cytophilic antibody: correlation of its distribution with activation of basophils and macrophages

364 M. Ferrarini, A. Munro and A.B. Wilson Eur. J . Immunol. 1973. 3: 364-370

M. Ferrarini*, A. Munro and Anne 8. Wilson

Immunology Division, Department of Pathology, University of Cambridge

Cytophilic antibody: correlation of its distribution with activation of basophils and macrophages

Immunoglobulin determinants on the surface of guinea pig basophils were detected using mono- and divalent antiglobulin reagents conjugated with fluorescein isothiocyanate. The fluorescent pattern obtained with monovalent antiglobulin was always that of uniformly stained rings. Divalent antiglobulin agglulinated immunoglobulin determinants t o form small fluorescent spots aubsequently collected at one end of the cell (“cap” formation), The formation of caps is temperature-dependent and is inhibited by sodium azide and cytochalasin B.

When basophils were incubated at 37 “C with divalent antiglobulin, morphological changes occurred, indicating that they had been activated. The same effect could be obtained with monovalent Fab’, but a t least one hundred times more of it was required than the corresponding F(ab’),.

Cytophilic antibodies on the surface of guinea pig peritoneal macrophages were also detected by immunofluorescence. In this case again, divalent antiglobulin reagents agglutinated the determinants t o form small fluorescent spots. Cap formation was not observed and pinocytosis started as the first aggregates were formed. I t was also found that cytophilic antibodies were not stable on the macrophages’ surface and were easily eluted at 37 OC. A reaction with cross-linking agents (divalent antiglobulin o r antigen) stabilized the cytophilic antibody and pinocytosis was initiated.

1. Introduction

In recent years several experiments have demonstrated that immunoglobulin molecules can move freely in the lymphocyte membrane [ 1-31. These immunoglobulins can be agglutinated by divalent antiglobulin reagents conjugated with fluorochrome so as t o form small fluorescent clusters (“spots”), which subsequently congregate at one end of the cell (“cap” formation). Mobility on the plane of the membrane has also been reported for histocompatibility antigens in lymphocytes [4] and fibroblasts [ 5 ] . These observations, together with consideration of the structure and the function of the cell membrane, have supported the hypothesis that mobility of proteins in a cell membrane can be a general phenomenon related t o the fluid structure of the membrane itself [6]. In the present experiments we have investigated the distribution of cytophilic antibodies on the membranes of basophils and macrophages using monovalent and divalent antiglobulin reagents conjugated with a fluorochrome. Indication that cytophilic antibodies in basophils can move in the plane of the membrane comes from the experiments of Sullivan et al. [7].

I t will be shown that cytophilic antibodies can be clustered on the cell membrane of basophils and macrophages by divalent antiglobulin reagents at 37 “C. The patterns obtained after such treatment in the two cell types are, however, different.

Cytophilic antibodies, passively absorbed by basophils, become the receptors for antigens of these cells [8]. Upon con- tact with antigen, basophils will become activated and will

* On leave from Clinica del Lavoro, University of Genova.

Correspondence: Manlio Ferrarini, Clinica del Lavoro, Universita di Genova, pd. 3 Ospedale S. Martino, Genova, Italy

Abbreviations: BSA: Bovine serum albumin albumin PBS: Phosphate buffered saline FITC: Fluorescein isothiocyanate DMSO: Dimethylsulfoxide DTT: Dithiothreitol

HSA: Human serum

release pharmacological mediators [9]. Activation of basophils can be obtained in vitro by exposing sensitized basophils t o the appropriate antigen [ lo] . An antiglobulin reagent can also mimic the effect of the antigen in virro [ 111. Morpho- logical changes ( d e g r a d a t i o n ) [ 121 o r release of histamine [ 131 are usually taken as evidence for the activation of basophils.

We have investigated the possibility of a correlation between clustering of cytophilic antibody and basophil activation. If ~

a corrrelation existed, then, only divalent antiglobulin could be expected t o activate basophils.

It will be shown that monovalent antiglobulin molecules can also activate basophils, although the concentration required for the reagent t o obtain such an effect is much higher than that required for the corresponding divalent antiglobulin.

2. Materials and methods

2.1. Animals

The guinea pigs used were adult male and female albinos, bred in this laboratory.

2.2. Immunization

Primary injections of 0.1 ml of an emulsion containing 100 pg of rabbit Fab’ in Freund’s complete adjuvant were given in- tradermally into both ears. After three weeks, the guinea pigs received 100 pg Fab‘ in PBS at the same injection sites.

2.3. Preparation of cell suspensions

2.3.1. Media

The medium used for basophils was Tris-albumin buffer, pH 7.3 [ lo] . This contanied 0.025 M Tris-(hydroxymethy1)- aminomethane, 0.12 M NaCl, 0.005 M KCI, 0.3 mg/ml human serum albumin (HSA), 0.2 mg/ml bovine serum

Eur. J. Immunol. 1973.3: 364-370 Distribution of cytophilic antibody 365

albumin (BSA), EDTA (4 x 10-3M);pH 7.3 was obtained by addition of 1 N HCl. For the experiments on degranu- lation, EDTA was omitted and calcium and magnesium at final concentrations of 6 x 10-4M and 1 x added.

The medium used for macrophages was Hanks’ Tris-citrate (pH 7.2) containing 0.2 % BSA.

2.3.2. Preparation of basophil/lymphocyte/Kurloff cell

M were

suspensions

The method described by Wilson and Coombs [ 141 was used. Briefly, the method involved treating defibrinated blood with methyl cellulose and carbonyl iron at 37 OC to remove the majority of erythrocytes and phagocytic cells, lysing remaining erythrocytes by short exposure to distilled water and washing the lymphocyte/basophil suspension with cold medium. Since the proportion of erythrocytes contaminating the buffy coat was found to be small in some of the experiments, the short exposure in distilled water was omitted.

2.3.3. Preparation of peritoneal macrophages

Peritoneal macrophages were obtained by washing the peritoneal cavity of the guinea pig with 20 ml of the appropriate medium containing 20 U/ml of heparin. The cells were subsequently washed twice by centrifugation (800 rpm) in medium.

2.3.4. Anti-guinea pig IgG

Several anti-guinea pig immunoglobulin antisera were used. They were all raised in rabbits by repeated injections of purified guinea pig IgG in Freund’s complete adjuvant. The antisera showed a strong activity against 71 and 72 guinea pig immunoglobulin when tested by immunoelectrophoresis. All antisera were heat-inactivated and absorbed with well- washed guinea pig red cells.

2.4. Purified immunoglobulin and immunoglobulin fragments

2.4.1. Rabbit and guinea pig IgG

Rabbit and guinea pig IgC were isolated from serum by pre- cipitation with 33 % ammonium sulfate followed by chromatography on DEAE-cellulose with 0.0175 M potassium phosphate buffer, pH 6.5.

2.4.2. (F(ab’)z fragment

F(ab’), fragment was obtained by incubation of purified IgC with 1.25 % (w/w) pepsin (Sigma Chemical Co., St. Louis, Mo.) in sodium acetate buffer (0.1 M, pH 4) for 6 h at 37 “c.

After this period, 5 N NaOH and 1.8 M Tris-HC1 buffer (one- tenth of the volume of the original protein solution) were added to bring the pH to 8. After dialysis against phosphate buffered saline (PBS), the F(ab‘), was separated by gel filtration through a Sephadex G200 column equlibrated in PBS.

2.4.3. Monovalent Fab‘ fragment

F(ab’), fragment and F(ab‘), fragment conjugated with fluorescein isothiocyanate were reduced with a final concentration of 10 mM of dithiothreitol (DTT) and 0.18 M

Tris-HC1 buffer (pH 8). After 1 h, the solution was cooled and iodoacetamide added to a final concentration of 30 mM. The mixture was left 1 h at 4 O C and then dialyzed against PBS. The monovalent Fab’ fragment was separated from any remaining F(ab’)z by gel filtration through a Sephadex GI00 column equilibrated in PBS. Reduction with DTT followed by alkylation with iodoacetamide were repeated once or twice for the Fab’ antiglobulin preparations used.

2.5. Conjugation of the antisera

Basically, the method described by Cebra and Golstein [ 151 was used to conjugate the rabbit IgC fraction and the F(ab’), fragments. To the protein solution, 0.5 M sodium carbonate buffer (pH 9.5) was added in order t o raise the pH to 9.5. Fluorescein isothiocynate (FITC)(Biological La- boratories, Baltimore, Md.) was dissolved in 0.5 M carbonate buffer, pH 9.5, and added t o the protein solution using a ratio of 1 mg of the fluorochrome to 100 mg of protein. The mixture was left 3 h at room temperature under stir- ring. The proteins were then purified by gel filtration on Sephadex G-25 followed by chromatography on DEAE- cellulose (eluting buffer: 0.01 75 M potassium phosphate, pH 6.5, with increasing NaCl concentration). Conjugates had an absorbance ratio (280 nm/495 nm) ranging from 1.2 to 2.0.

2.6. Staining of the cells

An equal volume of conjugated antiserum was added to a volume of cell suspension. The concentration of the cells in the suspensions was 10 x lo6 - 20 x lo6 per ml in the case of the basophil/lymphocyte/Kurloff cell suspensions, or 5 x l o6 - 10 x lo6 per ml in the case of peritoneal macrophages. The protein concentration of the conjugated antiglobulin was between 0.5 mg/ml to 1 mglml. After 30 min incubation, the cells were washed twice in the appropriate medium. Controls were set up by incubating cell suspensions with normal IgG, F(ab’), or Fab‘ fragments conjugated with FITC. Unless otherwise stated, all the above procedures were carried out at 4 “C.

2.7. Microscopy

A Leitz Orthoplan and a Vickers microscope, equipped with a vertical illuminator, were used. The cell preparations were observed alternatively under phase contrast or specific il- lumination for fluorescein.

3. Results

3.1. Distribution of immunoglobulin determinants on the surface of basophils

3.1.1. Distribution of immunoglobulin determinants as detected by IgC or F(ab’)2 antiglobulin reagents. Effect of temperature

These experiments were carried out in the absence of Ca++ and Mg++ to prevent degradation.

Basophils from normal (uninjected guinea pigs) had membrane immunoglobulin determinants which could be detected by immunofluorescence staining.

If precautions were taken to keep the basophils cooled, their membranes uniformly stained and the fluorescence appeared

366 M. Ferrarini, A. Munro and A.B. Wilson Eur. J . Immunol. 1973.3: 364-370

as a bright uniform ring. Conversely, when the cells were stained at 37 OC, all the immunoglobulin determinants were localized in one region of the cell; the cells then had the appearance of a bright fluorescent cap.

By staining the cellsin the cold and subsequently incubating them at 37 O C for different periods of time, it was possible t o follow the process of transformation of the rings into caps (Fig. 1).

Figure 1. Guinea pig basophils stained with rabbit IgG antiglobulin conjugated with fluorescein. (a) Cells were stained and washed in the cold. The cells appear like bright fluorescent rings. It is possible to recognize some aggregates (spots). (b) Cells were stained and washed in the cold and incubated for 15 min at 37 OC. The fluorescence is collected at one polar end of the cell (cap).

The stages leading t o the formation of the caps can be sum- marized as follows:

a) Formation of small fluorescent spots scattered on the entire surface of the cell. This stage can be obtained by just leaving the cells for a very short time a t room temperature.

b) Formation of medium-sized spots. These spots were sometimes, but not necessarily, localized at one end of the cells.

c) Formation of large spots. Usually they were localized at one end of the cell, but they were recognizable as separated entities and did not form a proper cap.

It is difficult to establish the length of the incubation time at 37 "C required for cap formation, because it varied with different cell suspensions and, also, it was possible t o find cells a t different stages (that is medium-sized spots, large spots, caps) in the one cell suspension after incubation at 37 OC. Generally, 15 min at 37 "C are sufficient for cap formation in the majority of basophils.

When basophils were incubated at 3 7 "C for periods of time longer than 30 min, the majority of them showed a large fluorescent spot located between the nucleus and the granules. This picture may suggest that the fluorescent antiserum was pinocytozed by the cells.

The stages of transformation from rings into caps described above were common t o the great majority of basophil cell suspensions tested. It is, however, of interest to note that in a small number of guinea pigs, it appeared that basophils had very few cytophilic antibodies absorbed onto the membranes. The rather faint ring of fluorescence observed after staining in the cold transformed to only small o r medium sized spots after incubation in the warm.

3.1.2. Requirement of divalency of the antiglobulin reagent for spot and cap formation

When basophils were stained with conjugated Fab' anti-guinea pig immunoglobulin and incubated in the warm for different periods of time, the appearance of the cells was always that of a bright fluorescent ring. The only change observed after long incubation a t 37 "C was a certain decrease in the brightness of the fluorescence. This can possibly be explained by the uncoupling of the antiglobulin from the surface determinants due to the higher dissociation constant of the Fab' as compared with that of the (Fab')z [ 161.

Thus, the mechanism of spot and cap formation on the surface of basophils is similar to that reported by other authors for lymphocytes [ 1-31 and can be compared to an agglutination reaction where cross-linkage of determinants by divalent reagents is required.

3.1.3. Inhibition of cap formation

It has been shown that cap formation on lymphocytes is a metabolically dependent process and is also associated with membrane flow and cell mobility [l-21. T o test whether this also holds true for basophils, inhibition experiments were performed using sodium azide and cytochalasin B [ 171. Table 1 shows the effect of sodium azide on cap formation on basophils. The inhibition was dose-dependent and could be obtained only if basophils were preincubated at 37 "C for a certain time ( 1 5-30 min) in the presence of the drug before staining.

Table 1. Effect of sodium azide on cap formation on basophils

Concentration of NaN3 in medium

lo-' M lom2 M

1 0 - 3 ~ 1 0 4 ~ 104M

Medium alone

Basophils Small Caps spot.\ (70 ) (5% )

96 4 100 0

10 90 a 92 5 95

16 a4


Table 2 shows the effect of cytochalasin B on cap formation. In these experiments, cells were incubated in medium containing 0.5 % dimethylsulfoxide (DMSO) with different concentrations of cytochalasin for 30 min at 37 OC, stained and washed at room temperature and further incubated at 37 OC for 20 min.

Table 2. Effect of cytochalasin B on cap formation on basophils

Concentration of cytochalasin B in Exp. 1 Exp. 2 mediuma)

Small Large Caps Small Large Caps spots spots spots spots

(%I (%I (%I (%I (%) (%)

100 pg/ml 40 22 38 59 25 16 50 ILglml 35 23 42 32 25 43 25 &/ml 17 13 70 32 28 40

Diluenta) 3 5 92 3 17 ' 80

a) The medium contained 0.5 % DMSO in order to dissolve cyto- chslasin B.

A certain degree of cap formation could be inhibited with cytochalasin B at a concentration of 100 pg/ml. These experiments were, however, complicated by the difficulties encountered in dissolving the cytochalasin B in medium containing a low amount of DMSO. High concentrations of DMSO were found to be toxic for basophils and inhibited cap formation.

It is interesting to note that spot formation was not inhibited by either of the drugs used, suggesting that this is a phenomenon produced by the agglutination of the immunoglobulin determinants by the divalent antiglobulin reagent and does not require active metabolic energy from the cell.

3.2. Activation of basophils by antiglobulin reagents

3.2.1. Morphological changes bf basophils incubated at 37 O C in the presence of antiglobulin reagent and Ca++ and Mg++

In these experiments, cells prepared and washed in medium not containing EDTA, were treated at 37 "C for 20 min with divalent antiglobulin reagents in the presence of Ca++ or Mg*.

Under these conditions, the majority of basophils observed under phase contrast showed fewer and enlarged granules inside the cell membrane. In addition, most of the granules appeared on the outside of the cell membrane, sometimes around a considerable part of the cell membrane, but more often collected on one side of the cell to form one or two large clumps. Basophils reacted with unconjugated antiglobulin in the presence of Ca++ and Mgf+ were resuspended, after washing, in medium containing diacetyl-fluorescein (0.5 pg/ml). The basophils were brightly fluorescent, which showed that they were still viable after activation by the antiglobulin reagent. The clear localization of the cell membrane obtained by diacetyl-fluorescein staining gave definite evidence that the majority of granules were situated outside the cell. The granules were firmly bound to the cell membrane. Mov- ing the cell in the microscopic field with gentle pressure on the coverslip did not separate the cell from the granules.

Figure 2. Basophils stained with rabbit F(ab')l antiglobulin at 37 O C in the presence of Ca++ and Mg++. (a) Phase contrast; the granules of the basophils are on the outside of the cell membrane. (b) Cells observed under UV: the granules are brightly fluorescent.

When the basophils were treated with a conjugated antiglobulin in the presence of Ca++ and Mg*, the granules on the outside of the cell were brightly fluorescent (see section 3.2.2.). In most the cases, this obscured the pattern of fluorescence on the cell membrane.

In our experiments, we never observed the complete loss of granules seen with human basophils [9], but the morpholc- gical changes show that the guinea pig basophils had been activated .

In control experiments, where basophils were incubated with conjugated normal (nonimmune) rabbit IgG or normal rabbit F(ab')2 in the presence of Ca++and Mg++ions, these particular changes in the morphology of the basophils were never observed and the granules were never stained.

3.2.2. Nonspecific absorption of proteins by activated basophils

In another series of experiments, basophils were washed in EDTA-free medium and incubated at 37 OC for 20 min with unconjugated antiglobulin reagents (IgG or F(ab'),) in the presence of conjugated normal (nonimmune) rabbit IgG or F(ab'), and Ca* and Mg". Basophils showed the same

368 M. Ferrarini, A. Munro and A.B. Wilson Eur. J. Immunol. 1973.3: 364-370

morphological changes and their granules appeared very brightly fluorescent when observed under UV light. These experiments show that the bright fluorescent staining of the granules was the result of absorption of fluorescent proteins from the surrounding fluids by activated basophils and was not due to a specific reaction between immunoglobulin contained in the granules and the antiglobulin reagent.

3.2.3. Activation of basophils by monovalent Fab'

In these experiments, aliquots of the same basophil suspension were incubated either with conjugated F(ab'), o r conjugated Fab' in the presence of Ca++ and Mg++.

The pattern observed in the cell preparation treated with either of the two reagents was similar. In each case, there was a high proportion of activated basophils.

Table 3 shows the results of the titration of the capacity of the two reagents to activate basophils. I t can be seen that at least one hundred times more Fab' than F(ab'), is required to activate basophils.

Table 3. Titration of the capacity of F(ab')2 and Fab' to activate basophils

An tiglobulin concentration

Neat4 lo-'

10" lo4 lo5

Activated basophils (k)

F(ab')z Fab' 90 a5 a0 62 9a 10 88 0

- 38 - 4

a) The protein concentration of the two antiglobulin reagents was 0.7 rng/rnl.

3.3. Distribution of immunoglobulin determinants on the surface of peritoneal macrophages

The mobility of cytophilic antibodies in the cell membrane of basophils could be a particular property of that type of cell, o r a more general phenomenon occurring in other cell types bearing cytophilic antibody. Therefore, the distribution of cytophilic antibodies on peritoneal macrophage membranes was also investigated. In these experiments, the antiglobulin reagents used were rabbit F(ab'), and Fab' anti- guinea pig IgG conjugated with fluorescein. Conjugated IgG was not used because it was absorbed nonspecifically by peritoneal macrophages, presumably as cytophilic antibody.

3.3.1. Distribution of immunoglobulin determinants detected by F(ab'), antiglobulin. Effect of temperature

In these experiments, peritoneal macrophages were obtained from normal guinea pigs. The cell suspensions were prepared, stained, and washed in the cold. Aliquots of the stained cell suspensions were then incubated at 37 "C for different periods of time, either in a tube, o r on a slide covered with a coverslip and sealed with nail polish. The latter procedure

was found preferable, because incubation in a tube resulted in a large loss of macrophages, presumably due t o their abi- lity to adhere to glass. One aliquot of the original cell suspension was stained, washed and finally incubated in the presence of 10- M NaN, .

Practically all macrophages from cell suspensions treated with conjugated rabbit F(ab')2 antiglobulin were brightly fluorescent. Conversely, macrophages from cell suspensions treated with normal (nonimmune) conjugated F(ab')? were completely unstained (Fig. 3).

Figure 3. Guinea pig peritoneal macrophages, stained in the cold with rabbit F(ab')2 antiglobulin and incubated at 37 O C for 30 min. The fluorescence is mainly localized in small vesicle5 in the cyto- plasm of the cells.

Cells which were stained and washed in the cold and not warmed, appeared like bright fluorescent rings. Short incubation periods in the warm (5- 15 min) resulted in the formation of small and medium-sized spots, as previously described for basophils. Lon- ger incubation times at 37 OC ( u p to 2 h) produced a distribution of the determinants, which was rather different from that observed on basophik The patterns observed can be summa- rized as follows: (a) Formation of large fluorescent spots. These spots were often, bu t not always, collected at one end of the cells. They were discrete entities and classical caps were rarely observed. (b) Appearance of granular formation. These granules were similar t o the autofluorescent granules observed in unstained macrophages. They were often collected on one side of the cell and showed a green fluorescence easily recognizable from the orange of the autofluorescent granules. The appearance of these granular formations could suggest that part of the cytophilic antibody-antiglobulin complexes were pinocytozed by the cells. (c) Fading of the brightness of the fluorescence. The brightness of the fluorescent staining decreased with the increase of the incubation time at 37 'C, and after 2 h, some of the macrophages were weakly stained or unstained. This phenomenon could be par- tially explained by the digestion of the pinocytozed fluorescent proteins. The uncoupling of the antiglobulin from the determinants and (or) the shedding of part of the antibody- antiglobulin complex from the membrane could have also contributed t o the observed fading of the fluorescence.

Macrophages treated with lo-' M NaN3 were covered over with small discrete fluorescent spots and the only changes observed after long incubation (up to 2 h) at 37 "C were a small amount of fading of the fluorescence and the formation of slightly larger spots.


3.3.2. Distribution of immunoglobulin determinants detected by Fab’ antiglobulin

In these experiments, cells prepared and washed as described above were stained with a conjugated rabbit anti-guinea pig (Fab’) and incubated at 37 O C for different periods of time. With this reagent, spot formation was never observed and fluorescence always appeared as a diffuse ring. Macrophages treated with conjugated normal rabbit Fab’ were always unstained. A rapid decrease in the brightness of the fluorescent cell was observed upon incubation for periods of time longer than 5 min. Cells incubated for more than 15 min were completely negative. This phenomenon was also observed in cell suspensions treated with lo-’ M NaN3.

3.3.3. Elution of cytophilic antibody from peritoneal macrophages incubated at 37 O C

The loss of fluorescence observed in the cells stained with F( ab’)2 and particularly with Fab’ antiglobulin after incubation at 37 O C could be explained either by uncoupling of the antiglobulin molecules from the membrane determinants or by elution of the antiglobulin-cytophilic antibody complex from the cell membrane. To solve this problem, it was first necessary t o establish whether cytophilic antibody could be released by unstained macrophages when incubated at 37 OC.

To this end, peritoneal macrophages, prepared and washed as previously described, were incubated for 20 min at 37 O C in medium and washed twice at 37 “C before staining with conjugated F(ab’)2 antiglobulin in the cold. The experiment was also carried out using diluent containing lo-’ M NaN3. Macrophages treated in this way were very weakly stained and, in the great majority, unstained. This was in sharp contrast t o the bright fluorescence obtained in the control preparations incubated for the same length of time in the cold and subsequently washed twice in the cold before staining,

These data, together with the observation that suspensions of lymphocytes and basophils lost little fluorescence after 2 h incubation at 37 OC when stained with Fab’ antiglobulin, show that the observed fading of the fluorescence was due t o the release of the cytophilic antibody-antiglobulin complex from the cell membrane.

3.3.4. Antigen binding b y peritoneal macrophages

In these experiments, peritoneal macrophages from guinea pigs injected with rabbit Fab’ were incubated in the cold with normal rabbit Fab’ of F(ab‘):! conjugated with fluorescein. After appropriate washing, the cells were incubated at 37 “C for different intervals of time.

The pattern observed was similar t o that observed for normal macrophages stained with F(ab’)z antiglobulin, however, the remarkable feature of these cell preparations was the rapid fading of the fluorescence. After 1 h incubation at 37 “C, the majority of the cells were unstained.

4. Discussion

Our experiments show that cytophilic antibodies on basophils and macrophages are mobile in the cell membrane and can be agglutinated by cross-linking with a divalent antibody. The observation that protein molecules can move freely on the plane of the cell membrane is not a new one.

In fact, immunoglobulin receptors [ 1-31 and histocompatibility antigens (41 can be agglutinated on the surface of lymphocytes; histocompatibility antigens have also been shown t o rearrange their distribution o n the fibroblast cell membrane [S]. These observations, together with other recent studies on the structure and the properties of cell membranes, have suggested that the cell membrane could be envisaged as a “fluid mosaic”, in which protein molecules are embedded in a matrix of phospholipids organized as a discontinuous fluid bilayer [ 6 ] . However, the uniform distribution of red cell antigens detected after staining with conjugated hetero- logous antisera reported by various authors [ 1-21, or the patchy distribution of H2 antigen in mouse red cells observed in this laboratory (A. Munro, unpublished results), may suggest that such membrane organization does not hold true for all types of cells.

Cytophilic antibodies, passively absorbed b y macrophages and basophils through their Fc fragment, become the antigen receptors for these cells. In order to be absorbed onto a hydrophobic cell membrane, a hydrophilic antibody molecule requires a molecule on the cell membrane, presumably a protein or a lipoprotein, having a complementary structure for the F c part of the antibody. This molecule, which we will call the “pro-receptor”, is the one which is mobile in the membrane.

The strength of the bond between the cytophilic antibody and the pro-receptor is probably different for macrophages and basophils. Cytophilic antibody is easily eluted from the macrophage membrane after incubation at 37 OC, while this phenomenon does not occur (or it occurs t o a lesser extent) on basophils. The distribution of immunoglobulin determinants on the surface of basophils and macrophages, when reacted with divalent antiglobulin, follows different patterns. In the case of basophils, the determinants are first collected at one end of the cell (cap); they then seem t o be pinocytozed. As regards macrophages, the determinants form clusters which d o not fuse together into a proper cap and the process of pinocytosis seems to take place when the first aggregates are formed. The explanation of these differences is not clear, but it could be that aggregates of different sizes are required on the membrane of the two cells t o start the process of pinocytosis. If the strength of the bond between pro-receptor and cytophilic antibody was different for macrophages and basophils, this could also ac- count for the different distribution of the immunoglobulin determinants. In the case of basophils, the immunoglobulin determinants would provide a rather stable matrix, easily agglutinated by the divalent antiglobulin to form a single large cap o n one end of the cell. In the case of macrophages, the elution of some of the cytophilic antibody-antiglobulin complexes would create some empty areas on the cell membrane, which make lattice formation more difficult, the result being a more patchy distribution of the determinants. The latter could be possible if the process of cap formation occurs by agglutination of spots, facilitated by random move- ment of the cell membrane. This explanation cannot be possible if cap formation occurs b y a systematic transport of the spots t o one end of the cell.

With macrophages, the cytophilic antibody is stabilized on the surface of the cells when it is cross-linked with antiglobulin. The spots of agglutinated cytophilic antibody are probably held by many combining sites t o the surface of macrophages, forming a complex much more firmly bound than a single cytophilic antibody molecule. This phenomenon is also seen when antigen is used to agglutinate the cytophilic antibody,

370 M. Ferrarini, A. Munro and A.B. Wilson Eur. J . Immunol. 1973. 3: 364-370

although t o a lesser extent. In macrophages, the signal t o start pinocytosis seems to be the formation of spots as, o n those cells, caps were never formed. The comparison of pinocytosis initiated with antiglobulin o r with monomeric o r dimeric antigens such as rabbit Fab’ or F(ab’)z shows that the amount of pinocytosis is correlated with the extent of aggregation of cytophilic antibody. This would allow us t o predict that antigens having repeated determinants will be more easily pinocytozed by macrophages.

In another series of experiments we investigated the possibility of a correlation between aggregation of cytophilic antibodies on the membrane (i .e. spot formation) and triggering of basophils. Basophils were incubated in the presence of Ca++ and Mg++ with F(ab’Iz and Fab’ antiglobulin. If aggregation of receptors was obligatory for the triggering of basophils, then the activation of basophils would only have been expected with the divalent antibody. A similar approach has already been used by Ishizaka and Ishizaka [ 181, who could detect histamine release from human basophils when treated with guinea pig IgG or F(ab‘)l anti-IgE, but not when treated with Fab‘ anti-IgE. The morphological changes that we have observed after incubation J f basophils with F( ab’)2 antiglobulin clearly indicated that the basophils were activated by such treatment. These changes were observed even at high dilutions of F(ab’)z.The activation of basophils was not observed with Fab’ unless the reagent was used at a high concentration. These concentrations of Fab’ were also the only ones able to stain basophils as a clear fluorescent ring. Incidently, the concentration of Fab’ at which activation of basophils was obtained was either near t o the hlghest concentration of Fab‘ used by the Ishizakas or higher. The interpretation of our results poses several problems. It is obvious that if a monovalent Fab‘ is capable of triggering a cell, a higher concentration of it would be needed than that of the corresponding F(ab’)z. This is because of the different dissociation constants of F(ab‘)2 and Fab’ [ 16,191. In this respect, fluorescent antibodies are particularly useful, as it is possible to see if the Fab’ antiglobulin has bound t o the surface of the basophils. Obvious- ly, when high concentrations of Fab’ are used, the observed activation of basophils could be explained by the presence of a small amount of contaminating F(ab’)z.

This possibility can never be excluded, particularly as the F(ab‘), is active at such a low concentration. However, all possible steps were taken t o d o so. The Fab‘ preparation failed to form spots on the surface of cells and also failed t o agglutinate sheep red cells coated with guinea pig antibody. Furthermore, the method of preparation of the Fab’ and the additional reduction and alkylation were designed t o exclude divalent molecules.

It is also possible that Fab’ is able to form microaggregates of immunoglobulin molecules on a cell membrane. When a

large number of Fab’ molecules are bound t o the immunoglobulin determinants of a cell membrane, microaggregates of a size not detectable by fluorescence may be formed as a result of a weak interaction between adjacent Fab‘ molecules. It has been suggested that a conformational change in one of the combining sites of a receptor molecule may be sufficient to change the state of a cell [20]. It is possible that the monovalent Fab’ activates basophils by a similar process. Unfortunately, we are, at the present, unable t o distinguish between a conformational signal and a lattice formation via Fab’-to-Fab’ interaction.

We thank ProK R.R.A. Coombs and Dr. A . Feinstein for very helpful discussions and Miss Yvonne Petit and Mrs. Rose-Maty Millar for their help. This work was supported by grants from theMedica1 Research Council o f Great Britain.

Received October 5 , 1972; in revised form March 2, 1973.

5. References

1 Taylor, R.B., Duffus, P . H . , Raff, M.C. and de Petris, S., Nature-

2 Loor, F., Forni, L. and Pernis, B., Eur. J. Immunol. 1972.2: 203. 3 Unanue, E.R., Perkins, W.D. and Karnovsky, M . J . , J. Immunol.

4 Preud’homme, J.L., Neauport-Sautes, C., Piat, S. , Silvestre, D. and

5 Frye, L.D. and Edidin, M., J. Cell. Sci. 1970. 7: 3 19. 6 Singer, S.J. and Nicholson, G.L.,Science 1972. 175: 720. 7 Sullivan, A.L., Grimley, P.M. and Metzger, H . , J . Exp. Med. 1971.

8 Wilson, A.B., Marchand, R.M. and Coombs, R.R.A., Lancet 1971.

9 Ishizaka,K. and Ishizaka,T., Clin. Alletxy 1971. I : 9. 10 Levy, D.A. and Osler, A.G., J. Immunol. 1966. 97: 203. 11 Ishizaka, T., Ishizaka, K., Johanson, S.G.O. and Bennich, H. ,

12 Hastie, R., Clin. Exp. Immunol. 1971.8: 45. 13 Lichtenstein, L.M. and Osler, A., J. Exp. Med. 1964. 120: 507. 14 Wilson, A.B. and Coombs, R.R.A., Int. Arch. Allergy Appl.

15 Cebra, J . J . and Golstein, G. ,J Immunol. 1965.95: 230. 16 Metzger, H.,Ann. N.Y. Acad. Sci. 1971. 190: 322.

17 Wessells, N.K., Spooner, B.S., Ash, J.F., Bradley, M.O., Ludvena, M.A., Taylor, E.L.,Wrenn, J.T. and Yamada, K.M.,Science 1971. 171: 135.

New. Biol. 1971.233: 225.

1972.108: 569.

Kourilsky, F.M., Eur. J. Immunol. 1972.2: 297.

134: 1403.

i: 1325.

J. Immunol. 1969.102: 884.

Immunol. 1971.40: 19.

18 Ishizaka, K. and Ishizaka, T., J. Immunol. 1969. 103: 588. 19 Greenbury, C.I., Moore, O.H. and Nunn, L.A.C.,Immunology

20 Cohn, M., Ann. N. Y . Acad. Sci. 1971. 190: 529. 1965.8: 420.

cytophilic antibody: correlation of its distribution with activation of basophils and macrophages

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