Capping, independent of metabolic energy, of surface immunoglobulin (S-Ig) on ‘Hairy cells’ from patients with Hairy cell leukaemia (HCL) is described. As controls leukaemic cells from a patient with a prolymphocytic leukaemia (PLL) and blood lymphocytes from healthy individuals were used. The specificity of the energy-independent capping of the HCL-cells as compared to the controls was tested by incubation of the cells at 4 °C in the presence of 01 M· sodium azide with different FITC-labelled ligands.

In order to find an explanation for this phenomenon, the influence of cytochalasin B, colchicine and the combination of both drugs on capping of S-Ig and concanavalin (Con A)-receptors at 37 °C was investigated. Furthermore the effect of Con A on S-Ig capping and vice versa was studied. The results show that only S-Ig on HCL cells could form caps at 4 °C in the presence of sodium-azide. Cytochalasin B alone induced a strong inhibition of Con A capping on all 3 cell types, whereas S-Ig capping was unaffected. Colchicine alone had practically no effect. Anti-Ig inhibited subsequent patch and cap formation with Con A on both HCL cells and PLL cells, whereas Con A caps and patches were redistributed by anti-Ig on PLL cells, but not on HCL cells. Conversely, Con A could link S-Ig to other receptors, leading to inhibition of S-Ig capping at 4 °C on HCL cells and to co-capping of S-Ig at 37 °C on both cell types. In addition Con A induced redistribution of S-Ig caps. The combination of cocapping of S-Ig by Con A, followed by redistribution of the caps by FITC-anti-Ig simulated inhibition of S-Ig capping by Con A on PLL cells.

The major conclusions are: in some cases inhibition of capping may actually be caused by redistribution of caps; the energy-independent capping cannot be explained by free diffusion of S-Ig in the membrane through lack of any connexion with receptor-mobility regulating systems. It is proposed that the energy requirement of capping is needed to inactivate a specific mechanism, which restrains receptor mobility and which is non-operative in HCL cells.

Capping is thought to result from a countercurrent flow of membrane components in which cross-linked clusters of receptors grow to patches that are transported towards one end of the cell, while the remaining membrane components flow in the opposite direction (De Petris & Raff, 1972). This phenomenon is considered to be energy-dependent, since low temperature or the presence of drugs, that inhibit metabolic activity, prevent capping (Taylor, Duffus, Raff & De Petris, 1971; Loor, Forni & Pernis, 1972). Futhermore, a multivalent ligand is needed (Taylor et al. 1971) and a correlation has been found between the density and distribution of surface receptor molecules and their fate after combination with the ligands (Kamovsky, Unanue & Leventhal, 1972).

Experiments, performed mainly with murine lymphoid cells, have shown that cytoskeletal components, such as microfilaments and microtubules may play an important role in the capping phenomenon (reviewed by Nicolson, 1976). It has been suggested that microfilaments are part of a contractile system, presumably containing actin and myosin (Schreiner, Fujiwara, Pollard & Unanue, 1977; Lazarides & Weber, 1974; Weber & Groeschel-Stewart, 1974), that is responsible for the displacement of membrane components (De Petris & Raff, 1972). Drugs, such as cytochalasin B, which disrupt microfilaments (Wessels et al. 1971), produce variable effects on capping of Con A receptors and surface immunoglobulin (S-Ig), ranging from negligible (Unanue, Karnovsky & Engers, 1973) to moderate inhibition (De Petris, 1974; Edelman, Yahara & Wang, 1973).

Microtubules seem to have an inhibitory effect on receptor mobility. Drugs, such as colchicine, which disrupt microtubules (Borisy & Taylor, 1967), either have little effect or even enhance capping of these receptors (De Petris, 1974; Yahara & Edelman, 1973). The use of both drugs together results in a striking inhibition of Con A-receptor capping and a similar or somewhat smaller inhibition of S-Ig capping.

Preincubation of cells with Con A inhibits the subsequent formation of patches and caps of other membrane receptors, such as S-Ig and θ antigen (Yahara & Edelman, 1972, 1973). This inhibition was only obtained when tetravalent Con A, but not divalent Con A, was used (Yahara & Edelman 1972). The inhibitory effect could be partially blocked (Edelman et al. 1973) or reversed (Schlessinger et al. 1977) by microtubule-disrupting drugs. In cells bearing Con A caps or S-Ig caps, treatment with cytochalasin B (De Petris, 1974), KCN or iodoacetic acid (Sällström & Alm, 1972) induced reversion of the caps to a ring and patch wise distribution.

From all these observations it has been postulated that microtubules and microfilaments are involved in the regulation and maintenance of the distribution of cellsurface receptors, which are supposed to be linked to these cytoskeletal elements.

This paper describes the occurrence of capping, independent of energy production, of Ig-anti-Ig complexes on ‘Hairy cells’, i.e. cells from 2 patients with a Hairy-cell leukaemia (Catovsky, 1977; Fu, Winchester, Rai & Kunkel, 1974), and compares this with capping of S-Ig and Con A receptors on other lymphoid cells. Since cytoskeletal elements are supposedly involved in receptor mobility, the effect of cytochalasin B, colchicine, and of both drugs together, on capping of S-Ig and Con A receptors on these cells was investigated. The inhibition of S-Ig capping and redistribution of preformed S-Ig caps by Con A and vice versa was also studied in the presence and absence of colchicine. The results are discussed and an explanation for the observed energy-independent capping of S-Ig is proposed.

Patients and controls

Two patients with a Hairy-cell leukaemia (HCL), one patient with prolymphocytic leukaemia (PLL) and 15 healthy individuals were used.

The first patient with a HCL (HCL-1) was a 43-year-old male. Clincial findings: anaemia, thrombocytopenia and a white-cell count of 18·4 × 109/l. with 98% lymphocytes; absence of palpable lymph nodes, a normal liver, and a very large spleen weighing 3-1 kg. The second patient with a HCL (HCL-2) was a 69-year-old male. Clinical findings: anaemia, thrombocytopenia and a white-cell count of 24·5 × 109/l. with 94% lymphocytes; absence of palpable lymph nodes and of hepatomegaly; splenomegaly. The blood smears and imprints of the spleen from both HCL patients showed large mononuclear cells with abundant cytoplasm and containing tartrate-resistant acid phosphatase. In the electron microscope numerous slender cytoplasmic projections were seen on most cells.

The patient with PLL was a 71-year-old male. Clinical findings: absence of palpable lymph nodes and hepatomegaly; a large spleen; haemoglobin content 3’9mmol/l; white-cell count 100·4 ×109/l. with 98% lymphocytes; the bone marrow consisted of 94% lymphoid cells with little strongly basophilic cytoplasm and 1 or 2 nucleoli; the lymphoblasts in the peripheral blood stained weakly with periodic acid-Schiff (PAS), whereas the blasts in the bone marrow did not; hypogammaglobulinaemia (5·3 g/1).

Preparation of cell suspensions

Mononuclear cell suspensions were isolated from defibrinated blood of the patients with HCL and with PLL, and of controls on a Ficoll-Isopaque layer (Pharmacia, Hygaard) with a density of 1 ·079 g/ml, according to the method described by du Bois, Schellekens, de Witt & Eysvoogel (1973). Next, the cells were resuspended in a culture medium, consisting of sterile RPMI 1640 (Flow Laboratories) supplemented with 20% (v/v) inactivated (30 min, 56 °C) foetal calf serum (FCS, Gibco), glutamine (0·03 % w/v, Merck), penicillin (100 IU/ml), streptomycin (100 μg/ml) and HEPES (12·5 mM).

The cell number was determined with a Coulter counter, model ZF, and viability was assessed by trypan-blue exclusion (0·1 % w/v).

Cell preservation

The cells, suspended in the culture medium containing 10 % (v/v) dimethylsulphoxide (DMSO) at a concentration of 25–50 × 106/ml, were frozen and stored in liquid nitrogen and thawed after various intervals as described by du Bois et al. (1976). After being rapidly thawed at 37 °C the cells were incubated in the culture medium for 2 h at 37 °C before experimentation.

Enrichment of B lymphocytes

After being thawed, the cells from the healthy controls were suspended at o °C in the culture medium at a concentration of 3–5 × 106/ml. Next, 10 ml of this suspension were layered upon 10 ml of the Ficoll-Isopaque mixture (d = 1·079 g/ml) and centrifuged for 20 min at 1000 g at 22 °C. The cells at the interface were collected and incubated for 2 h at 37 °C in the culture medium before experimentation.

Determination of rosette-forming cells

E. rosettes, serving as a marker for T cells, were formed with sheep red-blood cells. Cells bearing receptors for the Fc portion of immunoglobulin G were detected by the formation of rosettes with human erythrocytes, coated with anti-Rh(D) antibodies (EAIgG rosettes). The methods used have been described recently (Splinter, Bom-van Noorloos & van Heerde, 1978).

Membrane immunofluorescence

The percentage of cells bearing surface immunglobulin (S-Ig) and the type of S-Ig were determined with the following antisera. FITC-conjugated sheep antiserum polyspecific for human Ig (C.L.B. batch SH 17-1-F1 and SH 17-1-F3) FITC-conjugated rabbit antisera monospecific for the major heavy-chain classes (α, β and γ) and the light-chain classes (K and λ) of immunoglobulin (Dakopatts, batch F1091, F1092, F109K and F109L; C.L.B. batch KH 16-103-F3) and rabbit Fab anti-human γ (CLB). Concanavalin A (Con A) receptors were detected with FITC-labelled Con A (F-Con A, Miles-Yeda Ltd). A lymphocyte-specific antigen was demonstrated by indirect immunofluorescence with a rabbit anti-human lymphocyte globulin (ALG) (C.L.B. batch 17237-75-1) and an FITC-labelled swine anti-rabbit IgG (Dakopatts, batch F2190). Direct immunofluorescence was performed by incubation of a pellet containing 0·5–1·0 × 106 cells, for 30 min at 4 °C in 2 drops of antiserum. Next, the cells were fixed with a 1 % solution of paraformaldehyde prepared with PBS (pH 7·2), washed twice with PBS, supplemented with 0·2 % (v/v) of bovine serum albumin (BSA, Sigma), resuspended in 1 drop of a glycerol-PBS mixture (3:1, pH 7·8), mounted on a glass slide, covered and sealed. In this condition they were kept in the dark at 4 °C.

In the indirect-immunofluorescence method a second incubation for 30 min at 4 °C was performed with an FITC-labelled swine anti-rabbit IgG (Dakopatts, batch F2190) after washing the cells 4 times with PBS, supplemented with 0·2 % (v/v) of BSA. Then the cells were fixed. All the antisera were diluted with PBS, containing 0·1 M sodium azide (pH 7·2).

Capping of surface immunoglobulin and Con A receptors

General procedures

Before every experiment the cells were washed with cold (4 °C) PBS, supplemented with 0·2 % (v/v) BSA. All the washings were performed with the PBS-BSA mixture. Next, 1–5 × 107 cells were suspended in 1 ml PBS. For every capping experiment, 0·1 ml of this cell suspension was incubated with 0·1 ml of a solution of a ligand or drug. More details will be given in the section on Results. The following materials were used: FITC-labelled polyspecific sheep anti-human Ig and unlabelled sheep anti-human Ig (C.L.B. batch SH 19-01-Pol), concanavalin A (Con A, Calbiochem), FITC-labelled Con A, goat anti-Con A (anti-Con A, Nordic), succinyl Con A (succ. Con A) prepared according to Günther et al. (1973), colchicine (Merck) and cytochalasin B (Serva). When capping was investigated at 4 °C, all the materials were kept at o° C until the cell suspension and solution of the ligand were mixed and incubated at 4 °C.

Immediately after every experiment, the cells were fixed and stored as described above. A cap was defined as the accumulation of fluorescence on less than half the surface area of the cell, while at the same time the remaining surface was devoid of fluorescence. Capping was scored as a percentage of 200 fluorescent cells. Each of the percentages of capping reported in this article is the mean of at least 3 different experiments.

Electron-microscopical immunocytochemistry

The cells were washed twice with PBS and concentrated to about 10 × 106/ml 0·1 M PBS (pH 7·2). Next, they were incubated with ferritin-conjugated goat anti-human IgG (GAHIg-Fer, Nordic) with and without prefixation in 2 % glutaraldehyde. GAHIg-Fer was added at a concentration of 6 mg protein/ml. After incubation for 30 min at 37 or 4 °C, the cells were washed twice with the same buffer and postfixed for 15 min in 1 % OsO4. After conventional dehydration in a graded ethanol series, block staining with uranyl acetate in 100 % ethanol and embedding in a mixture of Epon and Araldite, the cells were sectioned and studied in a Philips 300 electron microscope.

Immunological characteristics of the cell suspensions

Table 1 shows the immunological membrane properties and viability of the 2 Hairy-cell leukaemias (HCL), the prolymphocytic leukaemia (PLL) and 3 mixtures of mononuclear blood cells from 15 healthy individuals (controls). The suspensions from the 2 HCL patients consisted mainly of B cells. The cells of HCL-1 carried a monoclonal surface immunglobulin (S-Ig), γK and the cells of HCL-2 had only a light-chain λ on their membrane. The suspension of cells from the PLL patient also consisted of monoclonal malignant B cells, and these cells, as will be shown later, also showed capping with Con A and anti-Ig, in contrast with cell suspensions from 12 CLL patients (unpublished observations). Therefore PLL-cells were chosen as a control in addition to the lymphocytes from healthy individuals. The latter cell suspensions consisted of a mixture of T and B cells and monocytes. The percentage of B cells was increased from 9 – 16% to 22 – 40% by density separation (see Methods) to facilitate the experiments with capping of S-Ig.

Percentage of capping with different ligands at 4 °C in the presence 0·1 M sodium azide

During the immunological investigations of the HCL cells it had been observed that incubation of the cells with anti-Ig, even at a temperature of 4 °C and in the presence of the 0·1 M sodium azide, resulted in the formation of caps. We, therefore, investigated whether (a) this abnormal property was confined to S-Ig capping, (b) polyvalency of the ligand was needed, and (c) freshly isolated HCL cells and the control cells would show the same property. For this purpose, 0·1 ml of each cell suspension was preincubated for 30 min at 4 °C in the presence of 0·1 M sodium azide, followed by the addition of 0·1 ml of a solution of different ligands, as shown in Table 2, and incubation for 45 min at 4 °C. Under these conditions approx. 50% of the Hairy cells were capable of capping only with a divalent antibody specifically directed against their S-Ig.

Antisera not directed against the light or heavy chain on the membrane, rabbit Fab-anti-γ, Con A and anti lymphocyte globulin (ALG) produced no caps. The PLL cells and normal mononuclear cells did not cap at all (Table 2). Examination of HCL cells before and after freezing and thawing led to identical results. Prefixation of the cells with paraformaldehyde prevented cap formation. It was concluded that capping of S-Ig on HCL cells is a phenomenon that occurs without production of energy and needs a divalent antibody, directed against the S-Ig.

Form and location of the caps on HCL cells at 4 and 37 °C

Unanue, Ault & Kamovsky (1974) showed that caps on stationary lymphoid cells occupy a larger area of the cell surface overlying the Golgi region than on mobile cells. Likewise, immobilization of polymorphonuclear leukocytes induced the formation of caps on the central region of the cells (Ryan, Borysenko & Karnovsky, 1974). Therefore, it was investigated whether S-Ig caps, formed at 4 °C on HCL cells, had similar properties. Comparison of the pattern of fluorescence obtained with anti-Ig and Con A on HCL cells at 4 and at 37 °C and on PLL cells at 37 °C showed (Fig. 1 A–H) that the forms of the caps were similar. However, electron microscopy of the cells, capped at 4 and 37 °C with ferritin-labelled anti-Ig, showed (Fig. 2 A – D) that at 4 °C the caps occupied a larger surface area than at 37 °C and that uropod formation occurred at 37 but not at 4 °C. These observations support the above-mentioned findings of Unanue et al. (1974).

Capping at 4 and 37 °C with different concentrations of FITC-anti-Ig and FITC-Con A

In the next series of experiments the concentration dependency of capping with anti-Ig and Con A was investigated at 4 and 37 °C without sodium azide. Anti-IgF was used at dilutions of 1:1, 1:5 and 1:10, and Con AF in 5 different solutions, ranging from 25 to 800 μg/ml. At 4 °C, only the HCL cells formed caps with anti-Ig. At 37 °C, capping with anti-IgF and Con AF was observed on the HCL cells, the PLL cells and the control lymphocytes. Prefixation of the cells prevented capping in all cases. Practically no differences in the percentages of capping at different concentrations of anti-Ig and Con A were observed. Therefore, it was arbitrarily decided to use anti-Ig at a final dilution of 1:5 and Con A at a final concentration of 400 μg/ml in subsequent experiments.

Effect of colchicine and cytochalasin B on capping at 37 °C

Since cytoskeletal elements, such as microfilaments and microtubules influence capping of membrane receptors (see Introduction), the effect of various concentrations of cytochalasin B, colchicine and combinations of both drugs on the percentage of cells, capped with anti-Ig and Con A, was investigated. For this purpose the cells were preincubated for 30 min at 37 °C with the drug(s), followed by a second incubation for 30 min at 37 °C in the presence of both the drug(s) and the ligand. Table 3 shows that cytochalasin B alone and in combination with colchicine induced a strong inhibition of Con A capping on all 3 cell types, whereas no inhibition was observed on the capping of S-Ig. Colchicine alone had practically no effect (not shown). The results obtained with HCL-1 were similar to those obtained with HCL-2. It was concluded that capping of Con A receptors is dependent on the presence of a functional cytochalasin B-sensitive system, whereas S-Ig capping is not.

Modulation of S-Ig capping by Con A and vice versa

The next series of experiments was undertaken to investigate whether S-Ig on HCL cells had any connexion with systems that regulate receptor mobility or could diffuse freely in the membrane through lack of this connexion. Therefore, the effect of Con A on the mobility of S-Ig and vice versa was investigated. The experiments were performed only with HCL cells and PLL cells, since the interpretation of the results obtained with normal lymphocytes, was difficult owing to the heterogeneity of these cells. The effect of anti-Ig capping of Con A receptors was investigated by incubating the cells with anti-Ig for 30 min, washing twice with PBS or fixing with paraformaldehyde and subsequent incubation with FITC-Con A for 30 min. As a control the incubation with anti-Ig was replaced by PBS. Table 4 (a)(b) shows that pre-incubation with anti-Ig completely inhibited subsequent capping of FITC-Con A on both cell types. Even patch formation of Con A receptors was inhibited. In the presence of colchicine (10−3 M) a similar inhibition of cap and patch formation of Con A receptors was observed (Table 4(c)(d)). Co-capping of Con A receptors by anti-Ig was not observed (Table 4 (e) (f)). Incubation with anti-Ig after preincubation with FITC-Con A (Table 4(g)(6)) induced no change in the percentage of Con A caps on HCL cells, whereas on PLL cells the percentage of the patches and caps was greatly reduced. The total percentage of fluorescent cells remained constant between 80 – 90.

It was concluded that anti-Ig inhibited subsequent capping and patch formation of Con A receptors, in the presence of colchicine on both cell types, and induced redistribution of Con A caps and patches on PLL cells only.

The effect of preincubation with Con A on subsequent S-Ig capping was investigated in experiments similar to those described above. As an additional control for the influence of Con A, the cells were also preincubated with divalent succinyl-Con A (250 μg/ml) and with succinyl-Con A followed by anti-Con A (undiluted). The results (Table 5 (a) (b)) indicate that preincubation with Con A apparently inhibited subsequent S-Ig capping on HCL cells at 4 °C and on PLL cells at 37 °C, but not on HCL cells at 37 °C.

The next experiments, in which preincubation of Con A was followed by fixation and incubation with FITC-anti Ig, showed (Table 5 (c) (d)) that Con A co-capped S-Ig under capping conditions at 37 °C. It is noteworthy that the percentage of S-Ig caps, formed after incubation with Con A without subsequent fixation (Table 5 (b)) and with subsequent fixation (Table 5 (d)) differed greatly on PLL cells but not on HCL cells. This may be explained as follows. Con A is capable of cross-linking Con A receptors with S-Ig. At 37 °C this interaction leads to co-capping of S-Ig, whereas at 4 °C capping of S-Ig is inhibited by cross-linking of S-Ig to other Con A-binding receptors (Table 5 (d)), which cannot form a cap at this temperature. Since anti-Ig effectively redistributes Con A caps on PLL cells, but not on HCL cells (Table 4 (h)), the percentage of Con A caps, visualized by FITC-anti Ig (Table 5 (b)), is decreased by subsequent incubation with anti-Ig on PLL cells but not on HCL cells. It should be emphasized that the apparent inhibition of S-Ig capping by Con A on PLL cells may be caused by redistribution of Con A caps by anti-Ig. Preincubation with succinyl-Con A did not inhibit subsequent S-Ig capping on HCL cells at 4 °C or on PLL cells at 37 °C (Table 5 (e)), whereas preincubation with succinyl-Con A followed by antiCon A produced an inhibition of S-Ig capping similar to that produced by Con A (Table 5 (f)). The results seem to imply that the inhibition of S-Ig capping by Con A is dependent on a higher valence and more extensive cross-linking of Con A receptors than can be obtained by divalent succinyl-Con A.

Incubation with Con A after the formation of S-Ig caps showed (Table 5 (g)(h)) that the percentage of caps was reduced to about half the original value on both cell types at 4 °C. However, in the presence of colchicine (10−3M) the percentage of S-Ig caps was substantially more reduced in both cell types at 37 °C (Table 5 (i)(j)). As Con A could redistribute S-Ig caps at 4 °C on HCL cells (Table 5 (h)), it seems that redistribution of caps is energy-independent. The observation that Con A cannot form caps at 4 °C indicates that binding of Con A is sufficient to redistribute S-Ig caps. Finally, the inability of anti-Ig to induce redistribution of Con A caps on HCL cells at 37 °C constitutes the only difference between HCL cells and PLL cells with regard to the modulation of receptor mobility.

The purpose of this investigation was to describe capping of S-Ig on ‘Hairy cells’, independent of energy production and to compare the different mechanisms by which receptor mobility is supposed to be influenced, in these cells with other lymphoid cells. This might produce more information about the mechanisms leading to capping and a possible explanation for the observed energy independency. From the first experiments it is concluded that capping, independent of energy production, can be induced by divalent anti-Ig on HCL cells. Support for this observation has been found in an article by Fu et al. (1974), in which abnormal capping of S-Ig at 4 °C on Hairy cells is mentioned, but no allusion is made to possible energy independency of this phenomenon. Our observation casts some doubt on the hypothesis that movement of the cell membrane is necessary to direct the cap to the trailing part of the cell (Unanue et al. 1974) and that prevention of patch formation at 4 °C may be due to an increase of the viscosity of the phospholipid layer of the cell membrane (Unanue & Kamovsky, 1973).

The major conclusions from the next series of experiments, designed to test in what way the abnormal mobility of S-Ig on HCL cells could be influenced, can be summarized as follows:

Cytochalasin B, colchicine and the combination of both drugs has no effect on S-Ig capping on all 3 cell types

On the other hand, Con A capping was strongly inhibited by cytochalasin B with or without colchicine on all 3 cell types. Investigations with murine lymphocytes have shown (De Petris, 1974, 1975; Unanue & Karnovsky, 1974) that the combination of cytochalasin B and colchicine strongly inhibits Con A capping and to a lesser extent S-Ig capping. This relative difference between our observations and those of De Petris & Unanue may reflect a different mechanism of S-Ig capping on human lymphocytes in comparison with mouse lymphocytes and may indicate that S-Ig capping is independent of cytochalasin-B-sensitive microfilaments.

Anti-Ig inhibits both patch and cap formation of Con A receptors on all cells, and this effect is not influenced by colchicine

Redistribution of Con A patches and caps is induced by anti-Ig on PLL cells, but not on HCL cells. Since these effects of anti-Ig have, to our knowledge never been described, it is impossible to conclude whether they are characteristic for normal human lymphocytes or leukaemic cells. Investigations to solve this question with purified normal lymphocytes and monoclonal B cells from non-Hodgkin lymphomas are in progress.

Con A causes co-capping of S-Ig, as already describedfor mouse lymphocytes (De Petris, 1975)

Co-capping of Con A receptors by anti-Ig was not observed. This is probably because the number of Con A receptors on the membrane is much greater than the number of S-Ig receptors. Therefore, an accumulation of a small number of Con A receptors, co-capped by anti-Ig will be overshadowed by the fluorescence of the remaining Con A receptors.

The inhibitory effect of Con A on S-Ig capping at 4 °C on HCL cells can be explained by cross-linking of S-Ig to other Con A receptors in agreement with the observations of De Petris (1975) 

The inhibitory effect at 37 °C on PLL cells seems to be caused by redistribution of the Con A caps by anti-Ig. In analogy with the effect of anti-Ig Con A induces redistribution of about 50% of the S-Ig caps on HCL cells at 4 and 37 °C, and on PLL cells at 37 °C. It seems that binding of Con A is sufficient to redistribute S-Ig caps (Table 5). Although Yahara & Edelman (1972) mention once that S-Ig patches and caps are not affected by Con A, our data suggest that redistribution of caps should also be carefully evaluated in any experiment on the modulation of receptor mobility in addition to co-capping and inhibition of capping. Furthermore, in agreement with Sallstrom & Alm (1972), who found that redistribution of caps can be induced by metabolic inhibitors such as KCN and iodoacetic acid, we demonstrated that redistribution occurs at 4 °C and is therefore independent of energy production. Since neither the inhibition of Con A-receptor capping by anti-Ig nor the redistribution of preformed S-Ig caps by Con A can be explained by simple cross-linking of one ligand by the other, these observations indicate that S-Ig is connected with Con A receptors through a system that regulates receptor mobility. This indicates that the energy-independent capping of S-Ig is not due to the ability of S-Ig to diffuse freely in or on the membrane through lack of any connexion with such a regulating system.

Therefore, it is proposed that the energy, normally required for capping, is necessary to inhibit actively a specific restraining mechanism, which is non-operative in HCL cells for S-Ig.

Since the only difference between HCL cells and PLL cells with regard to the modulation of receptor mobility is constituted by the inability of anti-Ig to redistribute Con A caps on HCL cells, it is possible that the above-mentioned specific restraining mechanism may be responsible not only for the inhibition of receptor mobility but also for the redistribution of caps.

Many more investigations have to be performed before we can define the nature of the mechanisms for regulating receptor mobility and their significance for growth control. The use of cells, such as HCL cells, which may lack some of the regulating mechanisms can be helpful in this respect and prompts the investigation of other malignant lymphoid cells. An additional reason to investigate capping of S-Ig and Con A receptors in lympho-proliferative diseases derives from the findings of Ben Bassat et al. (1977) that lymphocytes from patients with chronic lymphocytic leukaemia (CLL), Hodgkin’s disease or one of several non-Hodgkin lymphomas do not show capping with Con A. We also observed the absence of capping of S-Ig and Con A receptors on 12 samples of CLL cells (unpublished observations). In view of these findings it is rather surprising that HCL cells and PLL cells show Con A capping. This may suggest that Con A capping of leukaemic cells is a specific property, related to a certain phase of differentiation or that Con A capping is a property of a specific subpopulation of spleen lymphocytes, from which both HCL and PLL may be derived, as was recently suggested by Catovsky (1977). It certainly indicates that the usefulness of Con A and anti-Ig capping as an additional diagnostic marker of lymphoproliferative diseases should be further explored.

This investigation was supported financially by the Koningin Wilhelmina Fonds (KWF) grant nr. WLV 1–9.

We are very grateful to Dr H. de Langen, internist at the Central Hospital Alkmaar, The Netherlands, for kindly providing the material and clinical data of the two patients with Hairy cell leukaemia.

Special thanks are due to A. van Beek, A. Bom-van Noorloos and H. Spiele for their technical assistance.

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