Int. J. Cancer: 21, 561-569 (1978)
ALTERED MEMBRANE-ASSOCIATED FUNCTIONS I N CHRONIC LYMPHOCYTIC LEUKEMIA CELLS Tore GODAL1, 4, Aase HENRIKSEN l, Jens-Gustav IVERSEN z, Tone 0. LANDAAS and Tore LINDMO Laboratory for Immunology, Department of Pathology, Norsk Hydro's Institute for Cancer Research, The Norwegian Radium Hospital, Oslo 3; Institute for Physiology, University of Oslo, Karl Johans gate 47, Oslo I ; and Department of Biophysics, Norsk Hydro's Institute for Cancer Research, The Norwegian Radium Hospital, Oslo 3, Norway
Peripheral blood lymphocytes consisting mainly of neoplastic B cells from patients with chronic lymphocytic leukemia (CLL cells) showed a markedly reduced response to the human B-cell mitogens anti$, microglobulin, Sepharose-bound protein A and Sepharose-bound anti-human immunoglobulin (anti F(ab'),) in all of nine patients studied. O n the other hand, CLL cells from three out of eight patients tested responded well to the calcium ionophore A23187. Sepharose-bound protein A and anti-p2 microglobulin also failed to induce increased uptake of 'bRubidium (potassium analogue) in CLL cells as compared to B-cell-enriched preparations of normal peripheral blood lymphocytes. The capacity of CLL cells to cap various surface markers including 8, microglobulin was reduced. O n the other hand, surface concentrations of 8, microglobulin were not reduced as measured by fluorescein-labelled anti-j3,-micrbklobulin in singlccell cytofluorometry. It is concluded that various membrane-associated events elicited by ligandreceptor interactions are altered or blocked in CLL cells.
variety of mitogens, including anti Bz microglobulin (anti &M), Sepharose-bound protein A, and Sepharose-bound anti-F(ab), immunoglobulin, which have been reported to be B-cell mitogens (Ringden and Moller, 1975; Solheim, 1974; Forsgren et a/., 1976; Moller and Landwall, 1977; Godal (unpublished observations)). Since CLL cells responded poorly to these mitogens also, attempts were made to localize the apparent blockade of the mitogenic signal at the sub-cellular level. Comparative studies on anti-ifl,M binding capacity, capping and effect on membrane transport of ions in CLL cells as compared to normal PBL or B-cell-enriched preparations all suggested that the membrane-associated events elicited by ligand-receptor interactions may be altered or blocked in CLL cells. In CLL cells from some patients the putative membrane block could apparently be bypassed by the Ca++ ionophore, A23187.
Peripheral blood lymphocytes (PBL) from patients with chronic lymphocytic leukemia (CLL) have been found to give a poor or delayed response to phytohemagglutinin (PHA) (Bernard et al., 1964; Robbins, 1964; Oppenheim et al., 1965; Bouroncle et al., 1969; Han, 1973), concanavalin A (Con A) and pokeweed mitogen (PWM) (Novogrodsky et al., 1972; Smith et al., 1972, 1973; Catovsky et al., 1972; Konig et al., 1972; Frnrland et al., 1972). The delayed response could be mimicked by reducing the number of initially responding cells, indicating that the number of responding cells in CLL is reduced (Robbins and Levis, 1972 ; Schweitzer et al., 1973). By various separation procedures such as albumin gradients (Schweitzer et al., 1973) or the sheep red cell rosette technique (Wybran et a[., 1973), the responding cells could be separated out from the CLL cetls, suggesting that the responding cells were mainly normal PBL of CLL patients. This has also been clearly demonstrated in a patient in whom the CLL cells could be morphologically identified by characteristic crystalline IgA inclusions (Smith et al., 1973). However, these studies are complicated by the fact that Con A and PHA are mainly T-cell mitogens in man and PWM is mitogenic to both T and B cells (Greaves et al., 1974), whereas CLL cells usually have B-cell characteristics. In the present investigation the mitogenic responses of CLL PBL have been reinvestigated using a
Patients
MATERIAL AND METHODS
Blood from nine CLL patients, aged between 49 and 79 years, was obtained from various hospitals
in Oslo. None of the patients were receiving treatment at the time of the investigation. The findings for each patient with regard to leukocyte counts, surface characteristics of cells and proportion of T and B cells are set out in Table I. Control subjects
Healthy blood donors from the Red Cross Blood Center, Oslo, served as control subjects. Their ages ranged from 22 to 57 years. Cells
PBL were prepared from fresh heparinized blood (10 units preservative-free heparin/ml) by the FicollIsopaque method (Bnyum, 1968) as described elsewhere (Heier et al., 1977). B-cell-enriched preparations were made by using the sheep red cell rosette method as described by Greaves and Brown (1974). I n order to reduce the number of monocytes in the B-cell preparations, in Received: February 3, 1978. To whom requests for reprints should be sent.
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some experiments the lymphocytes were cultured in Falcon flasks at 37" C overnight (20% AB serum in RPMI 1640). Non-adherent cells were subsequently removed and B cells isolated by the sheep red cell rosette method. Such B-cell-enriched preparations were found to contain 58 1 4 % Ig-positive cells and 4 1 % E-rosette-positive cells. Mitogen stimulation
ri
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Cells were cultured in round-bottomed Linbro micro-titration plates with 2 x lo5 cells per well in 0.1 ml RPMI 1640 medium and 0.05 ml mitogen with a final concentration of human AB or inactivated fetal calf serum (FCS) (Gibco, Glasgow, Scotland) of 10%. The cultures were incubated at 37" C with 5 % CO, in air and 100% humidity. PHA (Difco, Detroit, Michigan, USA) Con A (Sigma, Saint-Louis, Missouri, USA) and PWM (Difco) were routinely tested in three dilutions: and 0.33 o/oo final concentration 3 o/oo (v/v), 1 for PHA, 0.1, 0.033 and 0.011 mg/ml of Con A and 1 % (v/v), 0.33% and 0.11% of PWM. 12-0-tetrade-canoyl phorbol- 13-acetate (TPA, MW.616) was obtained from Midland Corporation, Brewster, N.Y., USA. It was dissolved in DMSO 10 mg/ml and stored at -70" C in 50-pg aliquots. TPA showed an optimal stimulation over a wide range of concentrations. The final concentrations used were M, lo-@M, and lo-' M. A23187 (MW.523) was kindly provided as a gift by Dr. R. L. Hamill, Lilly Research Laboratories, Indianapolis. It was dissolved in DMSO 8 mg/ml and stored at -70" C in 40-pg aliquots. A23187 was found to give maximal responses over a dose range of 3-7 x M when applied for 30 min at 37" C (RPMI+10% AB serum) in microtitration plates. The cells were then spun down and the supernatant replaced with RPMI 1640+10% AB serum without A23187. For optimal responses to A23187 in CLL cells it appeared necessary to use AB serum stored for at least one month at f 7 0 " C. The mitogens to be described below were all tested in FCS. Concentrated ( 7 x ) anti p2 microglobulin (anti &M) was purchased from Dakopatts A/S, Copenhagen, dialysed against PBS and sterile filtered before use. Each batch was tested out in various concentrations. Maximal responses were found in 1/12-1/48 final dilutions, depending on the batch. Appropriate dilutions in RPMI 1640 were stored at -70" C until used. Sepharose protein A was purchased from Pharmacia Chemicals A/B, Uppsala. Dextran and lactose, which are added to the gel to preserve it under freeze-drying, were removed by washing during the swelling stage. One gram of gel was swollen and washed for 15 min on a glass filter M HCI solution (200 ml) and then with with RPMI 1640 medium until physiological PH was reached. The swollen gel was stored in small aliquots as 10% (v/v) dilutions at -70" C. Final concentrations of 3.3% were regularly found to give maximal responses. Sepharose-bound anti F(ab')i was prepared as follows: F(ab'), from human IgG (KABI, Stock-
MEMBRANE FUNCTIONS IN CLL CELLS
holm) was made by pepsin (Sigma) digestion as described by Turner et a f . (1970). Rabbits were immunized with 4 mg F(ab)2 in Freund's complete adjuvant (Difco) by five subcutaneous injections and boosted after 3, 4 and 5 weeks with 0.5 mg F(ab'), in 0.9 % NaCl intramuscularly. Blood was collected 8 and 10 days after the last injection. The immunoglobulin fraction was isolated by ammonium sulphate precipitation (50 % saturation) and ion exchange (DEAE) chromatography. Anti F(ab'), was dialyzed against coupling buffer (0.1 M NaHCO, 0.5 M NaCl PH 8.3) and mixed with swollen and washed CNBr-activated Sepharose 4B (7.5 mg per g dry weight Sepharose). The mixture was rotated end-over-end overnight at 4" C. Unbound material was washed away with the coupling buffer, and remaining active groups were reacted with I M ethanolamine at PH 8 for 2 h. To remove non-covalently adsorbed protein the gel was washed in three cycles, each cycle consisting of a wash at PH 4 (0.1 M acetate buffer containing 1 M NaCI) followed by a wash at PH 8 (0.1 M borate buffer containing 1 M NaCI). The gel was finally washed in RPMI 1640 until physiological PH was reached. Following this procedure, 90-95 % of the protein remained bound to the gel. The optimal concentrations for Sepharose anti F(ab')z stimulation were found with 0.8-0.2% (v/v). The final tests were carried out with 0.83, 0.17 and 0.033%. Sepharose rabbit IgG prepared indentical to Sepharose anti F(ab'),, served as control.
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from Dakopatts were used. Rabbit anti-human IgD was obtained from Behringwerke (code OTKH). All sera were ultracentrifuged 1 h 160,000 g prior to use to remove aggregated immunoglobulin and used in appropriate dilutions (l/lO-1/80) to avoid non-specific staining. Complement receptors were identified as described by Munthe-Kaas (1976) using IgM-coated sheep red cells treated with C6-deficient mouse serum. Red cells (E-IgMC) sensitized in this way are likely to contain both C3b and C3d. No attempt was made to distinguish between these two receptors. The test itself was carried out as described by Ross and Polley (1976). When 50% or more of PBL from CLL patients were positive, the CLL cells were considered to have a C receptor. Rosettes with E-TgM and E only were always below 2% with patient cells in this test system. Capping
Capping as described by Elson et af. (1973) and Solheim (1974) was carried out on immunoglobulin (Ig) and &M surface receptors. In addition, another surface marker, provisionally called JM, which is capping independently of Ig and PzM was tested. JM was originally found in the urine from a patient with Sezary's syndrome, and is present on both T and B lymphocytes, monocytes and HeLa cells (T. 0. Landaas and T. Godal, unpublished observations). Capping with BaM and JM was carried out with a double layer, i.e. first rabbit anti-/?,M and anti-JM D N A synthesis and then FITC labelled pig anti-rabbit Ig (F2190 Cell proliferation was assessed by [3H]thymidine from Dakopatts, Copenhagen). Three million lymincorporation as a measure of DNA synthesis. phocytes were incubated at room temperature for Four hours before harvest, 1.25 pCi meth~l-[~HI- 30 min with 0.1 ml anti B2M in dilution 1 :320 and thymidine (The Radiochemical Centre, Amersham, antiJM (1 :20). The cells were then washed twice England) was added to each culture. All cultures (MEM with Hepes buffer and 10% FCS) and subwere harvested at 70 h (i.e. on the third day) with a sequently incubated with anti-rabbit Ig (F2190 1 :40) Skatron Multiple Cell Culture Harvester and the at 4" C for 30 min. The cells were again washed incorporated radioactivity measured as outlined twice as above and then incubated at 37" C in 1 ml elsewhere (Heier et af., 1977). All tests were carried MEM/Hepes with 10% FCS to allow capping to out in triplicate and the median value used for take place. The cells were spun down and resusexpression of data. In order to allow direct compa- pended in 0.1 nil MEM/Hepes with 1 mg/ml NaN, risons to be made between responses to different and then examined for capping. Ig capping was mitogens, CLL responses were transformed to carried out as outlined above, but with one layer percentages of normal responses. All calculations only using polyvalent anti-Ig (F1009, Dakopatts) were based on net counts-i.e. mitogen-stimulated instead of F2190. cultures minus unstimulated (or Sepharose rabbit Ig-exposed) cultures. Determination of surface concentration of pa microglobulin Surface markers For determination of cell surface concentration T cells were identified by the E-rosette test with of BiM without interference with Fc receptors, sheep red blood cells, as described by Heier (1974), FITC-labelled anti-@,M (Dakopatts, code F112), and B cells by anti-Ig immunofluorescent staining prepared by pepsin digestion as described above, with a polyvalent fluorescein isothiocyanate (F1TC)- was used. labelled rabbit anti-human immunoglobulin (antiOne-tenth ml FITC labelled anti-p,M (F(ab')3 IgA, IgG, IgM, kappa and lambda) serum (code F1009, Dakopatts, Copenhagen) as described else- in dilution 1 :16 with 1 mg/ml NaN, was incubated where (Heier et al., 1977). For identification of with 3 x loe lymphocytes at room temperature for surface Ig on CLL cells FITC-labelled antisera 30 min. The cells were then washed once with (immunoglobulin fraction) specific for IgA (code MEM/Hepes and once with PBS, both containing F1092), TgM (code F1091), IgG (code F1090), 1 mg/ml NaN,. The cells were resuspended in 1 nil kappa (code F109K), and lambda (code F109L) PBS with 1 mg/ml NaNs.
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GODAL ET AL.
The amount of anti B2M F(ab’)s bound per cell was quantitated by measuring the fluorescence intensity on a single cell basis by use of flow cytofluorometry (Lindmo and Steen, 1977). The 488 nm wavelength of an Argon laser was used at 1,000 mW power for excitation of fluorescein, and the fluorescence intensity was measured at wavelengths longer than 515 nm. Unstained human lymphocytes as well as mouse bone-marrow cells stained with F112 did not give rise to pulses above the noise level seen in distributions of fluorescence from human cells stained with F112. IOl2,flUX
Cellular uptake of “Rb is assumed to reflect membrane transport of K t (Mills and Tupper, 1975). which has been observed to increase as early as 30 sec after mitogen stimulation (Averdunk, 1972). Ten to 2 0 ~ 1 0cells/ml ~ were incubated as described above in RPMI 1640 with 10% FCS at 37” C in two Falcon plastic tubes, one containing mitogen in optimal concentration, the other serving as control. After 10 min incubation 20 , ~ C i [ ~ ~ R b l(spec. Cl act. 30 Ci/niole, Institutt for Atomenergi, Kjeller, Norway) was added to each tube. Immediately, and after 10, 20, 30 and 40 min further incubation, 100-pl samples were taken from each tube. The cells were then rapidly separated from the incubation medium by centrifugation through a layer of Silicone oil (AR 200, Serva) at 10,000 g for 30 sec in a Beckman Microfuge B (Andreasen et al., 1974). The radioactivity in the cell pellet was determined in a Packard Gamma Scintiallation Counter. The K+ content of the cells was also determined, and used to estimate the total cell volume (Iversen, 1976). On the basis of these estimates the increase in intracellular concentration of Rb was calculated. The cellular uptake of isotope appeared to take place at a nearly constant rate in the 40-min period studied. The uptake rate in mitogen-treated and control cultures was compared in each experiment and differences of more than 10% were scored as significanr. RESULTS
As shown in Table I, all of the nine patients had IgM kappa or lambda chains. Six were scored as having CLL cells with Cs receptors (Patients 3,4, 6, 7, 8, 9). All the patients had more than 60% B cells in their PBL. But the exact proportion of CLL cells was often difficult to assess because of weak immunofluorescense and is likely to be higher than the figures given. The proportion of T cells ranged from 1 to 13%. The mitogen responses are shown in Figure 1. All patients tested showed suppressed responses t o the B cell mitogens PWM, anti-@,M, Sepharose protein A and Sepharose anti F(ab’)z as well as T-cell mitogens (PHA, Con A, TPA). However, a different picture was found with the ionophore A23187, where cells from three patients responded as strongly as normal cells. The identity of the blastoid cells was determined in one of the cases
+
(patient 9) and these were shown to have identical heavy and light chains to the patient’s CLL cells. Since one explanation for the suppressed responses could be a decrease or absence of surface receptors interacting with the ligands, the surface concen. tration of BzM using pepsin-digested fluoresceinlabelled anti-p,M was determined. As shown in Figure 2, a variable pattern of fluorescence distributions was found among the CLL samples tested. Two of the CLL samples (patients 7 and 9) show distributions similar to that found for normal cells (upper left, Fig. 2). Patient 3 shows a broader distribution which was found consistently in four independent samples taken from this patient. Broad distributions were, however, often seen also in normal samples (nine out of 18 cases). Thus, no consistent quantitative difference could be detected between normal and CLL cells by the use of single-cell cytofluorometry. The capping capability of CLL cells is shown in Figure 3. CLL cells revealed a markedly reduced capability to cap both BeM, Ig and J M surface receptors. Since the results with the ionophore as well as the capping experiments suggested that the blocking of mitogenesis in CLL cells was taking place at the membrane level, an early membrane event following mitogenetic stimulation, namely ion-flux, was studied, using rubidium as marker for potassium. Normal PBL preparations enriched in B cells by T-cell depletion responded in every experiment with increased ssRb uptake as early as 10 min after stimulation with Sepharose Protein A (Fig. 4 4 . Similar observations were made with anti-B,M both in B cells prepared by T-cell depletion (data not shown) and in B cells depleted of both T cells and monocytes (Fig. 46). In contrast, neither Sepharose protein A nor anti-&M altered the isotope uptake in CLL cells (Fig. 4a, b). DISCUSSION
The present study shows that peripheral blood lymphocytes from CLL patients respond poorly to the B-cell mitogens, PWM, Sepharose protein A, anti-&-microglobulin and Sepharose coupled F(ab’), antibodies. In fact, the response to these mitogens seemed to be as poor as, or poorer than, the response to the T-cell mitogens Con A and PHA. The response to the T-cell mitogens have been shown to be due to normal T cells present in PBL of CLL patients (Schweitzer et al., 1973; Wybran et al., 1973). Similarly, the responses found to B-cell mitogens in PBL from CLL patients could be due to “contaminating” normal B cells, but this possibility was not investigated in the present study. Various explanations may be considered for the suppressed responses to B-cell mitogens of CLL cells, in spite of the fact that the proportion of cells having 8-cell characteristics in PBL of CLL patients is greatly increased. First, the age distribution of the patient group and the normal group was not matched, although
MEMBRANE FUNCTIONS IN CLL CELLS
565
140 130
FIGURE1 - Mitogen responses of peripheral blood lymphocytes (PBL) from chronic lymphocytic leukemia (CLL) patients, 0 , as measured by DNA incorporation of [3H]thymidine 66-70 h after stimulation. The CLL responses are expressed as percentages of normal PBL responses (100 % SEM).The net absolute CpmhtsEM of the normal responses is also indicated. The response of each patient is shown by a circle. The number within the circle corresponds to the patient number given in Table 1. The mean value of CLL PBL responses to each mitogen is indicated by horizontal bars. The counts in unstimulated cultures were 1,673f675 (AB serum) and 2,575rt611 (FBS) of normal PBL as comDared to 605499 (AB serum) &d 488&170 (FBS)' of CLL PBL.
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FIGURE 2 - Histograms of fluorescein-labelled anti-b,M per cell measured by flow cytofluorometry for one normal sample (upper left) and three samples of peripheral blood lymphocytes from chronic lymphocytic leukemia patients. High counts were registered in channels close to the origin (No. 1-8) owing to noise pulses from impurities and cellular debris. More than 100,000 cells were measured for each histogram.
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CHANNEL NUMBER (PROPORTIONAL TO CELLULAR FLUORESCENCE INTENSITY)
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GODAL EF AL.
Third, at least some of the B cell mitogens, such as PWM and anti-p,-microglobulin, are known to require T cells .to give optimal responses (Insel and Merler, 1977; dstberg et al., 1976). Thus, in CLL PBL normal T cells and monocytes may be present in too low concentrations for CLL cells to respond to B-cell mitogens. Alternatively, suppressor cells might be present in CLL PBL. Neither of these possibilities can be excluded. However, various observations made in this study point to a fourth possibility, namely that CLL cells are altered in such a way that they fail to respond physiologically to ligand receptor interactions. This was clearly shown to be the case with regard to capping and cation uptake. The capacity of CLL cells to cap three cell membrane markers was studied. Various investigations have earlier shown that CLL cells show a reduced
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kl FIGURE 3 - Percentage of cells capping three different surface markers in normal, 0 , as compared to peripheral blood lymphocytes from patients with chronic lymphocytic leukemia, 0 . Horizontal bars indicate mean values of each group.
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they overlapped. However, the higher ages in the CLL patients cannot explain the large discrepancies observed here as the age-related decline in mitogenic responsiveness is much less pronounced (Pisciotta et al., 1967; Heier et al., 1977). Second, CLL may possibly be derived from a subpopulation of normal B cells which d o not respond to mitogens. This possibility cannot be entirely excluded, but would seem very unlikely since different surface markers such as Ig, Fc and C appear to occur in CLL at frequencies closely approaching those found in normal PBL B cells (Brouet et al., 1976; Salsano et al., 1976; Hellstrom et al., 1976; Pincus et al., 1972). These observations indicate, in fact, that CLL is induced randomly within the circulating pool of B cells.
FIGURE 4 - Effect of B-cell mitogens on 8sRb influx in PBL-B cells and CLL cells. Influx is measured as isotope uptake rate 10-50 min after stimulation with Sepharose protein A (a) and anti-p,M (b). The mean value of uptake rates of different cell populations stimulated with mitogen (M) and unstimulated controls (C) is shown in open bars (left ordinate). Each experiment is shown with paired comparisons between Rb flux in mitogen-stimulated and control cultures. Hatched bars (mean+sEM) show the mitogen-induced increase in sBRb uptake, given as percentage of control values (right ordinate). PBL-B cells: Peripheral blood lymphocytes enriched in B cells by the E rosette method without (Fig. 4a) or with (Fig. 46) monocyte depletion. CLL-cells: Peripheral blood lymphocytes from chronic lymphocytic leukemia.
MEMBRANE FUNCTIONS IN CLL CELLS
capability to cap (Menne and Flad, 1973; Huber et al., 1974; Cohen, 1975), while others have demonstrated capping under very similar experimental conditions (Salsano et al., 1974; Palencia et al., 1975). Our studies with Ig, PzM and J M all showed a markedly reduced capping capability of CLL cells. This finding, therefore, suggests that there are functional membrane-associated aberrations in CLL cells. Monovalent cation uptake was studied after exposure to Sepharose-bound protein A or anti-A microglobulin. Membiane transport of K-+or Rb+ has invariably been reported to increase after T-mitogen stimulation of lymphocytes (Lauf, 1975). Corresponding investigations of B-mitogenic effect on isolated B cells have not come to our knowledge. However, Freedman et al. (1975) measured Ca++ uptake in mouse spleen lymphocytes after stimulation by both T and B mitogens. They found increased uptake of Ca++in T cells, but not in B cells. Our study reveals that Rb+ transport increases after B-mitogen stimulation of normal human B cells, but not of CLL cells. Thus, the i o d u x studies also pointed to membrane-associated alterations in CLL cells. Attempts were made in two ways to bypass membrane-associated events in the mitogenic response. First, the mitogenic effect of TPA was studied. TPA, a tumor promotor, has been shown to increase intracellular levels of cyclic GMP and also to be mitogenic to lymphocytes (Estensen et al., 1974). CLL cells responded poorly to this mitogen, but the explanation may be trivial as recent studies suggest that TPA may be a selective T-cell mitogen (Touraine et al., 1977). Second, the mitogenic response to A23187 was studied. A231 87 is a ionophore with high specificity for Ca++ (Pressman, 1976) and its mitogenic effect is dependent on the presence of extracellular Ca++,
567
suggesting that its mitogenesis is due to a n influx of calcium ions into the cells (Luckasen et al., 1974; Maino et at., 1974; Hovi et al., 1976). We have shown that A23187 is mitogenic to both T and B cells (Godal, unpublished observations). Three out of the eight patients studied responded well to A23187, suggesting that the failure of CLL cells to respond to mitogens, at least in some cases, may be due to a membrane associated blockade of the mitogenic signal. Sheppard et al. (1977) have recently reported that the catecholamine receptors are reduced on CLL cells compared to normal PBL. In our study the quantity of PzM on the cell surface was studied by cytofluorometry. However, we failed to detect any consistent aberrations with regard to BzM between CLL cells and normal PBL, indicating that the failure of anti-p,M to induce capping, ionfluxes and mitogenesis is not due to reduced concentrations of BzM at the cell surface. On the other hand, we cannot exclude that a small fraction of surface receptors with special anchorage in the cell membrane or to sub-membraneous structures responsible for the functional responses, is absent from CL.L cells. The relationship between the cell membrane aberrations and the neoplastic and other properties of CLL cells remains at present a matter of speculation and is subject to further study. ACKNOWLEDGEMENTS
Drs. H. C. Godal, K. Laake, F. Wislnrff and U. Abildgaard kindly arranged for blood samples to be taken from patients. Mrs. Anne Grindflek and Miss Frances Dodman provided expert technical assistance. Miss Liv D. Arnes assisted in the preparation of the manuscript. This work was supported by the Norwegian Cancer Society.
ALTERATION DES FONCTIONS ASSOCIfiES A LA MEMBRANE DANS LES CELLULES DE LEUCEMIE LYMPHOCYTAIRE CHRONIQUE Les lymphocytes du sang pkriphkrique, consistant principalement en cellules B nkoplasiques, de neuf sujets atteints de leuckmie lymphocytaire chronique (cellules CLL) ont eu une reaction nettement affaiblie aux mitogtnes des cellules B humaines, l’anti-,9s-microglobuline, la protkine A/Sepharose et I’anti-F(ab’)s/Sepharose.Par contre, les cellules CLL de trois patients sur huit ont bien rCagi B I’ionophore A23187. La protkine A/Sepharose et I’anti-B,-microglobulinen’ont pas non plus accru I’incorporation de rubidium” (analogue du potassium) dans les cellules CLL, par comparaison avec les prkparations de lymphocytes normaux du sang pkriphkrique enrichis en cellules B. L’agrkgation de divers marqueurs de la surface, y compris la 0,-microglobuline, a dinrinuk sur les cellules CLL. Par contre, la concentration de 0,-microglobuline ti la surface n’a pas baissk, comme on I’a montrk en cytofluoromktrie avec I’anti-0,-microglobuline marqu6e A la fluoresckine. Les auteurs en concluent que divers processus associks A la membrane, dkclenchks par des interactions ligand-rkcepteur, sont altkr6s ou bloquks dans les cellules CLL.
REFERENCES
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AminosLure-Transport bei menschlichen Lymphozyten. Hoppe-Seyler’s Z. physiol. Chem., 353, 79-87 (1972). B ~ c., G ~ ~~A., and~~BOIRON, ~~M., Effects ~~ of , ~ phyto-hamagglutinin on b~ood~cu~tures of chronic lymphocytic leukzmias. Lancet, i, 667-668 (1964). J. F., BOURONCLE, B. A., CLAUSEN, K. P., and ASCHENBRAND, Studies of the delayed response of phytohemagglutinin
GODAL ET AL.
(PHA) stimulated lymphocytes in 25 chronic lymphatic leukemia patients before and during therapy. Blood, 34, 166-178 (1969). BROUET,J. C., PREUD’HOMME, J. L., and SELIGMANN, M., Lymphocyte membrane markers in B-cell proliferations and human non-Hodgkin’s lymphomas. In: J. Wybran and M. J. Staquet (ed.), Clinical tumor immunology, pp. 123-130, Pergamoii Press, Oxford, New York, Toronto, Paris, Sydney, Frankfurt (1976). BOYUM,A., Separation of leucocytes from blood and bone marrow. Scand. J. clin. Lab. Invest., 21, Suppl. 97 (1968). CATOVSKY, D., TRIPP,E., and HOFFBRAND, A. V., Response to phytohaemagglutinin and pokeweed mitogen in chronic lymphocytic leukaemia. Lancet, 1, 794-795 (1972). COHEN,H. J., Human lymphocyte surface iminunoglobulin capping. Normal characteristics and anomalous behavior of chronic lymphocytic leukemic lymphocytes. J. clin. Invest., 55, 84-93 (1975). ELSON,C. J., SINGH,J., and TAYLOR,R. B., The effect of capping by anti-~mmunoglobulinantibody on the expression of cell surface immunoglobulin and on lymphocyte activation. Scand. J. Immunol., 2, 143-149 (1973). ESTENSEN,R. D., HADDEN,J. W., HADDEN,E. M.. TOURAINE,F., TOURAINE, J.-L., HADDOX,M. K., and N. D., Phorbol myristate acetate: Effects of a GOLDBERG, tumor promoter on intracellular cyclic G M P in mouse fibroblasts and as a mitogen on human lymphocytes. In: B. Clarkson and R. Baserga (ed.) Control of proliferation in animal cells, Vol. 1, pp. 627-634, Cold Spring Harbor Conferences on Cell Proliferation (1 974). Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. FORSGREN. A., SVEDJELUND, A,, and WIGZELL,H., Lymphocyte stimulation by protein A of Staphylococcus aureus. Europ. J . Immunol., 6, 207-213 (1976). FREEDMAN, M. H., RAFF,M. C., and GOMBERTS, B., Induction of increased calcium uptake in mouse T lymphocytes by concanavalin A and its modulation by cyclic nucleotides. Nature (Lond.), 255, 378-382 (1975). FRBLAND, s. s., NATVIG,J. B., and STAVEM, P., Immunological characterization of lymphocytes in lymphoproliferative diseases. Restriction of classes, subclasses, and Gm allotypes of membrane-bound Ig. Scand. J. Immunol., 1, 351-360 (1972). GREAVES, M. F., and BROWN,G., Purification of human T and B lymphocytes. J. Immunol., 112, 420-423 (1974). GREAVES, M., JANOSSY,G., and DOENHOFF, M., Selective triggering of human T and B lymphocytes in ilitro by polyclonal mitogens. J. exp. Med., 140, 1-18 (1974). HAN,T., Studies of correlation of lymphocyte response to phytohemagglutinin with the clinical and immunologic status in chronic lymphocytic leukemia. Cancer, 31, 280-285 (1973). HEIER,H. E., The influence of mechanical force on the rosette test for human T lymphocytes. Scand. J . Immunol., 3, 677-681 (1974). HEIER,H. E., KLEPP,R., GUNDERSEN, S., GODAL,T., and NORMA”, T., Blood B and T lymphocytes and in vitro cellular immune reactivity in untreated human malignant lymphomas and other malignant tumors. Scand. J . Haematol., 18, 137-148 (1977). HELLSTROM, I., MELLSTEDT, H., PERLMANN, P., HOLM,G., and PETTERSON, D., Receptors for Helix pomatia A haemagglutinin on leukaemic lymphocytes from patients with chronic lymphocytic leukaemia (CLL). Clin. exp. Immunol., 26, 196-203 (1976). HOW,T., ALLISON, A. C., and WILLIAMS, S. C., Proliferation of human peripheral blood lymphocytes induced by A23187, a streptomyces antibiotic. Exp. Cell Res., 97, 92-100 (1976). HUBER, C., MICHLMAYR,G., BRAUNSTEINER, H., and HUBER,H., Redistribution of immunoglobulin determinants on human lymphocytes in lymphoproliferative disorders. Europ. J. Cancer, 10, 517-521 (1974). INSEL,R. A., and MERLER,E., The necessity for T cell help for human tonsil B cell responses to pokeweed mitogen: Induction of DNA synthesis, immunoglobulin, and specific
antibody production with a T cell helper factor produced with pokeweed mitogen. J. Immunol., 118, 2009-2014 (1977). IVERSEN, 3.-G., Unidirectional K + fluxes in rat thymocytes stimulated by concanavalin A. J. Cell Physiol., 89, 267-276 (1 976). KONIG,E., COHNEN,G., and BRITTINGER, G., Response t o phytohaemagglutinin and pokeweed mitogen in chronic lymphocytic leukaemia. Lancet, 1, 795 (1972). LAUF,P. K., Antigen-antibody reactions and cation transport in biomembranes: immunophysiological aspects. Biochem. Biophys. Acta, 415, 173-229 (1975). LINDMO, T., and STEEN,H. B., Flow cytometric measurement of the polarization of fluorescence from intracellular fluorescein in mammalian cells. Biophys. J., 18, 173-187 (1977). LUCKASEN, J. R., WHITE,J. G., and KERSEY, J. H., Mitogenic properties of a calcium ionophore, A23187. Proc. nut. Acad. Sci. (Wash.), 71, 5088-5090 (1974). MAINO,V. .C., GREEN.N . M., and CRUMPTON, M. J., The role of calcium ions in initiating transformation of lymphocytes. Nature (Lond.), 251, 324-327 (1974). MENNE,H.-D., and FLAD,H.-D., Membrane dynamics of HL-A-anti-HL-A complexes of human normal and leukaemic lymphocytes. Clin. exp. Immunol., 14, 57-67 1973). MILLS, B., and TUPPER,J. T., Cation permeability and ouabain-insensitive cation flux in the Ehrlich ascites tumor cell. J. Membrane Biol., 20, 75-97 (1975). MOLLER,G., and LANDWALL,P., The polyclonal B-cellactivating property of protein A is not due t o its interaction with the Fc part of immunoglobulin receptors. Scand. J. Immunol., 6, 357-366 (1977). MUNTHE-KAAS, A. C., Phagocytosis in rat Kuppfer cells in vitro. Exp. Cell Res., 99, 319-327 (1976). NOVOGRODSKY, A., BINIAMINOV,M., RAMOT, B., and KATCHALSKI, E., Binding of concanavalin A t o rat, normal human and chronic lymphatic leukemia lymphocytes. Blood, 40, 311-316 (1972). OPPENHEIM, J. J., WHANG,J., and FREI, E., Immunologic and cytogenetic studies of chronic lymphocytic leukemic cells. Blood, 26, 121-132 (1965). STB BERG, L., LINDBLOM, J. B., and PETERSON, P. A., Bpmicroglobulin on the cell surface. Specificity of inhibition of the mixed leukocyte reaction and mitogenic properties of antibodies against the two HLA antigen polypeptide chains. Europ. J. Immunol., 6, 108-113 (1976). PALENCIA, C., BAUMGARTNER, G., and KNAPP,W., Antibodyinduced redistribution of surface immunoglobulins in chronic lymphatic leukaemia. Europ. J. Cancer, 11, 193-197 (1975). PINCUS,S., BIANCO,C., and NUSSENZWEIG, V., Increased proportion of complement-receptor lymphocytes in the peripheral blood of patients with chronic lymphocytic leukemia. Blood, 40, 303-310 (1972). PISCIOTTA,A. V., WESTRING,D. W., DEPREY,C., and WALSH, B., Mitogenic effect of phytohaemagglutinin at different ages. Narure (Lond.j, 215, 193-194 (1967). PRESSMAN, B. C., Biological applications of ionophores. Ann. Rev. Biochem., 45, 501-530 (1976). RINGD~N O., , and MOLLER,E., B-cell mitogenic effects on human lymphocytes of rabbit anti-human p2-microglobulin. Scand. J. Immunol., 4, 171-179 (1975). ROBBINS,J. H., Human peripheral blood in tissue culture and the action of phytohemagglutinin. Experentia, 20, 164-168 (1964). ROBBINS,J. H., and LEVIS,W. R., Inherent inability ofchronic lymphocytic leukaemia lymphocytes to respond t o phytohaemagglutinin. Inr. Arch. Allergy, 43, 845-858 (1972). Ross, G. D., and POLLEY,M. J., Detection of compleinentreceptor lymphocytes (CRL). In: B. R. Bloom and J . R. David (ed.), In vitro methods in cell-mediated and tumor immunity, pp. 123-136, Academic Press, New York, San Francisco, London (1976). SALSANO,F., F R ~ L A N D S., S.,, NATVIG,J. B., and MANDELLI F., Chronic lymphocytic leukemia: studies on the effect of drug treatment on different lymphocytic subpopulations. Scond. J. Immunol., 5 , 1185-1190 (1976).
MEMBRANE FUNCTIONS IN CLL CELLS
SALSANO,F., FR0LAND. S . S . , NATVIC, J. B., and T. E., Same idiotype of B-lymphocyte memMICHAELSEN, brane IgD and IgM. Formal evidence for monoclonality of chronic lymphocytic leukemia cells. Scund. J . fmmunol., 3, 841-846 (1974). SCHWEITZER, M., MELIEF, C. J. M., and EIJSVOOGEL, V. P., The nature of the transforming lymphocyte in chronic lymphocytic leukemia. Europ. J. Immunol., 3, 121-126 (1973). SHEPPARD, J. R., GORMUS,R., and MOLDOW.C. F., Catecholamine hormone receptors are reduced on chronic lymphocytic leukaemic lymphocytes. Nulure (Lond.), 269, 693-695 (1917). SMITH,J. L., CAWLEY,J. C., and BARKER, C. R., The response to plant mitogens of two subpopulations of lymphocytes in a case of chronic lymphocytic leukaemia. Clin. exp. Immunol., 14, 397-408 (1973). SMITH, J. L.,COWLING, D. C., and BARKER, C. R., Response
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of lymphocytes in chronic lymphocytic leukaemia
to plant mitogens. Lancet, 1, 229-233 (1972). SOLHEIM,B. G., Association between the Br-m/HL-A molecule and membrane structures responsible for lymphocyte activation. Trunsplunt. Rev..21, 35-52 (1974).
TOURAINE, J.-L., HADDEN,J. W., TOURAINE, F.. HADDEN, E. M., ESTENSEN,R., and GOOD, R. A., Phorhol myristate acetate: a mitogen selective for a T-lymphocyte subpopulation. J . exp. Med., 145, 460-465 (1977). TU R N ERM. , W., BENNICH, H. H., and NATVIG, J. B., Pepsin digestion of human G-myeloma proteins of different subclasses. I. The characteristic features of pepsin cleavage as a function of time. Clin. exp. Immunol., I,603-625 (1970). WYBRAN, J., CHANTLER, S., and FUDENBERG, H. H., Isolation of normal T cells in chronic lymphatic leukaemia. Lancer. 1, 126-129 (1973).
ALTERED MEMBRANE-ASSOCIATED FUNCTIONS I N CHRONIC LYMPHOCYTIC LEUKEMIA CELLS Tore GODAL1, 4, Aase HENRIKSEN l, Jens-Gustav IVERSEN z, Tone 0. LANDAAS and Tore LINDMO Laboratory for Immunology, Department of Pathology, Norsk Hydro's Institute for Cancer Research, The Norwegian Radium Hospital, Oslo 3; Institute for Physiology, University of Oslo, Karl Johans gate 47, Oslo I ; and Department of Biophysics, Norsk Hydro's Institute for Cancer Research, The Norwegian Radium Hospital, Oslo 3, Norway
Peripheral blood lymphocytes consisting mainly of neoplastic B cells from patients with chronic lymphocytic leukemia (CLL cells) showed a markedly reduced response to the human B-cell mitogens anti$, microglobulin, Sepharose-bound protein A and Sepharose-bound anti-human immunoglobulin (anti F(ab'),) in all of nine patients studied. O n the other hand, CLL cells from three out of eight patients tested responded well to the calcium ionophore A23187. Sepharose-bound protein A and anti-p2 microglobulin also failed to induce increased uptake of 'bRubidium (potassium analogue) in CLL cells as compared to B-cell-enriched preparations of normal peripheral blood lymphocytes. The capacity of CLL cells to cap various surface markers including 8, microglobulin was reduced. O n the other hand, surface concentrations of 8, microglobulin were not reduced as measured by fluorescein-labelled anti-j3,-micrbklobulin in singlccell cytofluorometry. It is concluded that various membrane-associated events elicited by ligandreceptor interactions are altered or blocked in CLL cells.
variety of mitogens, including anti Bz microglobulin (anti &M), Sepharose-bound protein A, and Sepharose-bound anti-F(ab), immunoglobulin, which have been reported to be B-cell mitogens (Ringden and Moller, 1975; Solheim, 1974; Forsgren et a/., 1976; Moller and Landwall, 1977; Godal (unpublished observations)). Since CLL cells responded poorly to these mitogens also, attempts were made to localize the apparent blockade of the mitogenic signal at the sub-cellular level. Comparative studies on anti-ifl,M binding capacity, capping and effect on membrane transport of ions in CLL cells as compared to normal PBL or B-cell-enriched preparations all suggested that the membrane-associated events elicited by ligand-receptor interactions may be altered or blocked in CLL cells. In CLL cells from some patients the putative membrane block could apparently be bypassed by the Ca++ ionophore, A23187.
Peripheral blood lymphocytes (PBL) from patients with chronic lymphocytic leukemia (CLL) have been found to give a poor or delayed response to phytohemagglutinin (PHA) (Bernard et al., 1964; Robbins, 1964; Oppenheim et al., 1965; Bouroncle et al., 1969; Han, 1973), concanavalin A (Con A) and pokeweed mitogen (PWM) (Novogrodsky et al., 1972; Smith et al., 1972, 1973; Catovsky et al., 1972; Konig et al., 1972; Frnrland et al., 1972). The delayed response could be mimicked by reducing the number of initially responding cells, indicating that the number of responding cells in CLL is reduced (Robbins and Levis, 1972 ; Schweitzer et al., 1973). By various separation procedures such as albumin gradients (Schweitzer et al., 1973) or the sheep red cell rosette technique (Wybran et a[., 1973), the responding cells could be separated out from the CLL cetls, suggesting that the responding cells were mainly normal PBL of CLL patients. This has also been clearly demonstrated in a patient in whom the CLL cells could be morphologically identified by characteristic crystalline IgA inclusions (Smith et al., 1973). However, these studies are complicated by the fact that Con A and PHA are mainly T-cell mitogens in man and PWM is mitogenic to both T and B cells (Greaves et al., 1974), whereas CLL cells usually have B-cell characteristics. In the present investigation the mitogenic responses of CLL PBL have been reinvestigated using a
Patients
MATERIAL AND METHODS
Blood from nine CLL patients, aged between 49 and 79 years, was obtained from various hospitals
in Oslo. None of the patients were receiving treatment at the time of the investigation. The findings for each patient with regard to leukocyte counts, surface characteristics of cells and proportion of T and B cells are set out in Table I. Control subjects
Healthy blood donors from the Red Cross Blood Center, Oslo, served as control subjects. Their ages ranged from 22 to 57 years. Cells
PBL were prepared from fresh heparinized blood (10 units preservative-free heparin/ml) by the FicollIsopaque method (Bnyum, 1968) as described elsewhere (Heier et al., 1977). B-cell-enriched preparations were made by using the sheep red cell rosette method as described by Greaves and Brown (1974). I n order to reduce the number of monocytes in the B-cell preparations, in Received: February 3, 1978. To whom requests for reprints should be sent.
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GODAL ET AL.
some experiments the lymphocytes were cultured in Falcon flasks at 37" C overnight (20% AB serum in RPMI 1640). Non-adherent cells were subsequently removed and B cells isolated by the sheep red cell rosette method. Such B-cell-enriched preparations were found to contain 58 1 4 % Ig-positive cells and 4 1 % E-rosette-positive cells. Mitogen stimulation
ri
LY!
LY!
Y
3 b
Cells were cultured in round-bottomed Linbro micro-titration plates with 2 x lo5 cells per well in 0.1 ml RPMI 1640 medium and 0.05 ml mitogen with a final concentration of human AB or inactivated fetal calf serum (FCS) (Gibco, Glasgow, Scotland) of 10%. The cultures were incubated at 37" C with 5 % CO, in air and 100% humidity. PHA (Difco, Detroit, Michigan, USA) Con A (Sigma, Saint-Louis, Missouri, USA) and PWM (Difco) were routinely tested in three dilutions: and 0.33 o/oo final concentration 3 o/oo (v/v), 1 for PHA, 0.1, 0.033 and 0.011 mg/ml of Con A and 1 % (v/v), 0.33% and 0.11% of PWM. 12-0-tetrade-canoyl phorbol- 13-acetate (TPA, MW.616) was obtained from Midland Corporation, Brewster, N.Y., USA. It was dissolved in DMSO 10 mg/ml and stored at -70" C in 50-pg aliquots. TPA showed an optimal stimulation over a wide range of concentrations. The final concentrations used were M, lo-@M, and lo-' M. A23187 (MW.523) was kindly provided as a gift by Dr. R. L. Hamill, Lilly Research Laboratories, Indianapolis. It was dissolved in DMSO 8 mg/ml and stored at -70" C in 40-pg aliquots. A23187 was found to give maximal responses over a dose range of 3-7 x M when applied for 30 min at 37" C (RPMI+10% AB serum) in microtitration plates. The cells were then spun down and the supernatant replaced with RPMI 1640+10% AB serum without A23187. For optimal responses to A23187 in CLL cells it appeared necessary to use AB serum stored for at least one month at f 7 0 " C. The mitogens to be described below were all tested in FCS. Concentrated ( 7 x ) anti p2 microglobulin (anti &M) was purchased from Dakopatts A/S, Copenhagen, dialysed against PBS and sterile filtered before use. Each batch was tested out in various concentrations. Maximal responses were found in 1/12-1/48 final dilutions, depending on the batch. Appropriate dilutions in RPMI 1640 were stored at -70" C until used. Sepharose protein A was purchased from Pharmacia Chemicals A/B, Uppsala. Dextran and lactose, which are added to the gel to preserve it under freeze-drying, were removed by washing during the swelling stage. One gram of gel was swollen and washed for 15 min on a glass filter M HCI solution (200 ml) and then with with RPMI 1640 medium until physiological PH was reached. The swollen gel was stored in small aliquots as 10% (v/v) dilutions at -70" C. Final concentrations of 3.3% were regularly found to give maximal responses. Sepharose-bound anti F(ab')i was prepared as follows: F(ab'), from human IgG (KABI, Stock-
MEMBRANE FUNCTIONS IN CLL CELLS
holm) was made by pepsin (Sigma) digestion as described by Turner et a f . (1970). Rabbits were immunized with 4 mg F(ab)2 in Freund's complete adjuvant (Difco) by five subcutaneous injections and boosted after 3, 4 and 5 weeks with 0.5 mg F(ab'), in 0.9 % NaCl intramuscularly. Blood was collected 8 and 10 days after the last injection. The immunoglobulin fraction was isolated by ammonium sulphate precipitation (50 % saturation) and ion exchange (DEAE) chromatography. Anti F(ab'), was dialyzed against coupling buffer (0.1 M NaHCO, 0.5 M NaCl PH 8.3) and mixed with swollen and washed CNBr-activated Sepharose 4B (7.5 mg per g dry weight Sepharose). The mixture was rotated end-over-end overnight at 4" C. Unbound material was washed away with the coupling buffer, and remaining active groups were reacted with I M ethanolamine at PH 8 for 2 h. To remove non-covalently adsorbed protein the gel was washed in three cycles, each cycle consisting of a wash at PH 4 (0.1 M acetate buffer containing 1 M NaCI) followed by a wash at PH 8 (0.1 M borate buffer containing 1 M NaCI). The gel was finally washed in RPMI 1640 until physiological PH was reached. Following this procedure, 90-95 % of the protein remained bound to the gel. The optimal concentrations for Sepharose anti F(ab')z stimulation were found with 0.8-0.2% (v/v). The final tests were carried out with 0.83, 0.17 and 0.033%. Sepharose rabbit IgG prepared indentical to Sepharose anti F(ab'),, served as control.
563
from Dakopatts were used. Rabbit anti-human IgD was obtained from Behringwerke (code OTKH). All sera were ultracentrifuged 1 h 160,000 g prior to use to remove aggregated immunoglobulin and used in appropriate dilutions (l/lO-1/80) to avoid non-specific staining. Complement receptors were identified as described by Munthe-Kaas (1976) using IgM-coated sheep red cells treated with C6-deficient mouse serum. Red cells (E-IgMC) sensitized in this way are likely to contain both C3b and C3d. No attempt was made to distinguish between these two receptors. The test itself was carried out as described by Ross and Polley (1976). When 50% or more of PBL from CLL patients were positive, the CLL cells were considered to have a C receptor. Rosettes with E-TgM and E only were always below 2% with patient cells in this test system. Capping
Capping as described by Elson et af. (1973) and Solheim (1974) was carried out on immunoglobulin (Ig) and &M surface receptors. In addition, another surface marker, provisionally called JM, which is capping independently of Ig and PzM was tested. JM was originally found in the urine from a patient with Sezary's syndrome, and is present on both T and B lymphocytes, monocytes and HeLa cells (T. 0. Landaas and T. Godal, unpublished observations). Capping with BaM and JM was carried out with a double layer, i.e. first rabbit anti-/?,M and anti-JM D N A synthesis and then FITC labelled pig anti-rabbit Ig (F2190 Cell proliferation was assessed by [3H]thymidine from Dakopatts, Copenhagen). Three million lymincorporation as a measure of DNA synthesis. phocytes were incubated at room temperature for Four hours before harvest, 1.25 pCi meth~l-[~HI- 30 min with 0.1 ml anti B2M in dilution 1 :320 and thymidine (The Radiochemical Centre, Amersham, antiJM (1 :20). The cells were then washed twice England) was added to each culture. All cultures (MEM with Hepes buffer and 10% FCS) and subwere harvested at 70 h (i.e. on the third day) with a sequently incubated with anti-rabbit Ig (F2190 1 :40) Skatron Multiple Cell Culture Harvester and the at 4" C for 30 min. The cells were again washed incorporated radioactivity measured as outlined twice as above and then incubated at 37" C in 1 ml elsewhere (Heier et af., 1977). All tests were carried MEM/Hepes with 10% FCS to allow capping to out in triplicate and the median value used for take place. The cells were spun down and resusexpression of data. In order to allow direct compa- pended in 0.1 nil MEM/Hepes with 1 mg/ml NaN, risons to be made between responses to different and then examined for capping. Ig capping was mitogens, CLL responses were transformed to carried out as outlined above, but with one layer percentages of normal responses. All calculations only using polyvalent anti-Ig (F1009, Dakopatts) were based on net counts-i.e. mitogen-stimulated instead of F2190. cultures minus unstimulated (or Sepharose rabbit Ig-exposed) cultures. Determination of surface concentration of pa microglobulin Surface markers For determination of cell surface concentration T cells were identified by the E-rosette test with of BiM without interference with Fc receptors, sheep red blood cells, as described by Heier (1974), FITC-labelled anti-@,M (Dakopatts, code F112), and B cells by anti-Ig immunofluorescent staining prepared by pepsin digestion as described above, with a polyvalent fluorescein isothiocyanate (F1TC)- was used. labelled rabbit anti-human immunoglobulin (antiOne-tenth ml FITC labelled anti-p,M (F(ab')3 IgA, IgG, IgM, kappa and lambda) serum (code F1009, Dakopatts, Copenhagen) as described else- in dilution 1 :16 with 1 mg/ml NaN, was incubated where (Heier et al., 1977). For identification of with 3 x loe lymphocytes at room temperature for surface Ig on CLL cells FITC-labelled antisera 30 min. The cells were then washed once with (immunoglobulin fraction) specific for IgA (code MEM/Hepes and once with PBS, both containing F1092), TgM (code F1091), IgG (code F1090), 1 mg/ml NaN,. The cells were resuspended in 1 nil kappa (code F109K), and lambda (code F109L) PBS with 1 mg/ml NaNs.
564
GODAL ET AL.
The amount of anti B2M F(ab’)s bound per cell was quantitated by measuring the fluorescence intensity on a single cell basis by use of flow cytofluorometry (Lindmo and Steen, 1977). The 488 nm wavelength of an Argon laser was used at 1,000 mW power for excitation of fluorescein, and the fluorescence intensity was measured at wavelengths longer than 515 nm. Unstained human lymphocytes as well as mouse bone-marrow cells stained with F112 did not give rise to pulses above the noise level seen in distributions of fluorescence from human cells stained with F112. IOl2,flUX
Cellular uptake of “Rb is assumed to reflect membrane transport of K t (Mills and Tupper, 1975). which has been observed to increase as early as 30 sec after mitogen stimulation (Averdunk, 1972). Ten to 2 0 ~ 1 0cells/ml ~ were incubated as described above in RPMI 1640 with 10% FCS at 37” C in two Falcon plastic tubes, one containing mitogen in optimal concentration, the other serving as control. After 10 min incubation 20 , ~ C i [ ~ ~ R b l(spec. Cl act. 30 Ci/niole, Institutt for Atomenergi, Kjeller, Norway) was added to each tube. Immediately, and after 10, 20, 30 and 40 min further incubation, 100-pl samples were taken from each tube. The cells were then rapidly separated from the incubation medium by centrifugation through a layer of Silicone oil (AR 200, Serva) at 10,000 g for 30 sec in a Beckman Microfuge B (Andreasen et al., 1974). The radioactivity in the cell pellet was determined in a Packard Gamma Scintiallation Counter. The K+ content of the cells was also determined, and used to estimate the total cell volume (Iversen, 1976). On the basis of these estimates the increase in intracellular concentration of Rb was calculated. The cellular uptake of isotope appeared to take place at a nearly constant rate in the 40-min period studied. The uptake rate in mitogen-treated and control cultures was compared in each experiment and differences of more than 10% were scored as significanr. RESULTS
As shown in Table I, all of the nine patients had IgM kappa or lambda chains. Six were scored as having CLL cells with Cs receptors (Patients 3,4, 6, 7, 8, 9). All the patients had more than 60% B cells in their PBL. But the exact proportion of CLL cells was often difficult to assess because of weak immunofluorescense and is likely to be higher than the figures given. The proportion of T cells ranged from 1 to 13%. The mitogen responses are shown in Figure 1. All patients tested showed suppressed responses t o the B cell mitogens PWM, anti-@,M, Sepharose protein A and Sepharose anti F(ab’)z as well as T-cell mitogens (PHA, Con A, TPA). However, a different picture was found with the ionophore A23187, where cells from three patients responded as strongly as normal cells. The identity of the blastoid cells was determined in one of the cases
+
(patient 9) and these were shown to have identical heavy and light chains to the patient’s CLL cells. Since one explanation for the suppressed responses could be a decrease or absence of surface receptors interacting with the ligands, the surface concen. tration of BzM using pepsin-digested fluoresceinlabelled anti-p,M was determined. As shown in Figure 2, a variable pattern of fluorescence distributions was found among the CLL samples tested. Two of the CLL samples (patients 7 and 9) show distributions similar to that found for normal cells (upper left, Fig. 2). Patient 3 shows a broader distribution which was found consistently in four independent samples taken from this patient. Broad distributions were, however, often seen also in normal samples (nine out of 18 cases). Thus, no consistent quantitative difference could be detected between normal and CLL cells by the use of single-cell cytofluorometry. The capping capability of CLL cells is shown in Figure 3. CLL cells revealed a markedly reduced capability to cap both BeM, Ig and J M surface receptors. Since the results with the ionophore as well as the capping experiments suggested that the blocking of mitogenesis in CLL cells was taking place at the membrane level, an early membrane event following mitogenetic stimulation, namely ion-flux, was studied, using rubidium as marker for potassium. Normal PBL preparations enriched in B cells by T-cell depletion responded in every experiment with increased ssRb uptake as early as 10 min after stimulation with Sepharose Protein A (Fig. 4 4 . Similar observations were made with anti-B,M both in B cells prepared by T-cell depletion (data not shown) and in B cells depleted of both T cells and monocytes (Fig. 46). In contrast, neither Sepharose protein A nor anti-&M altered the isotope uptake in CLL cells (Fig. 4a, b). DISCUSSION
The present study shows that peripheral blood lymphocytes from CLL patients respond poorly to the B-cell mitogens, PWM, Sepharose protein A, anti-&-microglobulin and Sepharose coupled F(ab’), antibodies. In fact, the response to these mitogens seemed to be as poor as, or poorer than, the response to the T-cell mitogens Con A and PHA. The response to the T-cell mitogens have been shown to be due to normal T cells present in PBL of CLL patients (Schweitzer et al., 1973; Wybran et al., 1973). Similarly, the responses found to B-cell mitogens in PBL from CLL patients could be due to “contaminating” normal B cells, but this possibility was not investigated in the present study. Various explanations may be considered for the suppressed responses to B-cell mitogens of CLL cells, in spite of the fact that the proportion of cells having 8-cell characteristics in PBL of CLL patients is greatly increased. First, the age distribution of the patient group and the normal group was not matched, although
MEMBRANE FUNCTIONS IN CLL CELLS
565
140 130
FIGURE1 - Mitogen responses of peripheral blood lymphocytes (PBL) from chronic lymphocytic leukemia (CLL) patients, 0 , as measured by DNA incorporation of [3H]thymidine 66-70 h after stimulation. The CLL responses are expressed as percentages of normal PBL responses (100 % SEM).The net absolute CpmhtsEM of the normal responses is also indicated. The response of each patient is shown by a circle. The number within the circle corresponds to the patient number given in Table 1. The mean value of CLL PBL responses to each mitogen is indicated by horizontal bars. The counts in unstimulated cultures were 1,673f675 (AB serum) and 2,575rt611 (FBS) of normal PBL as comDared to 605499 (AB serum) &d 488&170 (FBS)' of CLL PBL.
23990
z
52
I
70i
40
8 20
0 -
10
&
-10 PHA
CON-A
TPA
PWM Anti-BzM
b
'otein A anti-F(ab1)2A23187
FIGURE 2 - Histograms of fluorescein-labelled anti-b,M per cell measured by flow cytofluorometry for one normal sample (upper left) and three samples of peripheral blood lymphocytes from chronic lymphocytic leukemia patients. High counts were registered in channels close to the origin (No. 1-8) owing to noise pulses from impurities and cellular debris. More than 100,000 cells were measured for each histogram.
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GODAL EF AL.
Third, at least some of the B cell mitogens, such as PWM and anti-p,-microglobulin, are known to require T cells .to give optimal responses (Insel and Merler, 1977; dstberg et al., 1976). Thus, in CLL PBL normal T cells and monocytes may be present in too low concentrations for CLL cells to respond to B-cell mitogens. Alternatively, suppressor cells might be present in CLL PBL. Neither of these possibilities can be excluded. However, various observations made in this study point to a fourth possibility, namely that CLL cells are altered in such a way that they fail to respond physiologically to ligand receptor interactions. This was clearly shown to be the case with regard to capping and cation uptake. The capacity of CLL cells to cap three cell membrane markers was studied. Various investigations have earlier shown that CLL cells show a reduced
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they overlapped. However, the higher ages in the CLL patients cannot explain the large discrepancies observed here as the age-related decline in mitogenic responsiveness is much less pronounced (Pisciotta et al., 1967; Heier et al., 1977). Second, CLL may possibly be derived from a subpopulation of normal B cells which d o not respond to mitogens. This possibility cannot be entirely excluded, but would seem very unlikely since different surface markers such as Ig, Fc and C appear to occur in CLL at frequencies closely approaching those found in normal PBL B cells (Brouet et al., 1976; Salsano et al., 1976; Hellstrom et al., 1976; Pincus et al., 1972). These observations indicate, in fact, that CLL is induced randomly within the circulating pool of B cells.
FIGURE 4 - Effect of B-cell mitogens on 8sRb influx in PBL-B cells and CLL cells. Influx is measured as isotope uptake rate 10-50 min after stimulation with Sepharose protein A (a) and anti-p,M (b). The mean value of uptake rates of different cell populations stimulated with mitogen (M) and unstimulated controls (C) is shown in open bars (left ordinate). Each experiment is shown with paired comparisons between Rb flux in mitogen-stimulated and control cultures. Hatched bars (mean+sEM) show the mitogen-induced increase in sBRb uptake, given as percentage of control values (right ordinate). PBL-B cells: Peripheral blood lymphocytes enriched in B cells by the E rosette method without (Fig. 4a) or with (Fig. 46) monocyte depletion. CLL-cells: Peripheral blood lymphocytes from chronic lymphocytic leukemia.
MEMBRANE FUNCTIONS IN CLL CELLS
capability to cap (Menne and Flad, 1973; Huber et al., 1974; Cohen, 1975), while others have demonstrated capping under very similar experimental conditions (Salsano et al., 1974; Palencia et al., 1975). Our studies with Ig, PzM and J M all showed a markedly reduced capping capability of CLL cells. This finding, therefore, suggests that there are functional membrane-associated aberrations in CLL cells. Monovalent cation uptake was studied after exposure to Sepharose-bound protein A or anti-A microglobulin. Membiane transport of K-+or Rb+ has invariably been reported to increase after T-mitogen stimulation of lymphocytes (Lauf, 1975). Corresponding investigations of B-mitogenic effect on isolated B cells have not come to our knowledge. However, Freedman et al. (1975) measured Ca++ uptake in mouse spleen lymphocytes after stimulation by both T and B mitogens. They found increased uptake of Ca++in T cells, but not in B cells. Our study reveals that Rb+ transport increases after B-mitogen stimulation of normal human B cells, but not of CLL cells. Thus, the i o d u x studies also pointed to membrane-associated alterations in CLL cells. Attempts were made in two ways to bypass membrane-associated events in the mitogenic response. First, the mitogenic effect of TPA was studied. TPA, a tumor promotor, has been shown to increase intracellular levels of cyclic GMP and also to be mitogenic to lymphocytes (Estensen et al., 1974). CLL cells responded poorly to this mitogen, but the explanation may be trivial as recent studies suggest that TPA may be a selective T-cell mitogen (Touraine et al., 1977). Second, the mitogenic response to A23187 was studied. A231 87 is a ionophore with high specificity for Ca++ (Pressman, 1976) and its mitogenic effect is dependent on the presence of extracellular Ca++,
567
suggesting that its mitogenesis is due to a n influx of calcium ions into the cells (Luckasen et al., 1974; Maino et at., 1974; Hovi et al., 1976). We have shown that A23187 is mitogenic to both T and B cells (Godal, unpublished observations). Three out of the eight patients studied responded well to A23187, suggesting that the failure of CLL cells to respond to mitogens, at least in some cases, may be due to a membrane associated blockade of the mitogenic signal. Sheppard et al. (1977) have recently reported that the catecholamine receptors are reduced on CLL cells compared to normal PBL. In our study the quantity of PzM on the cell surface was studied by cytofluorometry. However, we failed to detect any consistent aberrations with regard to BzM between CLL cells and normal PBL, indicating that the failure of anti-p,M to induce capping, ionfluxes and mitogenesis is not due to reduced concentrations of BzM at the cell surface. On the other hand, we cannot exclude that a small fraction of surface receptors with special anchorage in the cell membrane or to sub-membraneous structures responsible for the functional responses, is absent from CL.L cells. The relationship between the cell membrane aberrations and the neoplastic and other properties of CLL cells remains at present a matter of speculation and is subject to further study. ACKNOWLEDGEMENTS
Drs. H. C. Godal, K. Laake, F. Wislnrff and U. Abildgaard kindly arranged for blood samples to be taken from patients. Mrs. Anne Grindflek and Miss Frances Dodman provided expert technical assistance. Miss Liv D. Arnes assisted in the preparation of the manuscript. This work was supported by the Norwegian Cancer Society.
ALTERATION DES FONCTIONS ASSOCIfiES A LA MEMBRANE DANS LES CELLULES DE LEUCEMIE LYMPHOCYTAIRE CHRONIQUE Les lymphocytes du sang pkriphkrique, consistant principalement en cellules B nkoplasiques, de neuf sujets atteints de leuckmie lymphocytaire chronique (cellules CLL) ont eu une reaction nettement affaiblie aux mitogtnes des cellules B humaines, l’anti-,9s-microglobuline, la protkine A/Sepharose et I’anti-F(ab’)s/Sepharose.Par contre, les cellules CLL de trois patients sur huit ont bien rCagi B I’ionophore A23187. La protkine A/Sepharose et I’anti-B,-microglobulinen’ont pas non plus accru I’incorporation de rubidium” (analogue du potassium) dans les cellules CLL, par comparaison avec les prkparations de lymphocytes normaux du sang pkriphkrique enrichis en cellules B. L’agrkgation de divers marqueurs de la surface, y compris la 0,-microglobuline, a dinrinuk sur les cellules CLL. Par contre, la concentration de 0,-microglobuline ti la surface n’a pas baissk, comme on I’a montrk en cytofluoromktrie avec I’anti-0,-microglobuline marqu6e A la fluoresckine. Les auteurs en concluent que divers processus associks A la membrane, dkclenchks par des interactions ligand-rkcepteur, sont altkr6s ou bloquks dans les cellules CLL.
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