Eur. J. Immunol. 1990. 20: 2137-2140

Binding of monomeric IgG to macrophages

2137

Short paper Robert KellerO, Ruth Keisto, HervC BazinO, Peter Jollera and Peter H.Van der Meidev

Binding of monomeric immunoglobulins by bone marrow-derived mononuclear phagocytes; its modulation by interferon-y*

Immunobiology Research Group, Institute for Immunology and Virologyo, University of Zurich, Zurich, Experimental Immunology Unit", University of Louvain, Bruxelles, ANAWA Laboratories I w a , Wangen and Section Immunomodulationv, TNO Institute of Applied Radiobiology and Immunology, Rijswijk

The ability of resting and activated rat bone marrow-derived mononuclear phagocytes (BMMQ) to bind monomeric rat, mouse, and human IgG was determined by means of flow cytometry. Rat IgG2b bound with high affinity (Kd E 3 X M); binding was optimal at 4°C and was only little affected by trypsin treatment.The other IgG bound with only low affinity (rat IgGz,, mouse and human IgG) or not at all to rat BMMQ (rat IgGl, rat IgG2J.The binding of rat IgG2b was not affected by the presence of a surplus of low-affinity binding IgG, and vice versa, indicating that high- and low-affinity IgG bind to different sites. Binding of high- and low-affinity IgG as well as expression of MHC class I1 molecules and of tumoricidal activity by BMMQ was markedly enhanced by rat interferon-y in low concentration (0.1 to 1.0 IU IFN-y/ml). On the other hand, heat-killed Corynebacterium parvum organisms, that were equally potent in triggering tumoricidal activity, neither enhanced the binding of IgG nor the expression of MHC class I1 molecules by BMMQ, suggesting that these abilities are not necessarily closely related phenomena.

1 Introduction

2 Materials and methods

The ability of cells of the immune system to recognize foreign matter, to interact with each other and/or to respond to cytokines is fundamental for the operation of the immune defense; it depends largely on their specific structure, in particular the expression of specific receptors. The presence of receptors for the Fc moiety of antigenbound IgG on the surface of professional phagocytes, established since the late 1960s [l], facilitates the clearance of soluble and particular complexes in vivo and may have a role in the,initiation and regulation of the immune response and in the mediation of natural and antibody-dependent cellular cytotoxicity [2, 31. With the advent of mAb technique, the complexity of the FcyR family has become evident. In humans, mice, rats and guinea pigs, multiple FcyR have been identified but many questions regarding their structure and function remain open [3-81. Various studies indicate that monomeric and complexed IgG can bind to different receptors. For practical reasons the large majority of investigations dealt with binding of complexed IgG. Here, we have measured by means of FCM the direct binding of monomeric homologous and heterologous IgG to pure resting rat BM-derived mononuclear phagocytes (BMMQ) and its modulation by IFN-y and heat-killed bacteria.

2.1 IgG

[I 85751

*

The work of the Immunobiology Research Group was supported by the Swiss National Science Foundation (grants 3.336.86 and 31.25717.88) and the Canton Zurich.

Rat myeloma proteins IgG1,x (IR595), IgG2,,x (IR418), I g G t b , ~(IR863), and IgGz,,x (IR304), isolated from ascites fluid of LOU/M/Wls rats bearing the corresponding immunocytomas [9, lo], and rat IgGZb,x anti-DNP mAb, LO-DNP-11 [ ll] , were the rat IgG included in this study. Heterologous IgG were from Sigma Chemical Co., St. Louis, MO: purified mouse myeloma IgGl,x (M 9269), IgG2,,x (M 9144) and IgG2b,~(M 8894), and purified mouse (I 5381) and human IgG (I 4506). For some experiments, IgG were heat aggregated (60 min at 63 "C). 2.2 BMMm 2.2.1 Preparation of BMMQ,

BMMQ were obtained and cultured as previously described [12, 131. Briefly, BM cells separated from femurs of male inbred DA rats (RTla) and suspended in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% FCS (Gibco, Grand Island, NY), 5% horse serum (Gibco), and antibiotics (50 U/ml penicillin, 50 pg/ml streptomycin), and conditioned with SN (final concentration 10%) from strain L clone 929 cells (ATCC CCLl), were cultured in bacteriologic plastic petri dishes (diameter 100 mm). On day 6, the cells remaining adherent were removed with ice-cold Ca2+-, Mg2+-free PBS, washed, and suspended in IMDM supplemented with 5% FCS and antibiotics.These BMMQ that are homogeneous with regard to the cell lineage [12, 141, were the source of effector cells.

Correspondence: Robert Keller, Immunobiology Research Group, Institute for Immunology and Virology, University of Zurich, Birchstr. 95, CH-8050 Zurich, Switzerland

2.2.2 Activation of BMMm

Abbreviations: BMMa: Bone marrow-derived mononuclear phagocytes CP: Corynebacterium parvum rIFN-y: Recombinant rat interferon-y IMDM: Iscove's modified Dulbecco's medium

Resting d a y d BMMQ were incubated for various time intervals (standard 24 h) with medium alone or with medium supplemented with one of the MQ-activating agents before their abilities to bind IgG, to express MHC

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

0014-2980/90/0909-2137$3.50+ .25/0

2138

Eur. J. Immunol. 1990. 20: 2137-2140

R. Keller, R. Keist, H. Bazin et al.

class I1 molecules, and/or to manifest tumoricidal activity were assessed. In experiments in which incubation was for a prolonged period of time, medium (including supplements) was replaced every 48 h. Ma-activating agents (recombinant rat interferon-y, rRIFN-y; heat-killed Corynebacrerium parvum organisms, CP) were obtained as described [13, 151. 2.2.3 Trypsin treatment of BMMQ,

BMMQ were trypsinized for 20 min at 370c (5 mg/ml trypsin Gibm in ImM). Afterwards, the enzyme activity was neutralized with an equivalent amount of soybean trypsin inhibitor (Sigma), and were washed at oc with PBS before the binding assay.

3.2 Binding of monomeric IgG by resting BMMa

2.3 Binding of IgG Binding of IgG to the surface of B M M a was measured by means of FCM as described previously [14]. Under standard conditions, 105 BMMWml were incubated for 60 min at 4°C with IgG, washed twice, fixed with paraformaldehyde (1% ,20 min at 4 "C), washed twice, and incubated for a further 60 min at 4°C with affinity-purified PE- or FITC-conjugated rabbit anti-rat, anti-mouse or anti-human IgG F(ab')z (Serotec, Blackthorn, Bicester, GB), and washed twice. Binding was quantitated in a fluorescenceactivated cell system (FACS analyzer IFA, Becton Dickinson, Sunnyvale, CA). A minimum of 104 cells/sample was measured; electrical cell volume, 90" light scatter, log fluorescence 1 and log fluorescence 2 of each cell was determined. Data were collected in a list mode with a Hewlett Packard 9217 computer with harddisk data acquisition; statistical evaluation was done with the Consort 30 and the experimental software FACSDRAW (Becton Dickinson). For each experiment, the percentage of cells expressing a particular marker was determined using nonrectangular gates: these were comDared with the values from a negative control population. Three to five determinations were performed for each type of experiment. V

Y

identified as mononuclear phagocytes by their light and electron microscopic appearance, cytochemical staining for esterase, lysozyme secretion, the capacities to bind IgGcoated sheep erythrocytes, to ingest particles, and to evolve tumoricidal activity on incubation with I FN y or bacteria, by their autofluorescence and FCM characteristics [12-141. These cells were homogeneous with respect to the cell lineage; in particular, no lymphocytes could ever be detected. These resting day-6 BMMQ did not express spontaneous tumoricidal activity against P815 mastobytoma cells; however, their responsiveness to a variety of stimuli and their capacity to bind monomeric IgG could vary considerably from one experiment to another (not shown).

Binding of monomeric IgG by resting BMMQ also varied depending on the temperature during their interaction. As binding decreased with increasing temperature and was complete by 30 to 45 min (not shown), an incubation period of 60 min at 4 "C was chosen as a standard. The proportion of BMh4a that bound Ig varied depending on the IgG type and concentration. At low concentration (1-10 yg/ml), only rat IgG2b myeloma protein and/or mAb bound to a considerable although variable portion of resting BMMQ (range 21% to 66% positive cells). At much higher concentration, rat IgG2a,mouse and human IgG bound to a minor portion of resting BMMQ (Table 1). For monomeric rat IgGl and IgGzc, and for mouse IgGl, IgG2, and IgG2b myeloma proteins, no binding was ever detected. Binding of heat-aggregated IgG was in the same range as for monomeric IgG (not shown). 3.3 Binding of monomeric IgG to BMMQ,is enhanced by IFN-y but not by CP

I

2.4 Expression of MHC class I1 molecules BMMO were first incubated for 60 min at 4 "C in IMDM containing 2% FCS and saturating amounts of anti-MHC class I1 mAb (MRCOX6. Serotec). washed twice. and incubated for afurther 60 min at 4"Cwith affinity-purified PE-conjugated anti-mouse IgG F(ab')z before analysis by FCM [16].

Incubation of BMMQ with rRIFN-y enhanced binding of the monomeric IgG that were alreadv bound bv resting BMMQ but not %f other IgG. Rat igGzb again clearly differed from the other IgG as it readilv bound to a large proportion of IFN-y-activited BMMQ. i n BMMQ that hid been incubated for 24 h with 5 IU IFN-ylml, binding of rat Tab,e 1. Binding of monomeric rat,

2.5 Assay of tumoricidal activity

Type of IgG

Tumoricidal activity expressed by resting and by activated BMMQ derived from the same pool as utilized in the binding assays was determined in a 36-h [14C]dThd assay with prelabeled P815 murine mastocytoma cells as a target [13]. In some experiments, quantification of lactate dehydrogenase (LDH) release from tumor cells was performed in parallel with the [14C]dThd-release assay [17]. The two assays yielded corresponding results.

Rat IgG2b (IR863) Rat IgGZb (LO-DNP-11) Rat IgG?, (IR418) Rat 1gGzc (IR 304) Rat IgGl (IR595) Mouse IgG Human IgG

3 Results 3.1 General remarks The adherent cells harvested on day 6 after initiation of the culture of BM cells in L cell-conditioned medium were

and human IgG by

resting rat BMMQa) Percent positive cells

45 (k 24) 49 ( f 18) 23(+ 7) 1 ( + 1) O ( f 2) 21(+ 8) 19(+ 7)

a) Day6 BMMQ were cultured for 24h in IMDM, washed, interacted for 60 min at 4°C with IgG preparations, washed, and interacted for a further 60 min at 4°C with the corresponding PE-labeled anti-IgG F(ab')* antibody before cell-bound IgG was measured. IgG concentrations: 10 pg/ml for IgGzb, 100 pg/ml for all other IgG. Values are means ( f SD) from 4 to 10 experiments.

Eur. J. Immunol. 1990. 20: 2137-2140

Binding of monomeric IgG to macrophages

/

/ 0.5 1 2.5 5

10

50

25

Figure I. Comparative abilities of resting and IFN-y-activated BMMQ to bind monomeric IgG. Rat BMMQ were incubated for 24 h at 37°C in medium alone (resting BMh4@; open symbols) or in medium supplemented with rIFN-y (5 IU/ml; activated BMh4Q; closed symbols) before their interaction with various concentrations of IgG (60min at 4°C). (A) Rat IgGI; ( 0 ) rat IgG2,; (0)rat IgGzb (IR863); (R) rat IgG2c;(0)mouse IgG; ( 0 ) human IgG. IgG concentrations utilized in the experiments with resting BMMQ (controls; C): 5 pg/ml for rat IgGZb; 100 pg/ml for all other IgG. The values are derived from one typical experi-

HA

/ C

2139

l$pg

ment. 100

-

9080

-

70-

", 60Y Y

t

-

M-

w

f

40-

r

"

3020

-

I,

,

24

48

72

96

n

TIM€ OF INCUBATION

Figure 2. Kinetics of binding of monomeric IgG by resting (open symbols) andor IFN-y-activated BMM@ (closed symbols).

BMMQ were cultured for the time interval indicated before their interaction with IgG. (0)Rat IgGzb(IR863, 5 pg/ml); (0) human IgG (100 pg/ml); (0)mouse IgG (100 pg/ml). Medium change (with or without IFN-y; 5 IUlml) every 48 h.The values are derived from one typical experiment.

701 60

IgGZb was saturable at 10 pg/ml; half saturation of IgGzb receptors was achieved in various experiments at approximately 0.5 pg/ml rat IgG2b, indicating a Kd value in the range of 3 x M; it is noteworthy that binding of the two IgGZb preparations included, IgGzb myeloma protein (IR863, Fig. 1) and IgGZb mAb (LO-DNP-11; not shown), was within the same range. The binding of rat IgG2, and mouse and human IgG by BMMQ,was as well enhanced by IFN-y but the Ig concentrations required were much higher than for rat IgGZb (Fig. 1). I F N y (5 IU/ml) and CP (200 pg/ml) were equally potent of inducing in BMMQ, tumoricidal activity against a variety of tumor targets (not shown); however, incubation with CP did not augment the binding of monomeric IgG to B M Q , (not shown). In resting BMMQ, that were cultured for 24, 48, 72 and/or 96 h in normal medium, the ability to bind rat IgG2b and human or mouse IgG slightly increased as incubation was continued (Fig. 2). In the presence of rIFN-y, enhancement of binding of IgG reached a maximum after 48 h and, for rat IgGzb, remained for at least 96 h at a clearly elevated level (Fig. 2). Enhancement of IgG binding by IFN-y was clearly dependent on the actual IFN-y concentration. Concentrations of 0.5 to 1.0 IU/ml rRIFN-y sufficed to induce in B M Q , within 24 h submaximum to maximum binding of rat IgGZb and human IgG, to trigger tumoricidal activity (not shown) and the expression of MHC class I1 molecules on virtually all BMMQ,(Fig. 3). In most experiments, concentrations of 2.5 to 25 I U IFN-ylml were not more efficient in this respect, and concentrations of 50 IU/ml and more appeared to be toxic for BMMQ, (not shown).

t /,--I

3.4 Properties of the FcyR for monomeric rat IgGB on rat BMM@ The findings showing that 0.5 p g h l rat IgG2b suffice to obtain half saturation of IgG2b receptors (Fig. 1) are C 0.01 0.1 0.5 1.0 2.5 5.0 I U IFHr/ml evidence for the existence of a specific, high-affinity FcyR 0.05 for rat IgG2b on rat BMMQ,. In competition experiments, Figure 3. Dependence of the abilities of B W @to bind IgG or to the binding of monomeric rat IgG2b was not inhibited by enhance expression of MHC class I1 molecules on the concentra- monomeric or heat-aggregated human or mouse IgG tion of IFN-y. BMM@were incubated for 24 h at 37 "C in medium alone (controls, open symbols) or in medium supplemented with (100-250 pg/ml), and vice versa (data not shown). rIFN-y (closed symbols) in the concentrations indicated before binding of IgG and expression of h4HC class11 molecules was Pretreatment of BMMQ,with trypsin resulted in somewhat determined. (0)Rat IgGZb(5 pg/ml); (0) human IgG (100 pglml); diminished IgGZb binding; in five experiments, inhibition of (0) MHC class I1 expression. binding ranged from 15% to 39% (mean 28%).This rather

2140

Eur. J. Immunol. 1990. 20: 2137-2140

R. Keller, R. Keist, H. Bazin et al.

limited diminution in IgGzb binding after enzyme treatment suggests that the FcyR for IgGzb on rat BMMQ is not trypsin sensitive. Experiments, in which MHC class I1 molecules andor cell-bound IgG were cross-linked with anti-IgG F(ab’)2 antibodies, revealed that expression of MHC class11 molecules by BMMcD was not affected by cross-linking of cell-bound IgG, and vice versa suggesting independent regulation of the expression of these molecules (not shown).

4 Discussion Monomeric IgG bind rather weakly to MQ and have high rates of association and dissociation [MI; therefore, complexed IgG ( e . g rosette formation by antibody-coated red cells) have preferentially been used for the identification of FcR specificity [lo, 191. As the binding properties of monomeric and complex IgG for FcR on MQ are different [3,5], it appears unlikely that findings obtained with complexed IgG are also valid for monomeric IgG. In the present study, binding of monomeric IgG to rat BMMQ was determined by means of FCM. By this method, it is possible to quantitate binding of IgG to every single cell.The results clearly show that binding of monomeric rat IgG to rat BMMQ is subclass specific. Only rat IgGzb, either myeloma protein or mAb, bound with high affinity to resting BMMQ (Kd in the range of 3 x M). The other IgG examined bound with only low affinity (rat IgG2,, mouse and human IgG) or not at all to resting BMMQ (rat IgG, and IgGzc, mouse myeloma proteins; Table 1). The present FCM data on high-affinity binding of rat IgG2b to rat BMMcD, the conditions of its binding and the trypsin resistance of its FcyR coincide with results of recent measurements of binding of radiolabeled monoclonal rat IgG2b to rat splenic and peritoneal MQ [20]. Utilizing rosette formation by antibody-coated red cells, Medgyesi et al. [19] and Boltz-Nitulescu et al. [lo] investigated the subclass specificity of FcR that bind complexed IgG on rat alveolar and peritoneal MQ. Boltz-Nitulescu et al. [lo] identified on rat alveolar MQ a trypsin-resistant receptor binding rat IgGza myeloma protein but not the other subclasses, and a trypsin-sensitive receptor binding rat IgG2, or IgGZc. Medgyesi et al. [19] reported that in one rosetting assay system, rat IgG2, bound to peritoneal MQ and some cross-reactivity of IgG2, and IgGl was noted. These results differ from those of Denham et al. [20] and the present data. As we used the same myeloma proteins as Boltz-Nitulescu et al. [lo], and Medgyesi et al. [19], the observed differences can no longer be ascribed to different antibody preparations but rather to different methods andor the different binding properties of monomeric vs. complexed IgG for FcyR on MQ. On human monocytes, mouse peritoneal MQ, rat spleen and activated peritoneal MQ, the FcyRI is unique in its ability to bind monomeric IgG with high affinity [3, 6, 8, 201. The binding of monomeric IgG is subclass specific; in rats, only monomeric IgGZb, a major component of the plasma of normal adult rats (Peppard, cited in [20]), binds to the FcyRI. The physiological role of FcyRI-dependent binding of monomeric IgG2b in vivo remains to be determined. As highaffinity binding of rat IgG2b was not affected by a surplus of

low-affinity binding IgG, and vice versa, it appears likely that these different IgG bind to different sites on the MQ surface. After 24 h incubation of BMMQ with rRIFN-y, both high-affinity binding of monomeric rat IgG2b and lowaffinity binding of rat IgG2, and human and mouse IgG was clearly enhanced. It is noteworthy that for the enhancement of IgG binding and of the expression of MHC class I1 molecules (Fig. 3), as well as for induction of tumoricidal activity in BMMQ, low concentrations of IFN-y sufficed. In contrast, CP, as potent as IFN-y in inducing tumoricidal activity, neither affected binding of IgG nor expression of MHC class I1 molecules by BMMQ [16]. IFN-y-induced enhancement of IgG binding and of expression of MHC class I1 molecules persisted for at least 96 h (Fig. 2); in contrast, tumoricidal activity triggered in BMMQ by IFN-y is only short-lived [13]. These findings provide further support for the notion that the abilities of MQ to bind IgG and to express MHC class I1 molecules or tumoricidal activity are not necessarily closely related phenomena. We thank Dr. Michel Aguet for helpful discussions and Ms. Yvonne Eichholzer for expert technical assistance.

Received May 25, 1990.

5 References 1 Berken, A. and Benacerraf, B., J. Exp. Med. 1966. 123: 119. 2 Chang, T. W., Immunol. Today 1985. 6: 245. 3 Hogg, N., Immunol. Today 1988. 9: 185. 4 Unkeless, J. C., Sagliano, E. and Friedman,V H., Annu. Rev. Immunol. 1988. 6: 251. 5 Unkeless, J. C., J. Clin. Invest. 1989. 83: 355. 6 Burton, D. R., Mol. lmmunol. 1985. 22: 161. 7 Anderson, C. L. and Looney, R. J., Immunol. Today 1986. 7: 264. 8 Leu, R. W., Robinson, C. J., Wiggins, J. A., Shannon, B. J., Rummage, J. A. and Horn, M. J., J. Zmmunol. Methods 1988. 113: 269. 9 Bazin, H., Beckers, A. and Querinjean, P., Eur. J. Immunol. 1974. 4: 44. 10 Boltz-Nitulescu,G., Bazin, H. and Spiegelberg, H. L., J. Exp. Med. 1981. 154: 374. 11 Chassoux, D. M., Linarez-Cruz, L. G., Bazin, H. and Stanislawski, M., Immunology 1988. 65: 623. 12 Keller, R. and Keist, R., Exp. Cell Biol. 1982. 50: 255. 13 Keller, R., Keist, R., Van der Meide, P. H., Groscurth, P., Aguet, M. and Leist,T. P., J. Immunol. 1987. 138: 2366. 14 Keller, R., Joller, PW. and Keist, R., Cell. lmmunol. 1989.120: 277. 15 Keller, R., Keist, R. and Schwendener, R. A., Int. J. Cancer 1989. 44: 512. 16 Keller, R., Joller, I?, Keist, R., Binz, H. and Van der Meide, P. H., Scand. J. lmmunol. 1988. 28: 113. 17 Decker, T. and Lohmann-Matthes, M.-L., J. Immunol. Methods 1988. 115: 61. 18 Unkeless, J. C. and Eisen, H. N., J. Exp. Med. 1975. 142: 1520. 19 Medgyesi, G. A., Foris, G., Dezso, B., Gergely, J. and Bazin, H., Immunology 1980. 40: 317. 20 Denham, S., Barfoot, R.and Jackson, E., immunology 1987. 62: 69.