScan (Becton Dickinson, Mountain View, CA) flow cytometer using logarithmic amplifiers [22]. Specific fluorescence (SF) was obtained by subtracting the peak channel number of the negative control from the peak channel number of the corre- sponding experimental sample.

2.8 Immunohistochemical staining of human tissue sections

Specimens of human tonsil, thymus, lymph node, brain, lung, skin and kidney were obtained by surgical procedures. Tissue

sections were stained by an indirect one-step immunoperoxi- dase method [22, 321. For the double immunoperoxidase- alkaline phosphatase staining, we followed the sequential method reported by Masson et al. [33]. After the development of the peroxidase reaction, sections were incubated with the second mAb, then with a rabbit anti-mouse IgG, followed by a third incubation with the alkaline phosphatase-anti-alkaline phosphatase mAb complex (Dakopatts, Glostrup, Denmark). Each incubation was followed by three washes with Tris saline. Finally, the alkaline phosphatase reaction was developed, yielding a dark blue precipitate which contrasts with the brown colour of the peroxidase reaction. Sections were mounted in buffered gelatin for microscopic examination.

3 Results

3.1 Effect of several differentiation agents on the expression of the antigen recognized by the mAb HC1/6

The mAb HC1/6 was obtained by immunization with PMA- treated U-937 cells. The antigen recognized by HC116 is mod- erately expressed on untreated U-937 cells, but its expression is strongly increased after PMA-induced differentiation (Fig. 1). IFN-y, 1,25-(OH)2D3 and RA were also used to induce differentiation of U-937 cells and the expression of HC1/6 and several other cell surface markers was analyzed by flow cytometry (Fig. 1). The highest expression of the antigen recognized by the mAb HC1/6 was obtained in the presence of PMA. A certain augmentation of the HC1/6 antigen was also detected with IFN-y and 1,25-(OH)*D3 in contrast to the decrease observed in the presence of RA. Concomitantly with the loss of proliferation (data not shown), the expression of the transferrin receptor was markedly diminished in every differentiation protocol. The regulation of the HC1/6 antigen expression is accompanied by different antigenic changes depending on the differentiation agent used. In addition to HC1/6, PMA enhances the expression of CDl lc and CR3 (CDllb) antigens and diminishes the expression of the CD4 antigen. In the presence of 1,25-(OH)~D3 and RA, CR3 expression is also increased but the CDl lc antigen is almost unaffected. A feature common to F U and vitamin D3 but not to PMA is the marked decrease of the CD14 staining. On the other hand, CD4 expression was found to be affected only by RA and PMA. These results demonstrate that the expression of the antigen recognized by the mAb HC1/6 can be up-reg- ulated by PMA, IFN-y and 1,25-(OH)*D3. This regulation does not strictly follow any of the patterns shown by the anti- genic markers used as controls. Kinetic studies in the presence of PMA demonstrated that the antigen HC1/6 shows the high- est expression after 3 days in culture, similarly to the CDl lc antigen (data not shown).

In vitro, IFN-y induces activation of human Mo, which is associated with an increased expression of the high-affinity FcR for IgG (FcyRI) and major histocompatibility complex (MHC) class I1 antigens [15, 34, 351, as well as a decreased expression of CD14, CR3 and 63D3 antigens [36]. We then assayed the effect of IFNy on the expression of the HC1/6 antigen by human Mo (Table 1). Mo cultured in the presence of IFNy showed a marked decrease in the expression of HC116 as compared with the control culture. As expected, expression of MHC class I1 antigens increased during the culture of Mo but IFN-y and IFN-a up-regulated further their expression.

Eur. J. Immunol. 1989.19: 1373-1378 Regulated expression of a novel myeloid antigen 1375

I I

I I I

I I -

u - 9 3 7

u -937 + PMA

u-937 + D 3

u-937 + R.A.

u - 937 +6IFN

I I I I I

I I

I I I I I I

I I I I I

I I I I I I

I I -

CDllb I

I 1 I I 1 - I

y., I I h- I I , 1 -

I

I I 55 I 30 I 3 1 2

Vntreoted

I I 99 1 I ; I

PMAtreoted I

Transferrin CDllc C D 1 4 C D 4 ReceDtor C D 1 3

(6) Gronulocytes Lymphocytes Monocytes Platelets

11 I

I

I I I

I I I I I I I 1

I I I

Fluorescence Intensity

HC1/6 I I I

I I I I - I I I I I I

I

I I I

Figure 1. Effect of different stimuli on the expression of the antigen recognized by the mAb HC1/6. Unstimulated and PMA- (3 days), vitamin D3-(5 days), IFN-y-(5 days) or RA-(5 days) treated U-937 cells were incu- bated with Bear 1 (anti-CDllb), HCU1 (anti-CDllc), Bear 2 (anti-CD14), HC1/6 (anti-CD4), FG1/6 (anti-transferrin recep- tor), 8F3 (anti-CD13) or HC1/6 mAb and assayed for indirect immunofluorescence in an EPICS-C flow cytometer as de- scribed in Sect. 2.7. Arrows indi- cate the increase or decrease in fluorescence after treatment of cells with the differentiation agents as compared to unstimu- lated cells.

Figure 2. Reactivity of the mAb HC1/6 with different cell types. Figures at the upper right corners represent the percentage of positive cells. (A) Untreated or PMA-treated U-937 (promonocytic), HL-60 (myelomonocytic), K-562 (erythroblastoid), Jurkat (T leukemic), JM (T leukemic) and JY (B lymphoblastoid) cell lines. (B) Peripheral blood granulocytes, lymphocytes, Mo and platelets. Cells were incubated with the HC1/6 mAb and assayed for indirect immunofluorescence in an EPICS-C flow cytometer as described in Sect. 2.7.

Table 1. Effect of IFN-y and IFN-a on the expression of the antigen HC1/6 in Mo”)

Addition HC1/6 HLA-DR (a) SFb’ (%) SF

None

IFN-a ControlC)

IFN-y

~

% 4.7 98 26 26 3.2 97 41 95 4.4 97 52 96 5.1 93 9

Mo purified by the countercurrent elutriation method were incu- bated in the absence or in the presence of IFN-y (100 Ulml) or IFN-a (100 Ulml) for 2 days at 37°C in 5% COz. Cells were stained with either HC1/6 or the anti-HLA-DR mAb Q5/13 and their fluorescence was analyzed in a FACScan flow cytometer. SF values were obtained as described in Sect. 2.7. A control sample kept at 4°C was also included for comparison.

-3 I 2 3 4 5 M r X I 0

150-

95 -

45-

Figure 3. Immunoprecipi- tation analysis of the anti- gen recognized by the mAb HC1/6. PMA-treated U-937 cells were ‘2SI-labeled, lysed and immunoprecipi- tated with HCU6 (lanes 2 and 4), anti-CDllc (lane 3) or a negative control (lanes 1 and 5 ) mAb. Sam- ples were electrophoresed under either reducing (lanes 1-3) or nonreducing (lanes 4 and 5) conditions.

1376 C. Cabafias, F. Sanchez-Madrid, T. Bellon et al. Eur. J. Imrnunol. 1989.19: 1373-1378

Figure 4. Immunoperoxidase staining of tonsil (A), kidney (B) and lung (C) sections with the mAb HC1/6. (D) shows a double staining of a lung section with the mAb HC1/1 (peroxidase) followed by the mAb HC1/6 (alkaline phosphatase). The mAb HC1/6 recognizes the M@ present in all these tissues as well as the high endothelial venules (A). Staining of the renal glomerulus (B) shows an intense labeling of mesangial cells together with a diffuse reactivity of capillaries. Samples (A), (B) and (C) were hematoxylin counterstained. Magnification: (A, B; x 250); (C, D; x 400).

No changes in the level of expression of the antigen HC1/6 were obtained in the presence of IFN-a.

Other cell types were also assayed for reactivity with the mAb HC1/6 (Fig. 2). No staining was found with the erythroblastoid cell line K-562 nor with the lymphoblastoid B cell line JY and the T cell line JM. Similarly, a very weak reactivity was found with the T cell leukemic line Jurkat. In addition, granulocytes were weakly labeled by the mAb HC116, whereas peripheral blood Mo and platelets were clearly positive as opposed to the negative staining of lymphocytes. The relatively restricted expression of this antigen to cells of the myeloid lineage (U- 937, HL-60, Mo and platelets) becomes also apparent after study of different cell types treated with PMA (Fig. 2). Only the promonocytic cell line U-937 and the myelomonocytic cell line HL-60 showed an increased expression of the antigen in response to PMA. This contrasts with the negative response of T, B and erythroblastoid cell lines.

As determined by immunoprecipitation analysis under reduc- ing conditions, a broad band of 100-150 kDa was recognized by the mAb HC1/6 from cell surface radioiodinated U-937 cells after differentiation in the presence of PMA (Fig. 3, lane 2). The same relative molecular mass (M,) was obtained under nonreducing conditions (lane 4) demonstrating the absence of interchain disulfide bonds. The CD1 lc/CD18 antigen complex

was also immunoprecipitated for comparative purposes (lane 3).

3.2 Detection of the antigen recognized by the mAb HC1/6 in tissue M 8

The antigen recognized by the mAb HC1/6 was visualized by immunoperoxidase staining of frozen tissue sections. Thus, M@ from tonsil, lung and kidney were labeled by the mAb HC1/6 (Fig. 4). Scattered macrophages in the paracortical area (T cell zone) from tonsil (A), alveolar M a from lung (C) and mesangial M@ from kidney glomeruli (B) were positively stained with mAb HC1/6. In addition, Kupffer cells from liver as well as M@ from lymph node and thymus were labeled by mAb HC116, whereas microglial cells and dendritic epidermal cells were negative (data not shown). In most of these tissues, the presence of the HC1/6 antigen was evident in endothelial cells. Since the CDl lc antigen is an excellent marker for tissue M a , we carried out a double staining with HC1/6 and anti- CDl lc mAb (D). As shown by immunoperoxidaselalkaline phosphatase staining, the CDl lc (brown) and HC1/6 (dark blue) antigens are co-expressed by the majority of M@. How- ever, the HCU6 antigen was expressed on vascular structures whereas the CDl lc antigen was not.

Eur. J. Immunol. 1989.19: 1373-1378 Regulated expression of a novel myeloid antigen 1377

members of the integrin superfamily are also expressed by endothelial cells [44]. Interestingly, the ICAM-1 antigen (ligand for LFA-1 antigen) is also up-regulated by PMA in U-937 and HL-60 cells [45, 461. Since the antigens of the inte- grin family are involved in cell-matrix and cell-cell interac- tions, a similar function could be suggested for the antigen HC1/6. However, we have been unable to assign any func- tional role to the antigen HC1/6. In our hands, adhesion, mig- ration antigen presentation and antibody-dependent cell- mediated cytotoxicity of Mo are unaffected by the presence of the mAb HC1/6. The fact that this antigen is not only present on Mo/MO but also in platelets, suggests that the function of the antigen HC116 is shared by both lineages.

Different pathologies characterized by the presence of an increased number of activated MO were also analyzed by staining of skin sections with the mAb HC116. In lepromatous leprosy and sarcoidosis, most of the infiltrating MO, as well as the epithelioid and giant cells present in the granulomas ex- pressed the HC1/6 antigen (data not shown).

4 Discussion

The mAb HC116 described here recognizes a protein with a broad M, ranging from 100 to 150 kDa and which seems to be specific for the myeloid lineage. This antigen is highly ex- pressed on peripheral blood Mo, tissue M a , platelets, endothelial cells and, upon differentiation, on the MO-like U- 937 and HL-60 cells.

The expression of the antigen HC1/6 on Mo is not due to activation during the isolation protocol [37], since the same results were obtained when Mo were purified by the counter- current elutriation method [20]. In this sense, activation of Mo in the presence of IFN-y down-regulates the expression of the antigen HC1/6 in contrast with an increased expression of MHC class I1 antigens. Although IFN-y promotes differentia- tion of Mo into activated MO with cytotoxic activity and increased expression of MHC class I1 and FcR, certain myeloid differentiation antigens such as CD14 (Mo2) and 63D3 are down-regulated [36]. This is in agreement with the behavior of the antigen HC116 in the presence of IFN-y but not observed in the presence of IFN-a.

Other myeloid antigens with a similar M, as HC1/6 but differ- ent characteristics have been described [8,9,38,39]. Thus, the antigens CDl lb and C D l l c are associated with a second p subunit (CD18) and are not present on peripheral platelets, as opposed to HC1/6. On the other hand, mAb of the CD13 cluster which are specific for a 150-kDa protein, differ from the antigen HC1/6 in their strong reactivity for bile canaliculi and granulocytes, the lack of staining of platelets and the dis- tinct behavior upon stimulation with different agents (Fig. 1). Moreover, the CD31 antigen (sharp band at 130 kDa), although present on platelets,is highly expressed by peripheral granulocytes as well as by the T cell lines Jurkat and CEM as opposed to the antigen HC116 (broad band at 100-150 kDa).

A review of other unclustered antigens reveals that none is identical to the HC1/6 antigen. The p subunit of the VLA complex (110 kDa unreduced, 130 kDa reduced) which is identical to the gpIIa [30], is also present in platelets. How- ever, this antigen is different from HC1/6 since: (a) the M, of gpIIa changes between reducing or nonreducing conditions and (b) mAb directed against this antigen strongly react with the erythroblastoid cell line K-562 [41, 421. Moreover, mAb MoU26, which recognizes a 130-140-kDa antigen [38] is dis- tinct from the mAb HC1/6 due to the differential cellular reac- tivity since MoU26 strongly labels K-562 and does not react with Mo. Finally, the mAb CLB-HEC-75, which recognizes a 150-kDa antigen present on platelets and endothelial cells, is absent from Mo [43] and does not compete with the mAb HC1/6 for binding to PMA-treated U-937 cells (data not shown).

The mAb HC1/6 reacts with most of the vessels (venules, veins, small arteries and capillaries) and specially with high endothelial venules. Similarly, the ICAM-1 antigen and other

We are grateful to Drs. A . Sonnenberg, F. Hogervorst, V . Larraga,A. Marquet and R. Ramos for helpful dircussions.

Received January 19, 1989; in revised form April 22,1989

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Note added in proof: While this manuscript was in press we found out that the mAb HCY6 is reactive with transfectants of COS-7 cells ex- pressing the CD31 antigen.

Received July 17, 1989.