Modulation of neutrophil Fc and C3b receptors

Inflammation, Vol. 7, No. 2, 1983

MODULATION OF NEUTROPHIL Fc AND C3b RECEPTORS

Relationship with the Phagocytic Process and Activation of the Respiratory Burst 1

FRANCO PATRONE, FRANCO DALLEGRI, EZIO BONVINI, GUIDO FRUMENTO, ANGELO NOCERA,

MANLIO FERRARINI, and CARLO SACCHETTI

Departments of lnternal Medicine and Immunology University of Genova, Genova, Italy

Abstract--The effect of the interaction between human neutrophils and aggregated IgG on the expression of the receptors for the Fc portion oflgG (FcR) and for the C3b (C3R) has been investigated. Incubation of neutrophils with the appropriate concentrations of aggregated IgG at 37~ caused the loss of both the FcR and the C3R. This loss (modulation) was energy dependent (i.e., did not take place in cells incubated in the cold) and irreversible in that neutrophils did not reexpress either of the two receptors even upon prolonged incubation in vitro. The mechanisms leading to the modulation of FoR and C3R were different. FoR modulation was independent of the activation of the respiratory burst, since it occurred also in neutrophils from chronic granulomatous disease patients and was not induced by treatment of normal neutrophils with drugs such as phorbol myristate acetate (PMA), known to activate the respiratory burst. The FoR modulation was rather related to the redistribution ("capping") and endocytosis of the FcR induced by the interaction with aggregated IgG. This possibility was supported by the finding that FcR modulation was blocked by inhibitors of phagocytosis and by the observation that aggregated IgG, tagged with a fluorescent dye, were "capped" and subsequently endocytosed by metabolically active ceils. Mod- ulation of C3R was dependent upon the activation of the respiratory burst induced by the interaction of aggregated IgG with the neutrophils. This hypothesis was also supported by the finding that the modulation of C3R was induced by treatment of the cells with PMA and did not occur in chronic granulomatous disease neutrophils treated with aggregated IgG or PMA. Furthermore the modulation of C3R was inhibited by the addition of catalase, suggesting that such modulation was consequent to the damaging effect of the oxygen active by-products on the receptor structures. In addition to the C3R modulation described above, another type of C3R loss was observed. This occurred in chronic granulomatous disease (CGD) neutrophils following interaction with the appropriate antigen-antibody- complement complexes. In these ceils, phagocytocis of the complexes caused a concomitant modulation of the C3R that was possibly related to the redistribution and endocytosis of the C3R structures.

1Preliminary data were presented at the 9th International RES Congress, Davos, Switzerland, February 8, 1982.

155

0360-3997/83/0600-0155503.00/0 �9 1983 Plenum Publishing Corporation

156 Patrone et al.

INTRODUCTION

Human neutrophils, like those of other animal species, have surface receptors for the Fc portion of IgG (1, 2). These receptors (FcR) bind preferentially to the human IgG1 and IgG3 subclasses, and their avidity for the substrate is greatly enhanced when the IgG molecules are aggregated by the specific antigen or, in some experimental systems, by chemical or physical means (as reviewed in reference 3). The interaction between the neutrophil FcR and its ligand (presented in the appropriate molecular form) triggers a number of events such as the release of lysosomal enzymes, certain cytotoxic functions on nucleated cells and the attachment to and the ingestion of opsonized par- ticulate antigens (4-7).

Neutrophils also possess surface receptors for the cleavage products of the third complement component (C3b and C3bi) (as reviewed in reference 8). These receptors seem to subserve mainly the role of facilitating the adherence of the cells to the opsonized targets rather than that of triggering directly some or all of the functions mentioned above (5, 9-1i).

Previous in vitro studies on other cells equipped with FcR have demonstrated that the interaction between FcR and the appropriate ligands causes a temporary or sometimes irreversible loss of the FcR function. This phenomenon, generally referred to as "modulation," has been amply documented for human and murine monocytes or macrophages (12-15) and for some human lymphocyte subset populations (16, 17). The modulation of macrophage FcR (and in some instances of C3R) has been indicated as responsible for the defective clearance of circulating immune complexes (18) observed in some diseases such as, for example, systemic lupus erythematosus (19), nephritis (20), Sj6gren syndrome (21), vasculitis (20), or biliary cirrhosis (22).

In the present study, we have investigated the fa~e of both FcR and C3b receptor (C3R) of neutrophils upon interaction with the specific ligands. It will be shown that, following this interaction, there is an irreversible modulation of both the receptor functions. Furthermore, the interaction between the FcR and aggregated IgG leads to the irreversible loss of the C3R receptor function. Such a loss takes place only in neutrophils capable of initiating a respiratory burst and is likely to be consequent to the damaging effect that oxygen-active by-products, released by the cells, may have on the C3R structures.

M A T E R I A L S A N D M E T H O D S

Neutrophil Suspensions. Blood from healthy donors or chronic granulomatous disease (CGD) patients was used.

Neutrophii Fc and C3b Receptor Modulation 157

The diagnosis of CGD fulfilled the established clinical and laboratory criteria (23). The patient neutrophils did not reduce nitroblue tetrazolium upon stimulation with bacterial endotoxin (24) and were incapable of generating O~ in the presence of phorbol myristate acetate (PMA) as evaluated by the cytochrome c reduction assay (25). Furthermore, they did not display a phagocytosis-dependent metabolic burst, as evaluated by the [14C]glucose method (26). All of the patients were free of infection at the time of study and were not receiving medications.

Blood was collected in heparin (10 IU/ml), diluted with equal volumes of a 6% dextran (mol wt 70.000) solution in saline and allowed to sediment in Falcon plastic tubes (catalog no. 2051, Oxnard, California) at 22~ for 30 min.

The leukocyte rich buffy coats were fractionated further on FicoU-Hypaque density gradients (27). Neutrophils, which sedimented at the bottom of the gradients, were collected, washed three times in phosphate-buffered saline pH 7.3 (PBS) and resuspended in Dulbecco's modified Eagle medium (DME, Gibco-Biocult, Glasgow, Scotland) supplemented with 10% heat inacti- vated, filtered fetal calf serum (FCS, Gibco-Biocult). The suspensions contained 96% or more neutrophils with a cell viability greater than 97% as assayed by the ethidium bromide-fluorescein diacetate test (28).

Detection o f FcR and C3R. FcR were detected by measuring the binding to the neutrophil surface of heat-aggregated fluorescein-conjugated human IgG (FA-IgG) or by a rosette test. Briefly, human Cohn fraction II (Globuman Berna, Istituto Sieroterapico Berna, Como, Italy) was conjugated with fluorescein isothyocyanate (FITC, BDH, Italia S.p.A., Milano, Italy) as previously reported (29). The conjugated IgG were adjusted at a concentration of 20 mg/ml in PBS, pH 8, and aggregated by heating at 63~ for 2 h. The FA-IgG were subdivided in 0.2 ml aliquots and stored at -20~ Dilutions of FA-IgG were made in PBS (pH %3). The same batch of FA-IgG was used throughout. Neutrophils (1.6 • 106) were mixed with different concentrations of FA-IgG in 0.4 ml of DME supplemented with 10% fetal calf serum (FCS) in Falcon plastic tubes (catalog No. 2054). The tubes were incubated alternatively in a 37~ or in a 0 ~ C water bath. Neutrophils were subsequently washed three times in DME-FCS and examined in a Leitz Dialux fluorescent microscope (Leitz Italiana S.p.A., Milano, Italy). Ceils displaying bright fluorescent rings (when incubated in the cold) or one or more fluorescent "caps" (when incubated at 37~ were considered as positive. A minimum of two hundred cells were scored to determine the percent of positive cells.

The rosette test to detect FoR on neutrophils has been detailed elsewhere (27). Briefly 100 #1 of a neutrophil suspension at the concentration of 4 • 106 cells/ml in DME-FCS were mixed with an equal volume of a 1% suspension (in PBS) ofox red blood cells (ORBC) sensitized with purified rabbit IgG antibody (EAo) in plastic capillary tubes. The mixture was pelleted by cen- trifugation and incubated at 4~ for 30 min. The ceils were gently resuspended, mixed with one drop of a 0.5% solution of toluidine blue in PBS, and counted in a counting chamber. A minimum of 200 cells were scored to determine the percent of rosette-forming cells. Neutrophils that bound at least three indicator red cells were considered as rosettes.

C3R were detected as detailed previously (30). Briefly R3 prepared by reacting a pool of five normal human sera with zymosan was used as a source of complement. ORBCs were coated with rabbit-purified IgM antibody (EA M ) and reacted with R3 at 37~ (EA M C). The reaction was stopped after 75 see by adding Antrypol (Suramine BP, Bayer-Leverkusen, FRG) and the incubation carried out for another 2 min. With this method, C3 was fixed only in the C3b form, and the addition of Antrypol made any further cleavage of C3b very unlikely (31). All the pro- cedures were carried out in veronal-buffered saline supplemented with 0.1% gelatin (GVB-S), pH 7.4. That EAs~C had only C3b was proved by the positivity of the immune adherence reaction with human red cells (30) and by the absence of reactivity with DAUDI ceils which lack the C3b receptor and have the C3d receptor only (8). A neutrophil suspension, 100/~1, at the con- ceutration of 4 • 106 cells/ml in DME were admixed to an equal volume of a 1% EA M C suspension in GVB-S in plastic capillary tubes. The mixture was incubated at 37~ for 5 min, pelleted, incubated for another 30 min in the cold and examined for the percent of rosetting ceils as above. In some selected tests, EA~ rather than EA M were sensitized with complement using

158 Patrone et al.

the same procedure as above. In all of the experiments ORBC alone and EA M were used as control indicator cells. No rosette formation was ever detected.

Determination of Respiratory Burst Activity. The activity of the respiratory burst in neutrophils stimulated with various agents or in resting (control) cells was determined by measuring the activity of the hexose monophosphate shunt, using the [~4C]glucose oxidation method according to a modification of the technique of Skeel et al. (26, 32).

Drugs and Chemicals. Phorbol myristate acetate (PMA, Sigma Chemical Co~, St. Louis, Mo) used to activate the respiratory burst was dissolved in DMSO at the concentration of 2 mg/ml and stored in 0.1-ml aliquots at -20~ Dilutions of PMA were subsequently made in PBS. In each of the experiments with PMA, control nentrophil suspensions were treated with the same amount of DMSO present in the PMA-treated suspensions. At the concentration em- ployed, DMSO alone did not alter the neutrophil capacity of binding FA-IgG or of forming EA G or EA M C rosettes nor did it activate the respiratory burst. Colehieine and the scavengers of the oxygen-reactive by-products (33) cytochrome c type VI, superoxide dismutase (SOD) type I, and catalase were purchased from Sigma and dissolved in PBS immediately before use.

RESULTS

Modulation o f expression o f FcR and C3 R Induced by Aggregated lgG. FA-IgG were tested for their capacity to detect FoR on ~he surface of neutrophils. Preliminary titration tests of the reagent demonstrated that 90- 100% of the cells were brightly fluorescent up to the dilution of 400/~g/ml, whereas these percentages decreased progressively at higher dilutions. At 40 /zg/ml the reagent bound to approximately 50% of the cells, while at 4 #g /ml about 10% reactive neutrophils were detected. The concentrations selected for the subsequent experiments were 400/~g/ml, 40 ~g/ml and 4 #g/ml.

When incubated in the cold, the neutrophils displayed a fluorescent ring which changed to one or more polar caps when the cells were warmed at 37~ Upon prolonged incubation, these caps turned into fluorescent droplets that probably represented phagocytic vesicles. A staining pattern char- acterized by numerous fluorescent droplets (again interpreted as phagocytic vesicles) was observed for the cells incubated directly at 37 ~ C with FA-IgG (Figure 1).

Neutrophils from five different individuals were incubated with FA-IgG at the concentrations indicated above for 1 h at 37~ washed in the cold, and reacted with EA G or EAM C indicator cells. One aliquot of the cells was observed under the fluorescent microscope to check whether or not the cells had bound FA-IgG. Another aliquot was rosetted with EA or EAM C indicator cells.

Incubation with FA-IgG at 37~ resulted in a loss of the FoR that was dose dependent (Figure 2a). Neutrophils treated with FA-IgG (400/~g/ml at 37~ for 1 h) were subsequently cultured and harvested at 4-, 8-, 24-h inter- vals. No recovery of the FoR function was noted even after 24 h. These data cannot be explained by poor cell viability or cell loss since 90% of the neutrophils (with a viability of 80%) were recovered after 24 h in vitro.

Neutrophil Fc and C3b Receptor Modulation 159

Fig. 1. Staining pattern of neutrophils reacted with FA-IgG. (A) A cell stained in the cold and subsequently warmed for approximately 5 rain. A fluorescent ring turning into a polar cap is observed. (B) A cell stained in the cold and subsequently warmed for 20 rain. Two polar caps are observed. (C) A cell stained at 37 ~ C for 1 h. Several fluorescent droplets are seen.

Following incubation with FA-IgG as above, a loss of C3R was also observed, although on a smaller cell number (Figure 2b). The proportion of C3R § cells decreased to 60%, as opposed to the 30% observed for FcR § cells. This percent was, however, statistically different (P < 0.01) from that of the control cells not reacted with FA-IgG. Again the C3R loss was not re- versed by a 24-h incubation in vitro.

The loss of both FcR and C3R could be observed only with aggregated IgG and not with FITC-conjugated molecules in the native (i.e., deaggre- gated) form. In agreement with previous data (34), the incubation with the above concentrations of FA-IgG in the cold did not cause FcR or C3R loss

FA-Ig 6 [~ug/mll

,~OI)

r

(t

4 0 0

0

a

i , , ] i

b ,-+..,**

, !-I ~* , 1 ,

'0 . . . . . 8'0 ' 2 40 60 l()O % POSITIVE CELLS

Fig. 2, Modulation of neutrophil FcR (a) or C3R (b) induced by FA-IgG. The results are expressed as the mean values + 1 S E M of 5 different experiments. Statistical analysis was carried out by the Student's t test for paired data ( * P<~ 0,02, ** P < 0.01, *** P < 0.001).

160 Patrone et al.

(data not shown). Since FA-IgG bound equally well in the cold, this indicates that the FcR and C3R loss occurred only in metabolically active cells. Fur- thermore, these findings demonstrate that, at the concentrations used, FA- IgG did not inhibit EA or EAMC rosettes by blocking the receptors through a steric hindrance phenomenon. At higher concentrations (2 mg/ml), FA-IgG blocked FcR (but not C3R) on 80-90% of the neutrophils also in metaboli- caUy inactive cells (i.e., incubated in the cold) (data not shown and reference 27).

Relationship between Modulation of FcR or C3R and Respiratory Burst Activation. The treatment with aggregated IgG causes activation of the neutrophil respiratory burst (6). This activation was also observed in our experiments (Table 1), although it was not particularly efficient, owing to the special test conditions (i.e., a relatively low concentration of both cells and F,A-IgG and an incubation which omitted shaking of the tube). The modulation of both FoR and C3R could, therefore, be related to the activation of the respiratory burst. In order to test this hypothesis, the cells were treated with PMA, a substance that activates an efficient respiratory burst (reference 35 and Table 1) and subsequently rosetted with EAG or EAM C. The activation of the respiratory burst with PMA caused a profound decrease in the number of C3R § cells, but left the percent of cells with FeR unchanged (Figure 3).

Selective FcR Modulation Induced by FA-IgG in CGD Neutrophils. Neutrophils from three CGD patients were incubated with 400 #g/ml FA-IgG for 1 h at 37 ~ C and rosetted with EA6 or EAM C indicator cells. As shown in Table 2, a significant decrease in the percent of FeR § but not of C3R § cells was observed. These findings indicate that FoR modulation was independent of the activation of the respiratory burst, whereas the C3R loss observed in

Table 1. Activation of Neutrophil Respiratory Burst Induced by FA-IgG or PMA a

~4CO2 production/1.6 • t06 cells Cells treated with: (cpm) b

FA-IgG (400 #g/m1) 914.0 _+ 39.2 FA-IgO (40#g/ml) 759.3 + 16.1 FA-IgG (4 #g/m1) 436.0 -+ 5.5 PMA (0.1/ag/ml) 1541.0 _+ 55.9 PBS 177.0 _+ 6.5

a 1.6 • 106 neutrophils were incubated with FA-IgG, PMA, or PBS in the presence of 0.05/zCi of [ 1-14C]glucose for 1 h at 37 ~ C (32). The 14CO2 released by the cells was fixed to a glass micro- fiber paper and the radioactivity determined.

bMean values of 3 experiments _+ l SEM.


C[LLS TR[ATFD

WITH:

PMA ~ ' ~

PeS :::::::::::::::::::::::::::::::::::::::::::::::::

b 2'0 4'0 6'0 8'0 I~'~0 % POSITIV[ C[LL$

Fig. 3. Effect of PMA (0.I #g/ml) treatment on neutrophil FcR (open bars) or C3R (dotted bars). The results are expressed as the mean values + 1 SEM of 3 different experiments. Statis- tical analysis was carried out by the Student's t test for paired data ( *** P < 0.001).

the normal neutrophils could be consequent to this activation. This hypothesis was reinforced by the observat ion that P M A treatment did not cause any C3R loss in neutrophils f rom C G D patients (Table 2).

Relationship between FcR Modulation and the Process of Phagocy- tosis. Incuba t ion o f neutrophils with F A - I g G was followed by the phagocytosis o f a p ropor t i on o f the aggregates as could be seen by fluorescence microscopy (Figure 1). Therefore, the modula t ion o f F c R could be consequent to the internalization of the F c R which takes place during phagocytosis . Experiments f rom our own labora tory and others demonst ra ted that treatment with colchicine inhibits the capaci ty o f neutrophils to ingest opsonized

Table 2. Selective FcR Modulation Induced by FA- IgG in CGD Neutrophils

Cell treated with

Patients FA-IgG a PMA a PBS

% FcR § cells A 48 89 97 B b 25 41 55 C 44 n.d. c 77

% C3R + cells A 99 95 99 B 95 80 96

aFA-IgG, 400 pg/ml; PMA, 0.1 pg/ml. bpatient B had consistently a low proportion of FcR§ neutrophils with no apparent explanation.

Cn.d. = not done.

1 6 2 P a t r o n e e t a l .

Table 3. Effect of Colchicine on Neutrophil FoR Modulation

Cell treatment

FA-IgG Colchicine a (400 #g/ml) (10 -5 M) % FoR * cells b

+ + 71.0 + 1.2 + - 26.0 + 3.0 - + 86.0 + 3.6 - - 80.7 + 3.0

aColchicine was dissolved in PBS and added to the cell suspension in the appropriate amount to reach the concentration indicated. Cells were incubated with colchieine at 37~C for 1 h before being reacted with FA-IgG.

bThe results are expressed as the mean values +_ 1 SEM of 3 different experiments.

red cells or aggregated IgG (36). In order to investigate the relationship between phagoeytosis and FcR modulation, the cells were pretreated with colchicine at a concentration which in our experience blocks phagocytosis (10 -3 M) and incubated at 37~ with FA-IgG (400 #g/ml) in the presence of the drug. No significant loss of FcR was noted in the colchicine-treated neutrophils (Table 3). By contrast C3R was modulated by the colchicine-treated cells in the same manner as in the controls (data not shown). This finding was not unexpected since 10 -3 M colchicine was incapable of inhibiting the respiratory burst induced by FA-IgG (data not shown).

Additional information using cytochalasin B as an inhibitor of phagocytosis could not be obtained, because this substance, although it did not affect the binding of FA-IgG to neutrophils, greatly reduced their capacity of forming EA6 rosettes. This is in agreement with previously published data (37).

Substances Released following Respiratory Burst Activation May Cause Irreversible Damage of C3R. An irreversible alteration of the C3R structures caused by metabolically active substances released by activated neutrophils could have been responsible for the observed C3R modulation (38). This hypothesis was investigated by studying the effect of the scavengers of the oxygen-reactive by-products on the modulation of the C3R.

As shown in Table 4, catalase prevented the PMA-induced C3R loss, indicating that H202 may be responsible for the. damage of the receptor function. By contrast SOD and cytochrome c failed to prevent the C3R modulation (data not shown). The superoxide anions were not, per se, capable of damaging the C3R structures.

Neutrophil Fe and C3b Receptor Modulation

Table 4. Effect of Catalase on C3R Expression by PMA-Treated Neutrophils

Cell treatment

PMA Catalase (0.1 pg/ml) (4000 units/ml) % C3R + cells a

+ + 92.5 + 2.5 + - 20.0 + 14.0 - - 82.3 + 5,9

aThe results are expressed as the mean values + 1 SEM of 3 different experiments.

163

Modulation of C3R as a Consequence of Internalization of Receptor, In these exper iments we have investigated whether or not the internal izat ion o f the C 3 R could lead to receptor modula t ion . Neut rophi l s f rom a C G D patient were roset ted wi th E A ~ C indicator cells and incubated for 1 h at 37~ E A ~ C were preferred to EAM C because previous studies in o ther and our own l abora to ry had shown that sensit ization with IgG is essential for op- t imal phagocytos is o f the target (5, 9, 10). Fol lowing incubat ion, mos t of the neutrophi ls had ingested a var iable n u m b e r of indicator cells (Figure 4). The EAG C which had not been ingested were r emoved by hypo ton ic lysis and the neutrophi ls rerose t ted wi th EAM C indicator cells. As shown in Figure 4, such t r ea tmen t caused the modu la t ion of the C3R f rom the surface o f a con- sistent n u m b e r of neutrophils . As expected f rom the results repor ted above on C G D neutrophils , the cells t reated with E A c indicator cells a lone did not modu la t e the C3R.

CELLS REACTEO

WITH:

EAGC~ t E A G [ :i:i:i:i:i: :?:i:!:i:i:i:!:-:;:;:!:?~Yii~il ]

('; 2'0 4'0 "6}) 80 lO0

% POSITIVE C[LL$

Fig. 4. Neutrophil C3R toss induced by interaction with EAGC. CGD neutrophiis, reacted with EA G C or EA~ (control), were subsequently examined for phagocytosis, i.e., cells with ingested red cells (open bars), or the presence of C3R (dotted bars).

164 Patrone et al.

DISCUSSION

The present study demonstrates that there is an irreversible loss of both FcR and C3R upon interaction between human neutrophils and aggregated IgG. This loss occurs in metabolically active cells and cannot be explained by the blocking of the receptors by steric hindrance. Therefore, this phenomenon resembles the FoR loss which takes place in monocytes and in certain lymphocyte subsets upon interaction with immune complexes and is generally designated as modulation (11-17). The modulation of FcR and C3R follows different patterns. Consequently the two phenomena shall be discussed separately.

The modulation of the FcR is independent of the activation of the respiratory burst since it is also observed in CGD neutrophils and does not fol- low the activation of the respiratory burst of normal cells by PMA. The FcR modulation seems, rather, to be a consequence of the interaction between the FoR and the appropriate ligand that causes a redistribution ("capping") of the FcR followed by endocytosis of both the receptor and the ligand (12). Capping of FoR has been described previously in a variety of cells, such as monocytes, macrophages, and basophils (12, 29, 39-41). That the internalization of the aggregated IgG was a necessary step in the process of modulation is indicated by the inhibition of the modulation obtained with agents which block the phagocytic process. Furthermore the use of reagents tagged with fluorescein has permitted us to observe both the redistribution and the endocytosis of the aggregated IgG.

The fine mechanisms leading to the modulation of FoR are not com- pletely understood. The interaction with aggregated IgG (or immune complexes) in the appropriate amounts may cause the redistribution (possibly followed by endocytosis) of the majority of the FcR (12, 15, 42). The receptors remaining on the entire cell surface will not be sufficient to bind any other immune complexes. Alternatively, the contact with aggregated IgG (or immune complexes) could deliver a signal to the neutrophils which might in turn shed their FcR in a manner similar to that reported for the FcR of some lymphocyte subpopulations (16, 43).

I f compared to that of monocytes or macrophages, the FoR modulation of neutrophils displays a noticeable difference in that, whereas monocytes and macrophages can reexpress FoR in some experimental systems (14), neutrophils fail to do so. Unfortunately, in spite of extensive studies, not reported here, we have been unable to remove FoR from the neutrophil surface by enzyme treatment, and consequently it has not been possible to determine the turnover rate of these structures. Therefore, the possibility cannot be excluded that 24 h in culture do not represent sufficient time for the reex- pression of FoR. However, if this were the case, neutrophil FcR would differ from those of other cell types, which have an half-life on the cell membrane


much shorter than 24 h (44, 45). Another explanation may be that circulating neutrophils, being terminal cells, have lost the capacity of resynthesizing new FcR. It is of note that if this hypothesis were correct, neutrophils would have lost the capacity of synthesizing FcR but not C3R, since, in recent experiments, we have shown that trypsin-digested (46) C3R are reexpressed by the cells within 8 h. An alternative hypothesis could be that, upon interaction with aggregated IgG, a specific switching-off signal is delivered to the cells so that they do not reexpress FcR (13). A final possibility may be the incapacity of cells activated by the phagocytic process of resynthesizing any membrane structure.

The modulation of the C3R was induced by the activation of the respiratory burst and was probably related to the damaging effect that the oxygen- active by-products may have on the receptor structures (38). This hypothesis is supported by (1) the failure to induce C3R modulation in CGD neutrophils following activation with a variety of means, (2) the modulation of C3R occurring in normal neutrophils treated with PMA, and (3) the possibility of preventing the C3R loss with catalase.

Modulation of the C3R was also induced in the neutrophils from a CGD patient. In this case such modulation was obtained by the interaction with the appropriate ligand and was probably consequent to the redistribution and endocytosis of C3R caused by this interaction. In this respect the C3R modulation would not differ from that of the FcR. In agreement with previous data, phagocytosis of complement-coated red cells was obtained with indicator cells sensitized with IgG, but not with IgM antibody (5, 10). There- fore, it is possible that, in our experimental system, modulation of C3R was consequent to the interactions of both the C3R and the FcR with their re- spective ligands. At present this hypothesis is difficult to test experimentally.

Clearly the C3R modulation by FA-IgG that we have observed was not attributable to a cocapping of FcR and C3R. This is best demonstrated by the finding that interaction between FA-IgG and CGD neutrophils caused a selective modulation of FcR. Previously, FcR and C3R have been shown to cap independently on the surface of monocytes and macrophages (15, 47-49). More recently Fearon and associates have demonstrated that a divalent antibody directed to C3R structures is capable of selectively redistributing the C3R and of causing its endocytosis (50).

The finding that both FcR and C3R of neutrophils can be modulated upon contact of the cells with aggregated IgG has both practical and con- ceptual implications. For example, these data can explain the defective receptor functions observed in certain diseases with high levels of circulating immune complexes (51). Furthermore, modulation of the receptors can be envisaged as a device to limit the amount of particles that the neutrophils can ingest and have subsequently to process through their lysosomal system.

Acknowledgments--This work was supported by grants from the Italian C.N.R. to F. Patrone (PFCCN, No. 81.01432.96) and to M. Ferrarini.

166 Patrone et al.

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Modulation of neutrophil Fc and C3b receptors