Molecuiar Immunology, Vol. 27, No. 1, pp. 5747, Printed in Great Britain.

0161-5890/90 $3.00 + 0.00 Pergamon Press plc

1990

THE EFFECT OF CYTOKINES ON THE EXPRESSION AND FUNCTION OF Fc RECEPTORS FOR IgG ON HUMAN MYELOID CELLS D. V. ERBE,* J. E. COLLINS,* L.

SHEN,*

R. F. GRAZIANO* and M. W. FANGER*~

Departments of *Microbiology and ~Medicine, Da~mouth Medical School, Hanover, NH 03756. U.S.A. (First received 3 May 1989; accepted 20 June 1989)

Abstract-We examined expression and cytotoxic triggering capability of the three Fc receptors for IgG (Fey R) on human monocytes, PMNs and myeloid cell lines after in vitro culture with various cytokines. FcgR expression was evaluated using specific anti-FcyR monoclonal antibodies (mAb). The cytotoxic capability of each Fq R was examined after the effector cells were treated with the recombinant cytokines IFN-y, TNFcr, GM-CSF, G-CSF, M-CSF, IL-I, IL-2. IL-3, IL-4, or IL-6 Hybridoma cell lines (HC) bearing antibody directed to FcyRI (HC 32), FcyRII (HC IV.3) or FcyRIII (HC 3G8) were used as targets, as were chicken erythrocytes (CE) sensitized with heteroantibodies composed of anti-FeyR mAbs (32, IV.3, 3G8) linked to anti-CE antibody. Only IFN-y treatment significantly increased FcyR expression and then only Fey RI. IFN-y dramatically up-regulated Fey RI expression on all cells tested. However, ADCC was enhanced by treatment with a number of cytokines other than IFN-y. GM-CSF, TNF, and IFN-y treatment enhanced killing of HC 32 and HC IV.3 by in vitro cultured monocytes. G-CSF treatment enabled PMNs to kill HC through FeyRII, whereas PMN killing of HC through FeyRI11 could not be induced by any of the cytokines studied. Although only IFN-y treatment increased ADCC of CE by monocytes, GM-CSF treatment as well as IFN-y treatment augmented ADCC of CE by PMNs. In addition to IFN-y treatment, IL-6 treatment enabled U937 cells to lyse CE. Whereas IFN-y-treated U937 cells killed CE through both Fey RI and Fey RII, IL-6-treated U937 cells killed CE only through Fey RI. In addition to IFN-y treatment, G-CSF treatment enabled HL-60 cells to lyse CE through both Fey RI and Fey RII. These results demonstrate that although IFN-y appears unique in regulating Fey R expression on myeloid cells. cytokines other than IFN-y affect ADCC by these cells in a receptor-specific manner.

INTRODUCTION Monoclonal antibodies (mAb) to each of the three identified human Fc receptors for IgG have allowed

elucidation of their surface expression and the role of each in triggering cytotoxicity (see Fanger et al., 1989). Fey RI is recognized by mAbs 32 (Anderson et al., 1986), 10.1 (Dougherty et al., 1987), 22, 44, 62, and 197 (Guyre et al., submitted); FcyRII is recognized by mAbs IV.3 (Looney et al., 1986a, 19863) and KuFc 79 (Vaughn et al., 1985); whereas Fey RI11 is bound by mAb 3G8 (Fleit et al., 1982) and others (Unkeless et al., 1988; Perussia et al., 1983a, 1983c). FeyRI is expressed on the surface of human monocytes, macrophages and IFN-y -treated neutrophils, whereas Fey RI1 is found on monocytes, macrophages, neutrophils, eosinophils, B cells, and platelets. Fey RIII, on the other hand, is expressed on the surface of large granular lymphocytes (LGL) as well as macrophages, neutrophils and eosinoAbbrevjations: mAb, monoclonal antibody; ADCC, antibody-dependent cell cytotoxicity; PMNs, polymorphonuclear leukocytes; CE, chicken erythrocytes; HC, hybridoma cells; M199, medium 199; IFN-y, gamma interferon; IL, interleukin; CSF, colony stimulating factor; TNF, tumor necrosis factor; Fey R, Fc receptor for IgG; FITC-GAM, fluorescein isothiocyanate conjugated goat anti-mouse antibody.

phils (Anderson and Looney, 1986; Fanger et al., 1989). Using heteroantibodies composed of anti-Fey R and anti-chicken erythrocyte (CE) antibodies, we have shown Fey R-dependent lysis of CE by monocytes, polymorphonuclear cells (PMNs) and myeloid cell lines (Shen et al., 1986, 1987, 1989; Petroni et al., 1988). We have also shown lysis of hybridoma cells (HC) bearing antibodies to the Fc receptors by monocytes and granulocytes (Graziano and Fanger, 1987a, 19876). IFN-y has been shown to dramatically increase expression of Fey RI on human monocytes and myeloid cell lines (Guyre et al., 1983; Perussia et al., 1983b) and to induce Fey RI expression on PMNs (Petroni ef al., 1988). Moreover, IFN-1, treatment significantIy enhanced Fc)l R-dependent killing of CE mediated by monocytes, PMNs and myeloid cell lines @hen ef al., 1984, 1989). Furthermore, IFN-y treatment enabled PMNs to lyse HC bearing antiFey RI and anti-Fey RI1 antibodies (Graziano and Fanger, 19876). To a large extent, cytokines other than IFN-y have not been investigated for ability to affect FqR expression and killing potential triggered through Fey R. IL-l was found to inhibit the up-regulation of Fey RI on monocytes treated with IFN-), (Arend et al., 1987), whereas GM-CSF was reported to moderately increase expression of Fey RI1 on U937 cells

58

D. V.

ERBEet

(Liesveld et al., 1988). Additionally, we have shown that GM-CSF enhanced killing of CE and HC through Fc receptors on PMNs and eosinophils (Graziano et al., 1989). Cytokines other than IFN-;I have also shown effects on myeloid cell surface expression of antigens other than Fcl;R which may influence killing potential. In vitro culture of monocytes with IL-4 increased their expression of the adhesion molecules CD 1 I b and CD I I c (Te Velde er al., 1988). Culture of monocytes with M-CSF also led to increased CD1 Ic expression (Becker et al., 1987). Additionally, a number of cytokines other than IFN‘/ have been shown to enhance antibody-independent cell killing by monocytes, including IL-2 (Malkovsky et al., 1987), GM-CSF (Grabstein et al., 1986), and M-CSF (Sampson-Johannes and Carlino, 1988). Using conventional studies on the ability of various cytokines to regulate ADCC, it has been difficult to distinguish which and to what extent each of the three FcyR are involved. We have therefore used Fey Rspecific hybridoma cells and bi-specific antibodies to perform a systematic analysis of the effects of cytokines on expression and function of each of the Fey R and compared them with the well-established effects of IFN-y. Human monocytes, PMNs and the myeloid cell lines U937 and HL-60 were treated with TNF, GM-CSF, G-CSF, M-CSF, IL-l, IL-2, IL-3, IL-4, IL-6 or IFN-7 and compared for expression of Fey R and ability to mediate ADCC of both CE and HC. Only IFN-y significantly affected Fe) R expression, by up-regulating FQ RI expression on all cells tested. However, GM-CSF, TNF, and IFN-; enhanced the lysis of HC by in vitro cultured monocytes. G-CSF treatment enabled PMNs to kill HC through Fey RII, whereas no cytokine treatment enabled PMN lysis of HC through Fcl;RIII. Although only IFN-;I enhanced ADCC of CE by monocytes, GM-CSF or IFN-7 augmented ADCC of CE by PMNs. In addition to IFN-y, IL-6 enabled U937 cells to lyse CE, and G-CSF enabled HL-60 cells to lyse CE. MATERIALS

AND METHODS

EfSector cells Monocytes were purified from cytophoresis packs obtained from normal volunteers. Cells from cytophoresis packs were spun on Ficoll-Hypaque and the interface layer was collected. After three washes in RPM1 1640, the cells were resuspended in RPM1 containing fetal bovine serum (FBS) at 5 x lO’/ml in 15 ml polypropylene tubes and were rotated at 8 rpm for I hr at 4°C to induce monocyte aggregation. The aggregated cells were sedimented on ice at I g for 15-30 min, resuspended in 2 ml of medium and carefully layered onto an equal volume of ice-cold FBS. After sedimentation through the FBS for 20min at 4”C, the lower phase contained 75-95% monocytes, the remainder being lymphocytes. PMNs were isolated from the peripheral blood of healthy volunteers. Heparinized blood was diluted

al

1: 1 with RPM1 and 20 ml were layered over IO ml of Ficoll-Hypaque. After centrifugation at 400 R for 30 min, the uppermost fraction, consisting of plasma and RPMI, was diluted with 0.4 ml of 5% pyrogenfree dextran (mol.wt 100,000; United States Biochemical Corporation, Cleveland, OH) for every 1 ml of this upper fraction. This mixture was then added to the red blood cell and PMN pellet. The suspended cells were allowed to settle for 30 min at 4’C, the supernatant containing PMNs was removed, and contaminating erythrocytes were hypotonically lysed with water. PMN preparations contained no platelets and less than 2% lymphocytes and monocytes, based on differential staining. The myeloid cell lines U937 and HL-60 were obtained from The American Type Culture Collection (ATCC) and maintained in RPM1 supplemented with 10% FBS. CJatokines Human rIFN-7 (sp. act. 1.5 x lO’U/mg) and human rTNFu (sp. act. 3.7 x lO’U/mg) were kindly provided by Genentech, Inc., S. San Francisco, CA. The following cytokines were the generous gift of Immunex Corporation, Seattle, WA: human rIL- Ir and rIL-lb [specific activity for each was IO8 thymocyte mitogenesis U/mg protein (Kronheim et al., 1985)], human rIL-3 (4 x lo7 CFU/mg), human rIL-4 (10’ anti-p co-mitogenesis Ujmg), human rIL-6 (5 x IO4 U/ml yeast supernatant assayed in Cess cell human rM-CSF line Ig proliferation assay), (2 x 10hCFU/mg), human rG-CSF (10’ CFUimg) and human rGM-CSF (4 x 1O’Ujmg). Human rIL-2 (3 x lOh Ujmg) was kindly provided by Cetus Corporation, Emeryville, CA. Cytokine treatment of ej@ctor cells Following isolation, monocytes were cultured at 2 x IO’/ml in RPM1 containing 10% autologous serum for 24 hr with added cytokines. For the studies of in z)itro cultured monocytes, cells were cultured in RPM1 with 10% autologous serum alone for 3 days followed by 24 hr with added cytokines. After isolation, PMNs were cultured at 2 x IOh/ml in medium 199 (Grand Island Biological Co., Grand Island, NY) containing 20% FBS for 24 hr with added cytokines. For the cell lines U937 and HL-60, aliquots of cells at 0.5 x 106/ml were incubated in RPM1 with 10% FBS for 72 hr with added cytokines. In all cases, after incubation with cytokines, cells were washed twice before evaluation for receptor expression and ability to perform ADCC. Cytokine concentrations used for treatment of both the myeloid cells and cell lines were: 100 U/ml for IFN-y treatment, 500 U/ml for TNF treatment, 50 U/ml IL-la and 50 U/ml IL-l/j added together for IL-l treatment, 100 U/ml for IL-2 treatment, 50 U/ml for IL-3 treatment, 50 U/ml for IL-4 treatment, 200 U/ml for IL-6 treatment, 50 U/ml for GM-CSF treatment, 50 U/ml for M-CSF treatment, and 50 U/ml for G-CSF treatment. These arc

Cytokine effects on FcyR saturating concentrations of each cytokine, chosen to give maximal phenotypic and functional responses with minimal effects on cell viability. Antibodies and antibody fragments

The development and properties of 32, a mouse IgGl mAb to FcyRI (Anderson et al., 1986), IV.3, a mouse IgG2 mAb to Fey RI1 (Looney et al., 1986a, 19866) and 3G8, a mouse IgGl mAb to the granulocyte Fey RI11 (Fleit et al., 1982) have been reported. The IgG fraction of ascitic fluid from pristane-primed mice injected with the hybridoma cells was obtained by precipitation with 40% saturated ammonium sulfate. Ion exchange high-performance liquid chromatography (HPLC) on a protein-pak 5PW DEAE column (Waters Chromatography Division, Millipore, Milford, MA) was used to purify the antibodies. The F(ab’), fragments of 32 and 3G8 were made according to the method of Parham (Parham, 1983) by pepsin digestion at pH 3.5 and purified by HPLC gel filtration chromatography using a Bio-Sil TSK 250 column (Bio-Rad, Richmond, CA). Fab’ fragments were obtained by reduction with 1 mM dithiothreitol for 2 hr at 18°C followed by alkylation with 2 mM iodoacetamide for 1 hr at 18°C. The Fab fragment of IV.3 was made as previously described (Looney et al., 1986a) with minor modifications. Briefly, IV.3 antibody at a concentration of 2 mg/ml in PBS containing 10 mM cysteine and 2 mM EDTA was incubated with papain (Sigma, St Louis, MO) at an enzyme to substrate ratio of 1: 25 for 3 hr at 37°C. The reaction was stopped by addition of 20mM iodoacetamide, and the digest was dialyzed against 5 mM phosphate buffer (pH 8.0). The digest was then placed on a protein A-Sepharose 4B column to remove Fc fragments. Fab fragments were prepared from a polyclonal rabbit anti-CE IgG fraction (Cappel, Cooper Biomedical Inc., Malvern, PA) by pepsin digestion at pH 3.5, followed by absorption on protein A-Agarose at pH 8.0 and reduction with 1 mM dithiothreitol (2 hr, 18C) and alkylation with 2.0 mM iodoacetamide (1 hr, 18°C). Heteroantibody formation

Heteroantibodies of anti-Fey R Fab linked to antiCE Fab were made by the following method. AntiFcyR Fab at l-3 mg/ml was treated with an 8-fold molar excess of the bifunctional reagent N-succinimidyl-3-(2 pyridyldithiol) propionate (SPDP) (Pharmacia, Uppsala, Sweden) for 2 hr at 18°C and then dialyzed in PBS (pH 7.4). Anti-CE Fab in 50 mM phosphate and 5 mM EDTA (pH 7.5; phos/EDTA), was treated with a IO-fold molar excess of SATA (N-succinimidyl S-acetylthioacetate; Calbiochem, Behring Diagnostics, San Diego, CA), dissolved in dimethyl formamide, for 30 min at 18°C. After dialysis in phos/EDTA, the SATA-anti-CE Fab was deacetylated by adding hydroxylamine to 0.05 M and 1 hr incubation at 18°C. Hydroxylamine was

59

removed by passage through a 10 ml G-25 Sephadex column (Pharmacia, Piscataway, NJ), the SATAanti-CE Fab mixed at once with an equimolar amount of anti-Fey R Fab, and incubated at 18°C for 4 hr, after which cross-linking was terminated with 1 mM iodoacetamide. Heteroantibodies were dialyzed into PBS and were sterilized by 0.2 pm filtration. Preparations contained less than 15% non-cross-linked Fab, and were at a concentration of 0.7-1.5 O.D.,,,U/ml. Target cells

Hybridoma cell lines expressing on their surface antibodies directed to the human myeloid cell Fey Rs were labelled for 1 hr at 37°C with 200 pCi of [“Crlsodium chromate in normal saline (New England Nuclear, Boston, MA) and used as target cells. Hybridoma cells (HC) expressing high levels of surface Ig were obtained by flow cytometry as previously described (Graziano and Fanger, 1987a) and periodically checked to ensure that high surface Ig expression persisted. HC IV.3 was kindly provided by Drs Anderson and Looney, and HC 3G8 was kindly provided by Dr Jay Unkeless. Chicken erythrocytes (CE) were obtained from venous blood drawn from outbred roosters into preservative-free heparin. Cells were washed twice in medium 199 (GIBCO, Grand Island, NY) with 10% FBS, and 10 ~1 of packed cells were labelled for 1 hr at 37°C with 200 PCi of “Cr. Cells were washed three times in medium 199 with 10% FBS before use. CytoJluorograph analysis

Myeloid cells were washed in PBS containing 0.1% BSA and 0.05% sodium azide (PBS-BSA-AZ). Pelleted cells (106) were mixed with 25 ~1 of purified human IgGl(l0 mg/ml) and with mAb (100 ~1) in the form of culture supernatants. An IgGl antibody of undefined specificity produced by the P-3 myeloma cell line (Kohler and Milstein, 1975) was used as a control. After 1 hr incubation on ice, the cells were washed once with PBS-BSA-AZ and treated for 45 min on ice with 25 ~1 of a 1:20 dilution of FITC-conjugated affinity purified F(ab’), goat antimouse immunoglobulin (Boehringer-Mannheim, Indianapolis, IN). They were then washed, the cell pellet disrupted, and the cells fixed with 250~1 of ice-cold 2% paraformaldehyde in PBS. The Ortho (Westwood, MA) Cytofluorograph System 50H was used to quantify binding of mAb, which is expressed as the mean fluorescence intensity. Autofluorescence of the cells and fluorescence found with binding of the P-3 control mAb have been subtracted for each mAb and condition. Cytotoxicity assays

To quantify direct cytotoxicity of hybridoma cells, equal volumes of medium, “Cr-1abelled hybridoma cells and effector cells were mixed in round-bottomed microtiter wells. Plates were incubated for 6 hr at

D.

60

V. ERBE et al.

37°C after which half of the supernatant was removed and counted for release of 51Cr. Maximal lysis was obtained by addition of 2% sodium dodecyl sulfate in water. Per cent cytotoxicity was calculated as 100 x (counts released with effecters - spontaneous release)/(maximal lysis - spontaneous release). In all experiments. tests were conducted in triplicate and the results are expressed as the mean + standard deviation. To quantify rabbit anti-CE IgG-dependent or heteroantibody-dependent cytotoxicity of CE, equal volumes (50 ~1) of 5’Cr-labelled CE at 5 x 105/ml, effector cells at an E/T ratio of 5: 1, and antibody (either rabbit anti-CE IgG or the various heteroanti-

bodies) at the concentrations indicated were mixed in round-bottomed microtiter wells. Controls for the effects of antibody alone, and effector cells alone, were included in all experiments. Maximal lysis was obtained by the addition of 100 ~1 of 2% sodium dodecyl sulfate in water to 50~1 of CE. Plates were incubated for 18 hr at 37X, after which 50% of the supernatant was removed and then counted for release of “Cr. Per cent cytotoxicity was calculated as

100 x (counts

released

with

effecters

and

(A) 500 1

MAb 32

MAb 32

anti-

body - spontaneous release)/(maximum lysis spontaneous release). The data shown are expressed as the mean of triplicates with a standard deviation of less than 20% of the mean value. Results shown

MAb IV.3

MAb IV.3

MAb 3G8

Fig. 1. FcyR expression by monocytes and PMNs after cytokine treatment. Three-day cultured monocytes (A) or freshly prepared neutrophils (B) were cultured in the cytokines indicated for 24 hr as in Materials and Methods, washed and incubated on ice with 25 ~1 of human IgGl (12 mg/ml) and 100 ~1 of culture supematant from the mAbs indicated. After washing, the cells were treated with FITC-GAM F(ab’),. Data are shown as the mean fluorescence intensity and are representative of experiments with cells from at least three different donors.

Cytokine

are representative of experiments performed cells from at least three different donors.

effects on

with

61

FcyR

Effect of cytokines on the expression of the difSerent

FcyR on myeloid cells (Figs 1 and 2) As previously reported (Guyre et al., 1983; Perussia et al., 1983b; Petroni et af., 1988), IFN-y treatment

Statistical analysis

Each value represented is the average of two to three measurements. Statistical significance of differences in values obtained following different cytokine treatments was determined using an analysis of the variance and Student’s t-test. P < 0.05 was considered significant. RESULTS

Previous studies from our laboratory have described the expression and cytotoxic potential of the three types of Fey R on human myeloid cells and cell lines before and after treatment with IFN-y (Shen et al., 1989). In this report, we present data on a systematic evaluation of the effects of most of the other available recombinant cytokines, including IL-l, IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, M-CSF and TNF on the expression of each of the Fey R on human myeloid cells and on their cytotoxic capabilities.

markedly up-regulated FcyR1 on all myeloid cells and cell lines, but had no effect on the expression of Fey RI1 or Fey RIII. None of the other cytokines tested affected expression of any of the FcyR on monocytes (Fig. la). On PMNs, none of the other cytokines up-regulated FcyR expression. Although G-CSF treatment induced a small increase in expression of Fey RI on the PMNs of some donors, analysis of the variance showed that this effect was not statistically significant. Mendel et al. (1988) reported a marked drop in PMN expression of Fey RI11 following TNF treatment, which was seen to a lesser degree in these experiments (Fig. lb). Interestingly, of the cytokines examined, only TNF down-regulated Fey R expression and then only Fey RI11 on PMNs. Liesveld et al. (1988) reported increased expression of Fey RI1 on U937 cells after GM-CSF treatment, which was enhanced by co-culture with G-CSF. In our hands, slight increases in Fey RI1 on U937 cells were noted (Fig. 2a); however, these data were not

(4

300 1

MAb 32

200 ,

MAb IV.3

(W MAb IV.3

Fig. 2. Fey R expression by myeloid cell lines after cytokine treatment. Aliquots of cells from the U937 cell line (A) or HL-60 cell line (B) were cultured in the cytokines indicated for 72 hr as in Materials and Methods, washed and incubated on ice with 25 ~1 of human IgGl (12 mg/ml) and 100 ~1 of culture supernatant from the mAbs indicated. After washing, the cells were treated with FITC-GAM F(ab’),. Data are shown as the mean fluorescence intensity and are from at least three experiments.

D. V. ERBE et al

62

consistent and analysis of the variance they were not significant.

Donor 1 80 1

2

4

6

I 4

1 6

Donor 2 80 7

60 -

40 -

20 -

I 0

I 2

TlmeCdays)

Fig. 3. Hybridoma cell killing by monocytes decreases with time in culture. Monocytes from two donors (representative of 10) were placed in culture on day 0. On the days indicated, aliquots of cells were removed, washed and assayed for their ability to lyse HC 32 (closed squares) and HC IV.3 (open squares). 5’Cr-labelled HC were incubated

for 6 hr with monocytes at an E:T of 20: 1. Tests were performed in triplicate and values for each day are per cent cytotoxicity + SD.

We have previously shown that monocytes cultured for 24 hr or less are able to efficiently kill hybridoma cell lines bearing antibody to FcyRI and Fey RI1 (Graziano and Fanger, 1987b). In the present study on the ability of various cytokines to augment monocyte cytotoxicity through FcyR, HC lysis by monocytes treated for 24 hr with IL-I, IL-2, IL-3, IL-4, IL-6, M-CSF, G-CSF, GM-CSF or TNF was similar to killing by untreated or IFN-y-treated monocytes (data not shown). This high level of cytotoxicity of HC by untreated or cytokine-treated monocytes was consistent with the possibility that freshly obtained peripheral blood monocytes already expressed maximal killing ability, perhaps because of partial activation. Therefore. to evaluate cytokine effects on monocyte-mediated cytotoxicity of HC more adequately, we examined killing under suboptima1 conditions. One such approach resulted from our examination of the effect of in citro culture on the ability of monocytes to lyse HC through Fey RI and Fey RI1 (Fig. 3). Although freshly isolated monocytes (day 0) lysed both the HC line bearing antibody directed to Fey RI (HC 32) and the HC line bearing antibody to Fcl;RlI (HC IV.3), after 24 days in culture their ability to kill both HC 32 and HC IV.3 had decreased. The kinetics associated with this decrease in killing were donor-dependent-for some donors killing of HC 32 decreased sooner than that of HC IV.3; for others HC IV.3 killing decreased first. By

(B) Killing of HC IV.3

(A) Killing of HC 32

80 1

that

Effect oJ’ cytokines on the capability of the deferent FcyR on myeloid cells to mediate killing of selfdirected target cells

0

0

showed

Donor 1

Donor 2

6o 1

Donor 1

Donor 2

Fig. 4. Cytokines activate cultured monocyte killing of HC. Monocytes from two representative donors were cultured for a total of 4 days with the indicated cytokines added on day 3 of culture as in Materials and Methods. They were then washed and assayed for their ability to lyse (A) HC 32 or (B) HC IV.3, for 6 hr with compared with when they were freshly isolated (day 0). 5tCr-labelled HC were incubated monocytes at an E:T of 20: 1. Tests were performed in triplicate and values for each day are per cent cytotoxicity 5 SD. *P < 0.01 compared with untreated cells.

Cytokine

63

effects on FcyR

day 4 in culture, however, killing of both HC 32 and HC IV.3 had diminished significantly for all donors. Using cultured monocytes which mediated low levels of cytotoxicity, the ability of various cytokines to restore FqR-mediated killing was analysed. Monocytes placed in culture for 3 days were treated with or without cytokines for 24 hr and tested for their ability to kill through Fey RI and FqRII (Fig. 4). IFN-7~ treatment restored killing of both HC 32 and HC IV.3 to the level seen with freshly isolated cells (day 0). TNF and GM-CSF treatment also enhanced killing by cultured monocytes through both Fey RI and Fey RII, but to a lesser extent than IFN-y treatment. In contrast, IL-I, IL-4 and IL-6 treatment of monocytes did not restore their killing ability (Fig. 4) nor did treatment with M-CSF, G-CSF, or IL-3 (data not shown). Levels of expression of FcyRI and Fc?;RII were unchanged on treated cells except for Fey RI induction on IFN-y-treated cells (Fig. la). In previous studies, we have found that PMNs did not lyse HC unless activated with IFN-y, GM-CSF or TNF (Graziano et al., 1989; Fanger et al., 1989). IFN-y activated PMNs to lyse HC through Fey RI and FqRII, whereas GM-CSF and TNF activated PMNs to kill HC only through Fey RII. Here we report that G-CSF activated PMNs to kill HC through Fey RII, whereas M-CSF, IL-l, IL-2, IL-3, IL-4 and IL-6 did not activate PMNs to lyse HC through Fey RI1 (Fig. 5). Additionally, none of the cytokines examined enabled PMNs to lyse HC through Fey RI11 (Fig. 5).

In previous studies, we have found that the cell lines U937 and HL-60 did not lyse HC even after activation with IFN-y (Shen et al., 1989). In the present study we found that none of the other cytokines tested, including IL-l, IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, M-CSF and TNF, were able to induce the HL-60 or U937 cell lines to lyse HC (data not shown). Chicken

erythrocyte

killing

Considering the array of cytotoxic activities associated with myeloid cells, it is possible that different activities may be induced by different cytokines. As some evidence exists that Fey R-mediated cytotoxicity of HC targets may involve a mechanism different from that for killing of erythrocytes (Shen et al., 1989), we examined the ability of the various cytokines to modulate Fey R-mediated killing of chicken erythrocytes. To initially identify cytokines other than IFN-y which could enhance ADCC of CE by myeloid cells, killing assays were performed in the presence of rabbit anti-CE IgG. Of the cytokines tested only IFN-y enhanced monocyte killing of CE coated with rabbit anit-CE IgG (Fig. 6a). For PMNs, IFN-?/ or GM-CSF activation enhanced their lysis of CE, as before (Graziano et al., 1989), whereas TNF, M-CSF, G-CSF, IL-l, IL-2, IL-3, IL-4, and IL-6 treatment did not affect CE lysis by PMNs (Fig. 6b). For the cell lines, however, in addition to IFN-y, only IL-6 treatment permitted U937 cells to lyse CE coated with rabbit anti-CE IgG (Fig. 6c), whereas only G-CSF activated HL-60 cells to kill CE (Fig. 6d). This assay did not indicate which receptor on treated cells triggered lysis, however, as the rabbit UIlbW.td IFN-g IgG may bind all three human Fey R. E HC 32 Therefore, to determine which FcyR on IL-6n3 n4 treated U937 cells and G-CSF-treated HL-60 cells IL6 triggered lysis, heteroantibodies specific for each O-CSF M-CSF FcyR were used which consisted of anti-FcyR Fab untreated linked to anti-CE Fab. As Fig. 7 shows, IFN-ym-g treated U937 cells kill CE in the presence of either IL1 IL2 Fab 32 x Fab anti-CE or Fab IV.3 x Fab antiCE, Ei indicating that CE lysis occurred through both Fey RI IL6 G-CSF and Fey RI1 on IFN-y-treated U937 cells. On the M-CSF other hand, IL-6-treated U937 cells killed CE only in UnoeaIal the presence of Fab 32 x Fab anti-CE (Fig. 7). Thus, m‘l-S IL1 U937 cell lysis of CE following IL-6 activation occurs IL2 HC 3GS only through Fey RI. U937 cells killed CE through IL3 IL.4 Fey RI to a lesser extent following IL-6 activation L-6 C-CSF than IFN-y activation. This is consistent with the M-CSF I I I 1 observation that, whereas IFN-y treatment dramati0 10 20 30 40 cally up-regulated Fey RI expression on U937 cells, IL-6 treatment did not change FqRI expression Cytotoxicity (%) Fig. 5. G-CSF-activated PMNs kill HC through Fey RJI. (Fig. 2a). G-CSF-activated HL-60 cells, like IFN-y -activated PMNs isolated from a representative donor (of three) were cultured in the cytokines indicated for 24 hr as in Materials HL-60 cells, killed CE through both Fey RI and and Methods, washed and assayed for their ability to lyse Fey RI1 as killing occurred in the presence of either HC 32. HC IV.3 and HC 3G8. 5’Cr-labelled HC were Fab 32 x Fab anti-CE or Fab IV.3 x Fab anti-CE incubated for 6 hr with PMNs at an E:T of 20: 1. Tests were (Fig. 8). However, G-CSF-activated HL-60 cells did performed in triplicate and the standard deviations were less than 10% of the mean values. not kill CE through Fey RI or Fey RI1 to the level seen

F

I

&I

D. V. ERBE et ul.

upon IFN-y activation G-CSF enhancement of Fey RI1 occurred without either Fc receptor (Fig.

of these cells. Of interest, killing through Fey RI and changes in the expression of 2b).

DISCUSSION

Using monoclonal antibodies to each of the human IgG Fc receptors, the hybridoma cells producing them, and heteroantibodies made by linking them to anti-CE antibodies, we have investigated the effect of various cytokines on expression of Fey Rs on myeloid cells and cell lines and killing mediated through Fey R on these cells. The use of Fey R-specific reagents allowed dissection of cytokine effects on ADCC in a manner which elucidated which of the receptors was involved, and to what extent. The experiments presented here confirm and emphasize the unique role of IFN-7 in regulating FcyR expression. IFN-1/ treatment led to a marked increase in expression of Fey RI on monocytes, PMNs and the myeloid cell lines HL-60 and U937. In fact, of the

other cytokines examined, including IL-I, IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, M-CSF and TNF, only IFN-), markedly up-regulated Fey R expression and then only FcyRI. Also confirmed in these studies was the marked drop in Fey RI11 upon TNF stimulation of PMNs reported by Mendel ef al. (1988). This was the only significant decrease in FcyR expression following cytokine treatment noted in these studies. Although Fey R expression was not altered significantly by cytokines other than IFN-y, significant effects of other cytokines were seen in killing of both CE and HC. Though only IFN-y augmented ADCC of erythrocyte targets by monocytes, when HC lysis was evaluated, stimulation was seen with GM-CSF and TNF as well as IFN-y. To see cytokine effects on HC killing by monocytes, it was necessary to culture the monocytes for 3-4 days. Fresh or 24-hr cytokinetreated monocytes killed HC targets bearing high levels of surface Ig to the same (apparently maximal) level. Culturing monocytes for a short period (3-4 days) allowed killing to be evaluated under sub-maximal conditions-as monocytes began to differentiate

(A) Monocytes

(B) PMNs

80 1

60

0.01

1

0.1

Concn of sensitizing

10

Concn of sensitizing

Ab (pg/ml)

1

0.1

Concn of sensitizing

Ab @g/ml)

(D) HL-60

(C) u937

0.01

1

0.1

10

Ab (pg/ml)

Concn of serwtizing

Ab (pg/ml)

Fig. 6. Myeloid cell lysis of CE sensitized with rabbit anti-CE IgG. Monocytes (A), PMNs (B), U937 cells (C) and HL-60 cells (D) were cultured in medium alone (closed squares), IFN-y (open squares), IL-1 (closed diamonds),

IL-3 (plus signs), IL-4 (closed triangles), IL-6 (open triangles), (open circles), as in Materials and Methods. After washing, cells were tested for performance of ADCC using S’Cr-labelled CE sensitized with rabbit anti-CE IgG at the concentrations indicated. Effector to target ratio was 5 : 1.

GM-CSF (X

IL-2 (open diamonds),

marks), M-CSF (closed circles), or G-CSF

10

Cytokine

65

effects on FcyR

0.01

32 x anti-CE (O.D./ml)

32 x anti-CE (O.D./ml)

50 1

_

40-

,\"

O.Obl

O.dl

0.;

IV.3 x anti-CE (O.DJml)

h c .o-

30-

s

20 -

0 K 0

10 -

00

0.001

0.01

0.1

IV.3 x anti-CE (O.DJml)

Fig. 7. IL-6-treated U937 cells kill CE only through Fcp RI. U937 cells cultured for 72 hr in medium alone (plus signs), IFN-y ( x marks). IL-I (open circles) or IL-6 (closed circles), as in Materials and Methods, were washed and assessed for performance of ADCC against “Cr-labelled CE in the presence of Fab 32 x Fab anti-CE or Fab IV.3 x Fab anti-CE at the concentrations indicated. Effector to target ratio was 5 : I.

Fig. 8. G-CSF-treated HL-60 cells kill CE through FeyRI and FeyRII. HL-60 cells cultured for 72 hr in medium alone (plus signs), IFN-), ( x marks), IL-3 (open circles) or G-CSF (closed circles), as in Materials and Methods, were washed and assessed for performance of ADCC against 5’Cr-labelled CE in the presence of Fab 32 x Fab anti-CE or Fab IV.3 x Fab anti-CE at the concentrations indicated. Effector to target ratio was 5: I.

into macrophages in tlitro, their ability to mediate Fey R-dependent HC killing appeared to decrease. Consistent with this finding, a number of functional changes have been noted to occur as monocytes differentiate in vitro, including a drop in reactive oxygen species (superoxide anion and hydrogen peroxide) production by monocytes (Nakagawara et al., 1981). In these studies, IFN-y treatment consistently restored killing of the anti-Fey RI hybridoma (HC 32) and the antiFcyRI1 hybridoma (HC IV.3) by cultured monocytes to the (maximal) level seen with fresh monocytes. GM-CSF or TNF treatment also restored killing, though not as well as IFN-y. In the case of cultured monocyte killing of HC 32, the greater effect of IFN-y treatment over GM-CSF or TNF treatment could be a result, in part, of increased Fey RI expression on IFN-y-treated cells. However, in the case of cultured monocyte killing of HC IV.3, the greater effect of IFN-y treatment must occur at another level, as none of these cytokines altered Fey RI1 expression. Evaluation of cytokine effects on PMN-mediated ADCC revealed that only treatment with IFN-y induced killing of HC or CE through Fey RI on PMNs. This finding is consistent with the fact that

only IFN-y treatment led to significant expression of Fey RI by PMNs. On the other hand, G-CSF was shown to enable PMNs to kill HC through Fey RII, which adds it to the list of GM-CSF, TNF and IFN-y, which we have shown to induce PMNs to lyse HC through this receptor. Erythrocyte killing through Fey RI1 on PMNs, however, was enhanced only after treatment with IFN-y or GM-CSF, and not G-CSF or TNF. This finding is consistent with our observation that the mechanisms involved in CE killing appear to be distinct from those involved in HC killing. Along those lines, PMNs never killed HC through the phosphatidyl-inositol glycan-linked Fey RI11 (Simmons and Seed, 1988; Selvaraj et al., 1988) after treatment with any of the cytokines tested, although Fey RI11 on PMNs has been shown to trigger lysis of CE (Shen et al., 1989). The cell lines U937 and HL-60 were unable to lyse any of the Fey R-bearing hybridomas after treatment with any of the cytokines tested. These cells lines appear to lack the cytotoxic mechanism required for HC lysis, and none of these cytokines could independently induce this killing. Even when combinations of cytokines were used to treat the cell lines, such as

66

D. V. ERBE et al.

GM-CSF with IFN-y or M-CSF with IFN-7, no Iysis of HC occurred (data not shown). The cell lines U937 and HL-60 do have the capability to lyse erythrocyte targets, however, but only following cytokine activation. Previously, only IFN-y was thought to be capable of activating either U937 cells or HL-60 cells to lyse erythrocyte targets (Shen et al., 1989). Here, we demonstrated that IL-6 treatment enabled U937 cells to lyse CE, and that G-CSF treatment enabled HL-60 cells to lyse CE. IL-6-activated U937 cells killed CE only through Fcl;RI, whereas C-CSF-activated HL-60 cells killed CE through both Fc;) RI and Fey RII. Interestingly, in both cases (IL-6-treated U937 cells and G-CSF-treated HL-60 cells) the effector cell gained the ability to perform ADCC without altering Fey R expression. Thus, this system is ideally suited for dissecting induction of ADCC into mechanisms which either correlate with increased FcyR expression or not. ADCC through FciRI on U937 cells after IFN-y activation is concomitant with increased expression, whereas ADCC through Fey RI on U937 cells following IL-6 activation is not. The same is true for ADCC through Fey RI on IFN-),-activated HL-60 cells vs G-CSF-activated HL-60 cells. The molecular mechanisms of these two findings could shed interesting light on the requirements for mediating cytotoxicity. Are adhesion molecules up-regulated on IL-6-treated U937 cells or G-CSF-treated HL-60 cells? Is intracellular trafficking altered in these cells? Is more superoxide generated per FcyR bound? Findings with these cell lines can then be compared with cases of increased ADCC in the absence of receptor induction by normal peripheral blood cells-including ADCC by GM-CSF- or TNF-activated monocytes, and GMCSF-, G-CSF or TNF-activated PMNs. The cytokine treatment protocol used in this study deserves comment. One can argue that when a negative result was reported for a particular cytokine and effector cell, perhaps an effect would have been seen if a greater concentration had been used or the incubation period had been longer. However, saturating concentrations were used for each cytokine. Additionally, for all of the cytokines used, internal controls for activity were evaluated. For example, although IL-4-treated monocytes did not display increased FcyR expression or ADCC, they did display increased class II antigen expression (not shown) as has been reported (Te Velde rt al., 1988). Also, the time of treatment was kept relatively short to minimize secondary effects of cytokine treatment. This is especially pertinent with regard to monocyte treatment, as one cannot rule out the possibility of a secondary effect due to an induced cytokine from contaminating T cells. Whether the effects noted here were caused directly by the cytokine used in each treatment cannot be answered with these data. However, these effects were still independent of IFN-;I, as, if IFN-7 was involved, one would expect to see induction of Fe/RI expression. In fact, increased

Fey RI expression was not seen on GM-CSFor TNF-activated monocytes, IL-6-activated U937 cells or G-CSF-treated HL-60 cells. In conclusion, although only IFNy appeared to significantly affect Fey R expression on myeloid cells. other cytokines, including GM-CSF, G-CSF, TNF and IL-6, enhanced Fey R-dependent killing by myeloid cells. That cytokines other than IFN-; can induce killing by myeloid cells which is mediated through specific FcyR helps to define the range of conditions associated with activation, and is an important step in dissecting the molecular mechanisms involved in induction of cytotoxicity. Acknowledgemenrs-This work was supported by NIH grants AI 19053, CA 44794, and AI 22816. IFN-y and TNFa were the generous gift of Genentech, Inc. IL-2 was kindly provided by Cetus Corp. The following cytokines were kindly provided by Immunex Corp.: IL-la, IL-ID, IL-3. IL-4, IL-6, GM-CSF, G-CSF and M-CSF. The cytofluorograph was the generous gift of the Fannie Rippel Foundation, and is partially supported by the core grant of the Norris Cotton Center (CA 23108). REFERENCES

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