CELLULAR
IMMUNOLOGY
37, dog-421 (1978)
Separation of Rat Leukocytes by Countercurrent in Aqueous Two-Phase Systems 1. Characterization P. MALMSTR~M,”
Laboratory
of Subpopulations of Ceils l
K. NELSOIL’,~ P;. J~NSSON, H. 0. SJ~GREN, H. AND
Wallenberg
Distribution
and Department
WALTER,~~
5
P-PI. ALBERTSSON 5 of Biochemistry
1, University
of Lund, Lund, Swcdrn,
Received December 2,1977 Cells from rat spleen, lymph nodes, and thoracic duct were separated by countercurrent distribution in aqueous two-polymer phase systems containing dextran and polyethylene glycol. Lymphoid cells from the different organs gave distinct, highly reproducible distribution patterns. The yield of separated cells and their viability compared well with other methods of physical separation. The majority of the leukocytes was separated from erythrocytes. Cells with surface immunoglobulin were recovered in one side of the distribution, while thymus-derived lymphocytes as determined by indirect immunofluorescence and histochemical staining were found in all fractions. However, cells responding to PHA and Con A were concentrated in a small area of the distribution, indicating a separation of subpopulations of thymus-derived lymphocytes.
INTRODUCTION Heterogeneous populations of rodent lymphoid cells can be separated on the basis either of physical characteristics of the cells such as surface charge, size, or density or of functional characteristics such as phagocytosis or adherence. Separation procedures can also be based on specific characteristics of the cells such as distinctive antigen expression or cell-surface receptors. Frequently used physical methods of separation include cell electrophoresis, velocity sedimentation, and buoyant density separation. The theory and application of these techniques have been reviewed extensively (l-3). Free-flow electrophoresis has been applied to human and a variety of rodent lymphoid cells and separates cells according to their surface charge. Velocity sedimentation at unit gravity separates cells according to size. Buoyant density gradient centrifugation and equilibrium density centrifugation separate cells according to cell density. 1 This investigation was in part supported by Public Health Service Grant CA-14924 from the National Cancer Institute through the Large Bowel Cancer Project and by grants from the Swedish Cancer Society and John and Augusta Person’s Foundation. ‘Author to whom requests for reprints should be addressed: Department of Tumor Immunology, Wallenberg Laboratory, University of Lund, Fack, S-220 07 Lund, Sweden. 3Karen Nelson was supported by a National Institute of Health Fellowship CA-05299 from the National Cancer Institute. ’ H. W. was on leave from the Veterans Administration Hospital, Long Beach, California. 5 Department of Biochemistry 1, Chemical Center, University of Lund, Lund, Sweden. 409 0008-8749/78/0372-0409$02.00/O Copyright 0 1978by AcademicPress,Inc. All rights of reproductionin any form reserved.
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ET
AL.
Aqueous solutions of dextran and polyethylene glycol (PEG)G mixed above critical concentrations form immiscible, liquid two-phase systems (4). When buffered and rendered isotonic, they are suitable for the separation of viable cells. Cells introduced into a two-phase system usually partition between one of the phases and the interface (4). The partition of the cells, i.e., their relative affinity for the top or bottom phase or their adsorption at the interface, depends both upon polymeric and ionic composition of the phases and upon the membrane properties of the cells. Using changes in phase system composition, one can effect cell separations based on surface charge-associated or on membrane lipid-related properties or on biospecific affinity (5). In the present experiments, we used phase systems which separated cells primarily on the basis of charge, a charge not necessarily the same as that measured by cell electrophoresis. Electrophoresis reflects the charge of the cells at the shear plane, while partition appears also to gauge charge deeper in the membrane (6). When the differences in cell partition coefficients 7 are large, separations can be obtained in a single partition step. As the differences in the partition coefficients of subpopulations of cells often are small, a multistep procedure is necessary to achieve a useful separation. Murine plaque-forming cells have been separated from colony-forming cells (7), rat erythrocytes from leukocytes (5), horse granulocytes from lymphocytes (5), and human tonsillar lymphoid cells forming spontaneous rosettes with sheep erythrocytes from nonrosetting cells (8) using countercurrent distribution. In the present study, we investigated the feasibility of separating subpopulations of rat lymphoid cells by partition in aqueous two-phase systems. MATERIALS
AND METHODS
Aniwzals. Rats of the WF (Ag-B2) and BN (Ag-B3) inbred strains were bred in this laboratory by strict sibling mating. They were fed a standard pellet diet and water ad libitunz. Preparation of lymphoid cells for separation by countercurrent distribution (CCD). Spleens and lymph nodes were aseptically removed from rats anesthetized with ether. Single cell suspensions were prepared in cold Waymouth’s medium plus 5% fetal calf serum (wash medium), either by gently pressing lymph nodes through a fine wire mesh or by expressing cells from the splenic capsule using bent needles. Tissue fragments were sedimented at unit gravity. The thoracic duct was cannulated in its cervical portion (9)) and lymph was collected in 0.5 ml of Waymouth’s medium plus 10% rat serum. (The thoracic duct cells were kindly supplied by Dr. B. Husberg, Malmo General Hospital, Malmii, Sweden.) Cells from the three organ sources were washed twice and pelleted in graduated conical tubes (2095, Falcon, Oxnard, California). They were suspended at 10% (v/v) in top phase (see below) for loading into the CCD apparatus. In one experiment (Fig. 3)) spleen cells with surface immunoglobulin ( SIg + cells) were removed prior to separation by CCD ( 10). Cells, 3 X 108, in 0.5 ml of wash medium plus 5 mM EDTA were passed rapidly at 4°C through a 4-ml column of Sephadex 6 Abbreviations used concanavalin A ; PBS, glutinin ; RBAA, rat SIg+ cells, cells with 7 Partition coefficient:
: BSA, bovine serum albumin ; CCD, countercurrent distribution ; Con A, phosphate-buffered saline; PEG, polyethylene glycol ; PHA, phytohemarabbit antiserum to RBAA ; brain-associated antigens ; R-anti-RBAA, membrane-bound immunoglobulin. Percentage of cells in the top phase of total cells added.
CCD
SEPARATION
OF RAT
LEUKOCYTES
411
G-100 (Pharmacia Fine Chemicals, Uppsala, Sweden) coupled with the IgG fraction of a rabbit antiserum to rat IgG. SIg+ cells were reduced from 42% to less than 2% of total spleen cells by this procedure. After passage, the cells were washed twice to remove EDTA before separation by CCD. Phase systems and CCD apparatus. We used phase systems composed of 5% (w/w) dextran T-500 (Lot No. 5556, Pharmacia Fine Chemicals, Uppsala, Sweden), 3.9 or 3.7% (w/w) polyethylene glycol (Carbowax 6000, Union Carbide, New York), 0.094 M sodium phosphate buffer pH 7.4, and 5% (w/w) heatinactivated fetal calf serum. The phases were mixed and equilibrated in a separatory funnel at 4°C overnight. Top (PEG-rich) and bottom (dextran-rich) phases were then separated. We used an automatic countercurrent distribution apparatus with circular plates as described by Albertsson (4) with 120 cavities and a bottom plate capacity of 0.67 ml. Cavities 1 and 2 and 61 and 62 were loaded with 0.5 ml of bottom phase and 0.9 ml of cells suspended in top phase (50-100 x IOG leukocytes per cavity). Remaining cavities received 0.6 ml of bottom phase and 0.8 ml of top phase (4, 5). Cells were loaded in this way to achieve a stationary interface. The cycle of operation was: shaking for 25 set and settling for 7 min, followed by a transfer. Fifty-nine transfers were completed at 4°C. Collection and counting of cells. The cells from each cavity of the countercurrent plates were collected directly into plastic centrifuge tubes and kept at 4°C. Cells from two to five adjacent tubes were pooled and phosphate-buffered saline (PBS) added to convert the two-phase system to a single phase. Cells were washed by centrifugation three times with wash medium. The number of cells in each pooled fraction was determined by counting cells diluted in PBS with an automatic counter (Mode1 ZB, Coulter Electronics, Harpenden, Hertsfordshire, England). Erythrocytes were lysed by adding Zapoglobin (Coulter Electronics) prior to automatic counting of leukocytes. The number of cells in each pool was counted, and the number of cells recovered per cavity was calculated as percentage of total cells recovered. The viability of the cells was determined by trypan blue exclusion. Direct immunofhorescence assay. To analyze the distribution of cells with surface immunoglobulin (SIg+), cells from each fraction were incubated with fluoresceinlabeled antibody to rat immunoglobulin (Miles-Yeda, Rehovoth, Israel j , Cells, 5 X 105, in 0.1 ml of PBS plus 2% bovine serum albumin and 0.2y0 sodium azide (PBS-BSA) were mixed with an equal volume of diluted antiserum. After 30 min on ice, the cells were washed three times with PBS-BSA and the percentage of stained cells was determined by counting at least 200 cells in each fraction. All samples were run in duplicate. A Leitz Dialux microscope fitted with a 100 W 12 V tungsten-halogen lamp, a Ploemopak 3 filter block, BG38 and PK500 excitation filters, and K515 and S525 supression filters was used, Indirect immunofluorescence assay. The distribution of thymus-derived lymphocytes was determined in part by indirect immunofluorescence using a rabbit antiserum to rat brain-associated antigens (R-anti-RBAA) (11, 12), which was further absorbed to remove antibodies cross-reacting with stem cells (13). In complement lysis assays at dilutions which lysed 100% of WF thymocytes, there was no lysis of SIg+ lymphocytes. There was also no apparent cytotoxicity to direct plaque-forming cells as detected in a modified Jerne assay (14). In addition, equal numbers of lymph node cells were positive for RBAA antigens by indirect immunofluorescence and complement lysis assays using this antiserum. Spleen cells
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ET
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from the pooled fractions were incubated with diluted R-anti-RBAA antiserum as above. Cells were washed twice after 30 min of incubation, mixed with fluoresceinlabeled antibody to rabbit IgG (SBL, Stockholm, Sweden) incubated again for 30 min, and washed, and the number of stained cells was determined. As a control for nonspecific staining by the conjugated antiserum, cells were mixed with PBS-BSA only during the first incubation. Mitogen stimulation of lymphoid cells. PHA-P (Difco, Detroit, Michigan) was reconstituted with 5 ml of distilled water per vial. Concanavalin A (Con A) (Pharmacia Fine Chemicals, Uppsala, Sweden) was dissolved in distilled water. Spleen cells were cultivated for 72 hr in 96 well, U-bottom microplates (Cooke Engineering, Alexandria, Virginia) at a concentration of 6 x lo5 cells per well in RPMI1640 supplemented with 2% r.-glutamine, 20 rnM HEPES, and 5% (BN x WF) Fl serum. PHA at a final concentration of l/1000 (15) and Con A, 2.5 fig ml-l (16), were added at the onset of cultures. One &i of [methyl-3H] thymidine (sp act., 5 Ci mmol-I) (Radiochemical Centre, Amersham, England) was added to each well 24 hr before termination of the cultures. Cultures were harvested with a
0
.:lj
2@ CAVITY
30
4G
50
60 -
NUMBER
1. Distribution of rat leucocytes (-) and erythrocytes (---) from different organs separated by countercurrent distribution in a 5.0/3.7 phase system. See Fig. 5 for complete composition of the phase system. All separations include 59 transfers at +4”C. The distributions presented are an average of four separations of spleen cells, an average of two separations of lymph node cells, and one separation of thoracic duct cells. FIG.
CCD
SEPARATION
OF RAT
413
LEUKOCYTES
semiautomatic multiple sample harvester (Otto Hiller, Madison, Wisconsin) onto glass-fiber filters. Radioactivity of the samples was determined with an Intertechnique Liquid Scintillation Counter (Nanotechnique, Tgby, Sweden). Cytochemical staining of cells. The distribution of cells with chloroacetate esterase (granulocytes ) ( 17) and a-naphthyl esterase [ granulocytes, monocytes, and some thymus-derived lymphocytes ( 18) ] was determined by a combined staining procedure (19). Smears of cells from the pooled fractions were stained within 24 hr. The number and morphology of cells with deep blue or dark red granules were determined for at least 300 cells in each preparation. RESULTS Partition of rat leukocytes from diferent organs. Single cell suspensions from WF rat spleen, lymph nodes, and thoracic duct lymph were separated by countercurrent distribution (CCD) in a phase system containing 5.0% dextran and 3.7% PEG (See Materials and Methods for complete composition of phase system). Cells in adjacent cavities were pooled and counted, The percentages of leukocytes and erythrocytes recovered in each cavity are shown in Fig. 1. Leukocytes from the spleen separated into three peaks: The major peak of cells had the lowest partition coefficient (cavities 10-25). This distribution curve is an average of four individual separations. The maximum variation of the median cavity of each peak was five cavities: peak I (cavities 16-21), peak II (cavities 28-33), and peak III (cavities 45-50). The majority of lymph node leukocytes were found in a large peak corresponding to the second peak of splenic leukocytes. Leukocytes from thoracic duct lymph separated into a major and a minor peak, the major peak occurring between the second and third peaks of splenic leukocytes. Erythrocytes were located in one peak (cavities 35-60) regardless of source. The viability of splenic leukocytes after CCD separation is shown in Table 1. Damaged cells were mainly recovered in the first peak. Cells excluding trypan blue
TABLE Viability
1
of Splenic Leukocytes
Cavities
pooled
After
Mean percentage viable cells f SEb
04 5-9 10-14 15-19 20-24 25-29 30-34 35-39 4044 45-49 SO-59 a Viability was determined polymers. b Mean of three separations
by trypan
CCD Separationa
blue exclusion
in a 5.0% dextran/3.9%
64.6 72.0 74.3 76.0 69.0 87.0 91.6 85.0 87.0 86.5 91.6
f f f f f f f f f f f
3.9 7.6 5.4 7.1 6.3 3.7 2.2 6.4 6.5 2.5 5.2
after
washing
to remove
PEG phase system.
phase system
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8
CAVITY
ET
AL.
NUMBER
FIG. 2. Countercurrent distribution of spleen cells carrying different surface markers as defined by immunofluorescence. Percentage per cavity of total white blood cells recovered (-). Percentage of cells in each fraction stained with anti-rat IgG, (direct immunofluorescence) (~---a ). Percentage of cells in each fraction stained with antiserum to rat brain, (indirect immunofluorescence) (0-O). Average of two experiments in a 5.0/3.7 phase system.
in peaks II and III averaged 90%. The average recovery of viable cells from a CCD separation was 71% of loaded, viable cells (range, 55-100, n = 15). Imvnunofluorescence studies. In the unseparated spleen cell population, 49% of the cells had surface immunoglobulin (SIg+ cells ; range, 44-56%). When CCDseparated cells were assayed for surface immunoglobulin by direct immunofluorescence, almost 50% of the cells with the lowest partition coefficient (the first peak) were SIgf (Fig. 2). The percentage of SIg+ cells constantly declined along the CCD train until fewer than 2% of the cells with the highest partition coefficient (third peak) expressed SIg. As is apparent from the figure there was a selective loss of SIg+ cells, an average of 35% (range, 25-40) of the SIg+ cells placed into the CCD apparatus were lost during the separation. The proportion of thymus-derived lymphocytes was estimated by indirect immunofluorescence using an antiserum to rat brain-associated antigens (RBAA) (12, 13). The unseparated spleen cell population contained 38% stained cells
CAVITY
NUMBER
FIG. 3. Distribution of SIg+ and SIg- spleen cells. SIgS cells were removed from one spleen cell suspension by affinity chromatography prior to CCD separation. A. Distribution of spleen cells (O-O) and spleen cells depleted of SIg+ cells (O---O) in a 5.0/3.9 phase system. B. Comparison of the percentages of SIg+ cells in the different fractions from the whole spleen cell population (O--O) and spleen cells depleted of SIgS cells by affinity chromatography (O- - -0).
CCD
SEPARATION
OF RAT
LEUKOCYTES
41.5
FIG. 4. Distribution of spleen cells responding to mitogens. Pooled fractions of cells from the CCD train were stimulated with PHA at l/100 dilution (top) and Con A at 2.5 pg ml-’ (bottom), cultivated for 72 hr, and labeled with [nzetJryZ-3H]thymidine 24 hr prior to harvest. Results of mitogen stimulation are expressed as percentage stimulation compared to unseparated control spleen cell cultures. The figures include the results of five different experiments on cells separated in a 5.0/3.7 phase system. Filled symbols represent tumor-immunized rats and unfilled symbols represent untreated rats.
(range, 3142%). F rom Fig. 2 it can be seen that RBAA+ cells account for 68--74% of the cells with a high partition coefficient (peak III) and 41-45% of the cells with a low partition coefficient (peak I). The fractions at the right end of the CCD train were thus virtually free of SIg+ cells, but no fraction was completely devoid of RBAA+ lymphocytes. No selective loss of RBAA+ lymphocytes was observed. Partition of spleen cells after removal of SIgf cells. To delineate further the distribution of SIg+ and SIg- spleen cells, SIg+ cells were removed from the spleen cell suspension prior to CCD separation (10). Figure 3A compares the distributions of spleen cells and spleen cells depleted of SIg+ cells in a 5.0/3.9-phase system. Figure 3B compares the percentage of SIg+ cells recovered in the pooled fractions. The removal of SIg+ cells decreased the percentage of leukocytes re-
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ET
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covered in the first peak. The 2% SIg+ cells remaining after affinity chromatography were all recovered in the first fractions. Lymphocytes with optimal response to T-cell PHA and Con A resjonsiweness. mitogens were concentrated in cavities 32 to 42, corresponding to the right half of the second peak of splenic leukocytes. A peak of stimulation was most distinct for PHA, while the response to Con A showed a somewhat broader profile (Fig. 4). Cells on either side of these cavities showed stimulation that was lower than that of unseparated control cells. These differences cannot be accounted for by a lower number of RBAA+ lymphocytes in these fractions (compare with Fig. 2). Spleen cells from both normal control rats and from rats immunized to colon carcinomas were tested and no difference in reactivity to PHA and Con A was seen. Histochenzical demonstration of intracellular esterase. Populations of rodent lymphocytes have been identified by cytochemical staining of intracellular esterases. Chloroacetate esterase is present in granulocytes but not in cells of the monocyte or lymphocyte series (17). a-Naphthyl esterase has been reported in granulocytes, monocytes, and a subpopulation of thymus-derived lymphocytes (17, 18). The distribution of these cells from rat spleen was evaluated by staining smears of cells from pooled fractions for both enzyme activities (19). The size, morphology, and number of cells with deep blue granules (chloroacetate esterase) or dark red granules (a-naphthyl esterase) was determined for at least 300 cells in each preparation. Small mononuclear cells with one to three red granules were not considered positive in this study (18). In two separations of spleen cells, small mononuclear cells heavily stained for a-naphthyl esterase accounted for fewer than 10% of the cells in peak I and 10 to 20% of the cells in the left half of peak II and rose to 45 to 5070 of the cells in the right half of peak II and peak III. As SIg+ cells are reported to lack this enzyme, we also investigated the enzyme activity of separated SIg- cells (Fig. 3). As shown in Table 2, removal of SIg+ cells increased the percentage of esterase-positive small mononuclear cells in peak I and the left half of peak II, indicating that esterase-positive cells were not retained on the anti-Ig TABLE
2
Distribution of Spleen Cells with ol-Naphthyl Chloroacetate Spleen cell peak
Cells from cavities
Percentage
Neither enzyme
I I I II II III
5-9 10-14 15-19 23-25 29-31 4549
Acetate
Esterase or
Esterase Activity
93 72 IO 66 67 47
of leukocytes immunoglobulin
with no surface with
c+Naphthylacetate esterase mononuclear cells Small
Large
2 23 21 30 31 48
3 0 2 2 3 5
Chloroacetate esterase polynuclear cells
2 4 7 2 0 0
(LSpleen cells depleted of SIg+ cells were separated in a 5.0/3.9 phase system (Fig. 3). Smears of cells from pooled fractions were stained using a combined method for both enzymes (19).
CCD
SEPARATION
OF RAT
417
LEUKOCYTES
I
, 1
mm 6. 5. 4 3. 2. 1.
8 5 4 3 2 1 0
10
20
30
CAVITY
40
50
60
NUMBER
5. Effect of changes in phase system composition on the distribution of rat spleen leucoFIG. cytes (-) and erythrocytes (---). Phase systems used were 5.0% dextran T-500 and 3.7% polyethylene glycol 6000 (5.0/3.7) or 5.0% dextran T-500 and 3.9% polyethylene glycol 6000 (5.0/3.9). Both phase systems contained 0.094 M sodium phosphate buffer, pH 7.4, and were supplemented with 5% (w/w) fetal calf serum.
affinity columns. Esterase-positive cells again accounted for 48% of the leukocytes in peak III. The distributions of large mononuclear cells with a-naphthyl esterase and polynuclear cells with chloroacetate esterase were similar in the three separations. The latter were recovered in the first peak (Table 2). a-Naphthyl esterase positive large mononuclear cells were found in all three peaks. Comparison of two diflerent phase systewzs. In this study we compared two phase systems both containing 5.0% dextran but with different polyethylene glycol (PEG)
CAVITY
NUMBER
6. Distribution of rat spleen leucocytes with surface immunoglobulin, SIg, in two different phase systems, 5.0/3.7 ( 0-0 ) and 5.0/3.9 (O---O). A. SIg+ cells in each cavity expressed as a percentage of total white blood cells recovered. B. Percentage of SIg+ cells in each fraction. FIG.
418
MALMSTRCM
ET
AL.
concentrations: 3.7 or 3.9%. The difference in the distribution of spleen cells separated in the two phase systems, 5.0/3.7 and 5.0/3.9, is shown in Fig. 5. A great part of the leukocytes was moved to the left when the concentration of PEG was increased. The distribution of red cells was essentially unaffected. The distribution of SIg+ cells was compared in both phase systems. Increase in the concentration of PEG shifted the SIgf cells to the left end of the CCD train along with the whole cell population (Fig. 6A). However, the relative percentage of the cells in the different peaks was virtually unchanged (Fig. 6B). A small fraction of the SIgf cells seemed not to be affected by this change in the phase system, as the distribution curves of these cells in both phase systems from cavities 25 through 40 were almost identical. DISCUSSION The present investigation demonstrates the feasibility of using countercurrent distribution in aqueous two-phase systems for separation of rat lymphoid cells. Lymphoid cells from different organs give distinct, highly reproducible distribution patterns. The average recovery of 70% of the cells subjected to separation is quite satisfactory when compared to other techniques and in view of the length of the procedure and the numerous tube transfers involved. The viability of the recovered cells also compares favorably with other separation methods. Leukocytes from the spleen were separated into three peaks, Lymphocytes expressing SIg were found mainly in the first peak. Lymphocytes expressing rat brain-associated antigens (RBAA), the majority of the thymus-derived lymphocytes in the spleen (20), distributed all across the CCD train and constituted most of the cells of peak II and III. Lymphocytes giving a maximum response after PHA stimulation were recovered in the right half of the second peak. The maximum response to Con A was found in the same region, but the distribution of the responding cells was somewhat broader. Small mononuclear cells with intracellular esterase were mainly recovered in peaks II and III. These cells reportedly represent a subpopulation of thymus-derived lymphocytes (18). Granulocytes as defined morphologically and histochemically were recovered in the first peak. The wide distribution of thymus-derived lymphocytes indicates great variety in surface characteristics of these cells. This might reflect the existence of several subsets of thymus-derived lymphocytes in the spleen or, alternatively, cells at various stages of maturation. We realize that the enumeration of thymus-derived lymphocytes by indirect immunofluorescence carries the risk of nonspecific staining and overestimation of the number of positively stained cells. All percentages of stained cells were corrected for nonspecifically stained cells. The frequency of the latter was determined by incubation of cells with BSA followed by the anti-rabbit Ig serum. No other attempt was made in the present investigation to rule out staining of cells with Fc receptors by, e.g., use of F(ab)z fragments of the antisera. However, our recorded frequencies of SIg+ cells and thymus-derived (RBAA+) cells in the rat spleen correspond closely to those reported by others (20, 21). In view of the wide distribution of RBAAf cells, the narrow peak of cells responding to T-cell mitogens is intriguing. The peak stimulation seen in cavities 37 to 42 cannot be accounted for by a higher number of thymus-derived lymphocytes in these fractions, as they vary from 42 to 78% along the CCD train. However, the mitogen stimulation assays are also affected by the presence or absence of other
CCD
SEPARATION
OF RAT
LEUKOCYTES
419
cell pop&tions. For example, an optimal response to mitogens requires accessory cells (22). In the lymphocyte culture system used, it has been shown that both adherent cells (removable by Sephadex G-10 columns) and normal rat serum are necessary for an optimal response (P. Malmstriim, unpublished results). However. no apparent concentration of large mononuclear cells could be detected in the region of optima] mitogen responsiveness. Large mononuclear cells with a-naphthyl esterase thus distributed all along the CCD train. It has been reported that an admixture of erythrocytes may increase the response to PHA in human systems (23) but does not affect the response to Con A. In the cavities where optimal lymphocyte stimulation is found, the ratio of erythrocytes to lymphocyes varies from 5 :l to 15 : 1. Control experiments with an admixture of rat erythrocytes from peripheral blood to lymphocytes in proportions from 1: 1 to 20 : 1 have shown slight inhibitory effects on the response to PHA and Con A except at the highest dose. 20 : 1, where a slight enhancement was seen. (P. Malmstrom, unpublished results). Moreover, the peak of erythrocytes in the 5.0/3.7 phase system was found in cavity 48. Suppressive effects on lymphocyte activation by macrophages (24) and by cooperation between macrophages and a subpopulation of thymus-derived lymphocytes (25,26) have been found to influence profoundly the response to mitogens. The existence of possible suppressor populations in this system has not been investigated. There appeared to be a selective loss of about 35% of the SIg+ cells during the separation. A reason for this might be that some SIg+ cells of the unseparated cell population, which have passively acquired Ig by their Fc receptors, lose this Ig during the separation procedure. Another possibility is that S1g-t cells are lost during the separation because of a greater tendency to adhere to plastic surfaces (27). Although many other physical separation methods also yield viable, functional cells, a distinct advantage of CCD separation is that the cell fractions are not exposed to differences in pH, osmolarity, or ion concentration during the separation procedure. Although surface charge-associated properties form one basis of cell separation in two-phase systems, the distribution of SIg+ cells and thymus-derived lymphocytes in our system differs from that reported using cell electrophoresis. With free-flow electrophoresis, SIg+ cells and thymus-derived cells in mice are separated into two distinct peaks (2). The overlap of populations in our system may simply reflect differences in the original cell composition, as other investigators removed adherent and phagocytic cells prior to separation (2). Unfortunately, cells other than monocytes and macrophages are also lost during these procedures (3). Alternatively, two-phase systems may efficiently separate subpopulations of the two main groups of lymphoid cells by virtue of ce]] characteristics other than the surface charge such as hydrophobic and other nonionic interactions between the cells and the phases. Similar subpopulations have hiterto been separated only by use of density gradient centrifugation or density gradient electrophoresis (1, 28). The phase systems used in this study were chosen on the basis of cell partition experiments in single tubes, with systematic changes in phase composition (for a discussion see Refs. 4 and 5). Systems with about 5070 leukocytes in the top phase were considered suitable. The decrease in partition coefficient of the majority of the leukocytes, when the PEG concentration is raised from 3.7 to 3.9$%, is probably due
420
MALMSTRiiM
ET
AL,
to the increase in interfacial tension (29). The reason that the leukocytes in the current experiments are shifted to a greater extent than the erythrocytes is a consequence of the very high affinity of the latter for the top phase. The polymer concentration would have to be increased appreciably to effect a decrease in the partition coefficient of rat erythrocytes. Thus, changes in phase composition do not affect the partition coefficients of different cell types to the same extent. This may be used to design phase systems for particular purposes with the different factors, electrical charge, interfacial tension, hydrophobicity, etc. interacting in a desired manner. CCD separation can thus be considered a reliable and reproducible method for the separation of rat leukocyte populations. It is also possible to make affinity phase systems specific for certain cell surface receptors. Using specific ligands coupled to one of the polymers, it has been possible to shift the partitioning of proteins (30), cell membrane fragments (31, 32), and cells (33, 34). This technique may be further developed for affinity partition of intact lymphoid cells. Also, a combination of CCD separation with other separation methods based on defined cell surface receptors may yield a finer discrimination of lymphocyte subpopulations. ACKNOWLEDGMENTS We thank Ms. Adine Karlsson, Ms. Ingar Nilsson, Ms. Mariana Nilsson, Ms. Ingela Stadenberg, and Ms. Lena Thiman for skillful technical assistance.
REFERENCES 1. Moller, G. (Ed.), Transpant. Rev. 25, 1975. 2. Natvig, J. B., Perlmann, P., and Wigzell, H. (Eds.), Stand. J. ZmmunoZ., SupjZ. 5, 1976. 3. Bloom, B. R., and David, J. R. (Eds.), “In Vitro Methods in Cell-Mediated Immunity. Academic Press, New York, 1976. 4. Albertsson, P.-A., “Partition of cell particles and macromolecules.” Almqvist and Wiksell, Stockholm, 1971. 5. Walter, H., In. “Methods of Cell Separation” (N. Catsimpoolas, Ed.), Vol. 1, pp. 307-354, Plenum,~New York, 1977. 6. Brunette, D. M., McCulloch, E. A., and Till, Y. E., Cell T&s. Kin&. 1, 319, 1968. 7. Walter, H., and Nagaya, H., Cell. Immunol. 19, 158, 1975. 8. Saldeen, T., and Linder, E., Acta Pathol. Microbial. Scand. 49, 433, 1960. 9. Schlossman, S. F., and Hudson, L., J. Immunol. 110, 313, 1973. 10. Golub, E. S., Cell. Immunol. 2, 353, 1971. 11. Peter, H.-H., Clagett, J., Feldman, J. D., and Weigle, W. O., J. ImmunoZ. 110, 1077, 1973. 21, 124, 1976. 12. Filippi, J. A., Rheins, M. S., and Nyerges, C. A., Transplantation 13. Cunningham, A. J., and Szenberg, A., Immzmology 14, 599, 1968. 14. Johnston, J. M., and Wilson, D. B., Cell. Immunol. 1, 430, 1970. 15. Chauvenet, A. R., and Scott, D. W., J. Zmmunol. 114, 470, 1975. 16. Fey, F., and Kuntze, A., Folia Haematol. (Leipzig) 93, 241, 1970. 17. Mueller, J., Keller, H. U., Brun de1 Re, G., Buerki, H., and Hess, M. W., Advan. Exp. Med. BioZ. 66, 117, 1976. 18. Yam, L. T., Li, C. Y., and Crosby, W. H., Amer. J. CZin. Pathol. 55, 283, 1971. 19. Roy, R., McNicoll, J., and Daguillard, F., Int. Arch. Allergy ApgZ. Immunol. 52, 32, 1976. 20. Go!dschneider, I., and McGregor, D. D., J. Exp. Med. 138, 1443, 1973. 21. Yoshinaga, M., and Waksman, B. H., Ann. Immunol. (Inst. Pasteur) 124C, 97, 1973. 22. Yachnin, S., Clin. Exp. Immunol. 11, 109, 1972. 23. Raff, H. V., and Hinrichs, D. J., Cell. ZmmunoZ.29, 109, 1977. 24. Folch, H., and Waksman, B. H., Cell. Zmmunol. 9, 12, 1973. 25. Raff, H. V., and Hinrichs, D. Y., Cell. Immunol. 29, 118, 1977. 26. Hogg, N., and Greaves, M. F., Zmmmtology 22, 959, 1971.
CCD
27. 28. 29. 30. 31. 32. 33.
SEPARATION
OF RAT
LEUKOCYTES
Platsoucas, C. D., Griffith, A. L., and Catsimpoolas, N., J. ~wlzz~nol. MctR. 13, 145, 1976. RydCn, J., and Albertsson, P.-A., .I. Colloid Interface Sci. 37, 219, 1971. Flanagan, S. D., and Barondes, S. H., J. Biol. Chcm. 250, 1484, 1975. Walter, H., and Krob, E. J., FEBS Lett. 61, 290, 1976. Flanagan, S. D., Barondes, S. H., and Taylor, P., J. Biol. Chem. 251, 858, 1976. Eriksson, E., Albertsson, P.-A., Johansson, G., Mol. Cell. BiocRem. 10, 123, 1976. Walter, H., Krob, E. J., and Tung, R., Exp. Cell. Rcs. 102, 14, 1976.
421
IMMUNOLOGY
37, dog-421 (1978)
Separation of Rat Leukocytes by Countercurrent in Aqueous Two-Phase Systems 1. Characterization P. MALMSTR~M,”
Laboratory
of Subpopulations of Ceils l
K. NELSOIL’,~ P;. J~NSSON, H. 0. SJ~GREN, H. AND
Wallenberg
Distribution
and Department
WALTER,~~
5
P-PI. ALBERTSSON 5 of Biochemistry
1, University
of Lund, Lund, Swcdrn,
Received December 2,1977 Cells from rat spleen, lymph nodes, and thoracic duct were separated by countercurrent distribution in aqueous two-polymer phase systems containing dextran and polyethylene glycol. Lymphoid cells from the different organs gave distinct, highly reproducible distribution patterns. The yield of separated cells and their viability compared well with other methods of physical separation. The majority of the leukocytes was separated from erythrocytes. Cells with surface immunoglobulin were recovered in one side of the distribution, while thymus-derived lymphocytes as determined by indirect immunofluorescence and histochemical staining were found in all fractions. However, cells responding to PHA and Con A were concentrated in a small area of the distribution, indicating a separation of subpopulations of thymus-derived lymphocytes.
INTRODUCTION Heterogeneous populations of rodent lymphoid cells can be separated on the basis either of physical characteristics of the cells such as surface charge, size, or density or of functional characteristics such as phagocytosis or adherence. Separation procedures can also be based on specific characteristics of the cells such as distinctive antigen expression or cell-surface receptors. Frequently used physical methods of separation include cell electrophoresis, velocity sedimentation, and buoyant density separation. The theory and application of these techniques have been reviewed extensively (l-3). Free-flow electrophoresis has been applied to human and a variety of rodent lymphoid cells and separates cells according to their surface charge. Velocity sedimentation at unit gravity separates cells according to size. Buoyant density gradient centrifugation and equilibrium density centrifugation separate cells according to cell density. 1 This investigation was in part supported by Public Health Service Grant CA-14924 from the National Cancer Institute through the Large Bowel Cancer Project and by grants from the Swedish Cancer Society and John and Augusta Person’s Foundation. ‘Author to whom requests for reprints should be addressed: Department of Tumor Immunology, Wallenberg Laboratory, University of Lund, Fack, S-220 07 Lund, Sweden. 3Karen Nelson was supported by a National Institute of Health Fellowship CA-05299 from the National Cancer Institute. ’ H. W. was on leave from the Veterans Administration Hospital, Long Beach, California. 5 Department of Biochemistry 1, Chemical Center, University of Lund, Lund, Sweden. 409 0008-8749/78/0372-0409$02.00/O Copyright 0 1978by AcademicPress,Inc. All rights of reproductionin any form reserved.
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MALMSTRGM
ET
AL.
Aqueous solutions of dextran and polyethylene glycol (PEG)G mixed above critical concentrations form immiscible, liquid two-phase systems (4). When buffered and rendered isotonic, they are suitable for the separation of viable cells. Cells introduced into a two-phase system usually partition between one of the phases and the interface (4). The partition of the cells, i.e., their relative affinity for the top or bottom phase or their adsorption at the interface, depends both upon polymeric and ionic composition of the phases and upon the membrane properties of the cells. Using changes in phase system composition, one can effect cell separations based on surface charge-associated or on membrane lipid-related properties or on biospecific affinity (5). In the present experiments, we used phase systems which separated cells primarily on the basis of charge, a charge not necessarily the same as that measured by cell electrophoresis. Electrophoresis reflects the charge of the cells at the shear plane, while partition appears also to gauge charge deeper in the membrane (6). When the differences in cell partition coefficients 7 are large, separations can be obtained in a single partition step. As the differences in the partition coefficients of subpopulations of cells often are small, a multistep procedure is necessary to achieve a useful separation. Murine plaque-forming cells have been separated from colony-forming cells (7), rat erythrocytes from leukocytes (5), horse granulocytes from lymphocytes (5), and human tonsillar lymphoid cells forming spontaneous rosettes with sheep erythrocytes from nonrosetting cells (8) using countercurrent distribution. In the present study, we investigated the feasibility of separating subpopulations of rat lymphoid cells by partition in aqueous two-phase systems. MATERIALS
AND METHODS
Aniwzals. Rats of the WF (Ag-B2) and BN (Ag-B3) inbred strains were bred in this laboratory by strict sibling mating. They were fed a standard pellet diet and water ad libitunz. Preparation of lymphoid cells for separation by countercurrent distribution (CCD). Spleens and lymph nodes were aseptically removed from rats anesthetized with ether. Single cell suspensions were prepared in cold Waymouth’s medium plus 5% fetal calf serum (wash medium), either by gently pressing lymph nodes through a fine wire mesh or by expressing cells from the splenic capsule using bent needles. Tissue fragments were sedimented at unit gravity. The thoracic duct was cannulated in its cervical portion (9)) and lymph was collected in 0.5 ml of Waymouth’s medium plus 10% rat serum. (The thoracic duct cells were kindly supplied by Dr. B. Husberg, Malmo General Hospital, Malmii, Sweden.) Cells from the three organ sources were washed twice and pelleted in graduated conical tubes (2095, Falcon, Oxnard, California). They were suspended at 10% (v/v) in top phase (see below) for loading into the CCD apparatus. In one experiment (Fig. 3)) spleen cells with surface immunoglobulin ( SIg + cells) were removed prior to separation by CCD ( 10). Cells, 3 X 108, in 0.5 ml of wash medium plus 5 mM EDTA were passed rapidly at 4°C through a 4-ml column of Sephadex 6 Abbreviations used concanavalin A ; PBS, glutinin ; RBAA, rat SIg+ cells, cells with 7 Partition coefficient:
: BSA, bovine serum albumin ; CCD, countercurrent distribution ; Con A, phosphate-buffered saline; PEG, polyethylene glycol ; PHA, phytohemarabbit antiserum to RBAA ; brain-associated antigens ; R-anti-RBAA, membrane-bound immunoglobulin. Percentage of cells in the top phase of total cells added.
CCD
SEPARATION
OF RAT
LEUKOCYTES
411
G-100 (Pharmacia Fine Chemicals, Uppsala, Sweden) coupled with the IgG fraction of a rabbit antiserum to rat IgG. SIg+ cells were reduced from 42% to less than 2% of total spleen cells by this procedure. After passage, the cells were washed twice to remove EDTA before separation by CCD. Phase systems and CCD apparatus. We used phase systems composed of 5% (w/w) dextran T-500 (Lot No. 5556, Pharmacia Fine Chemicals, Uppsala, Sweden), 3.9 or 3.7% (w/w) polyethylene glycol (Carbowax 6000, Union Carbide, New York), 0.094 M sodium phosphate buffer pH 7.4, and 5% (w/w) heatinactivated fetal calf serum. The phases were mixed and equilibrated in a separatory funnel at 4°C overnight. Top (PEG-rich) and bottom (dextran-rich) phases were then separated. We used an automatic countercurrent distribution apparatus with circular plates as described by Albertsson (4) with 120 cavities and a bottom plate capacity of 0.67 ml. Cavities 1 and 2 and 61 and 62 were loaded with 0.5 ml of bottom phase and 0.9 ml of cells suspended in top phase (50-100 x IOG leukocytes per cavity). Remaining cavities received 0.6 ml of bottom phase and 0.8 ml of top phase (4, 5). Cells were loaded in this way to achieve a stationary interface. The cycle of operation was: shaking for 25 set and settling for 7 min, followed by a transfer. Fifty-nine transfers were completed at 4°C. Collection and counting of cells. The cells from each cavity of the countercurrent plates were collected directly into plastic centrifuge tubes and kept at 4°C. Cells from two to five adjacent tubes were pooled and phosphate-buffered saline (PBS) added to convert the two-phase system to a single phase. Cells were washed by centrifugation three times with wash medium. The number of cells in each pooled fraction was determined by counting cells diluted in PBS with an automatic counter (Mode1 ZB, Coulter Electronics, Harpenden, Hertsfordshire, England). Erythrocytes were lysed by adding Zapoglobin (Coulter Electronics) prior to automatic counting of leukocytes. The number of cells in each pool was counted, and the number of cells recovered per cavity was calculated as percentage of total cells recovered. The viability of the cells was determined by trypan blue exclusion. Direct immunofhorescence assay. To analyze the distribution of cells with surface immunoglobulin (SIg+), cells from each fraction were incubated with fluoresceinlabeled antibody to rat immunoglobulin (Miles-Yeda, Rehovoth, Israel j , Cells, 5 X 105, in 0.1 ml of PBS plus 2% bovine serum albumin and 0.2y0 sodium azide (PBS-BSA) were mixed with an equal volume of diluted antiserum. After 30 min on ice, the cells were washed three times with PBS-BSA and the percentage of stained cells was determined by counting at least 200 cells in each fraction. All samples were run in duplicate. A Leitz Dialux microscope fitted with a 100 W 12 V tungsten-halogen lamp, a Ploemopak 3 filter block, BG38 and PK500 excitation filters, and K515 and S525 supression filters was used, Indirect immunofluorescence assay. The distribution of thymus-derived lymphocytes was determined in part by indirect immunofluorescence using a rabbit antiserum to rat brain-associated antigens (R-anti-RBAA) (11, 12), which was further absorbed to remove antibodies cross-reacting with stem cells (13). In complement lysis assays at dilutions which lysed 100% of WF thymocytes, there was no lysis of SIg+ lymphocytes. There was also no apparent cytotoxicity to direct plaque-forming cells as detected in a modified Jerne assay (14). In addition, equal numbers of lymph node cells were positive for RBAA antigens by indirect immunofluorescence and complement lysis assays using this antiserum. Spleen cells
412
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ET
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from the pooled fractions were incubated with diluted R-anti-RBAA antiserum as above. Cells were washed twice after 30 min of incubation, mixed with fluoresceinlabeled antibody to rabbit IgG (SBL, Stockholm, Sweden) incubated again for 30 min, and washed, and the number of stained cells was determined. As a control for nonspecific staining by the conjugated antiserum, cells were mixed with PBS-BSA only during the first incubation. Mitogen stimulation of lymphoid cells. PHA-P (Difco, Detroit, Michigan) was reconstituted with 5 ml of distilled water per vial. Concanavalin A (Con A) (Pharmacia Fine Chemicals, Uppsala, Sweden) was dissolved in distilled water. Spleen cells were cultivated for 72 hr in 96 well, U-bottom microplates (Cooke Engineering, Alexandria, Virginia) at a concentration of 6 x lo5 cells per well in RPMI1640 supplemented with 2% r.-glutamine, 20 rnM HEPES, and 5% (BN x WF) Fl serum. PHA at a final concentration of l/1000 (15) and Con A, 2.5 fig ml-l (16), were added at the onset of cultures. One &i of [methyl-3H] thymidine (sp act., 5 Ci mmol-I) (Radiochemical Centre, Amersham, England) was added to each well 24 hr before termination of the cultures. Cultures were harvested with a
0
.:lj
2@ CAVITY
30
4G
50
60 -
NUMBER
1. Distribution of rat leucocytes (-) and erythrocytes (---) from different organs separated by countercurrent distribution in a 5.0/3.7 phase system. See Fig. 5 for complete composition of the phase system. All separations include 59 transfers at +4”C. The distributions presented are an average of four separations of spleen cells, an average of two separations of lymph node cells, and one separation of thoracic duct cells. FIG.
CCD
SEPARATION
OF RAT
413
LEUKOCYTES
semiautomatic multiple sample harvester (Otto Hiller, Madison, Wisconsin) onto glass-fiber filters. Radioactivity of the samples was determined with an Intertechnique Liquid Scintillation Counter (Nanotechnique, Tgby, Sweden). Cytochemical staining of cells. The distribution of cells with chloroacetate esterase (granulocytes ) ( 17) and a-naphthyl esterase [ granulocytes, monocytes, and some thymus-derived lymphocytes ( 18) ] was determined by a combined staining procedure (19). Smears of cells from the pooled fractions were stained within 24 hr. The number and morphology of cells with deep blue or dark red granules were determined for at least 300 cells in each preparation. RESULTS Partition of rat leukocytes from diferent organs. Single cell suspensions from WF rat spleen, lymph nodes, and thoracic duct lymph were separated by countercurrent distribution (CCD) in a phase system containing 5.0% dextran and 3.7% PEG (See Materials and Methods for complete composition of phase system). Cells in adjacent cavities were pooled and counted, The percentages of leukocytes and erythrocytes recovered in each cavity are shown in Fig. 1. Leukocytes from the spleen separated into three peaks: The major peak of cells had the lowest partition coefficient (cavities 10-25). This distribution curve is an average of four individual separations. The maximum variation of the median cavity of each peak was five cavities: peak I (cavities 16-21), peak II (cavities 28-33), and peak III (cavities 45-50). The majority of lymph node leukocytes were found in a large peak corresponding to the second peak of splenic leukocytes. Leukocytes from thoracic duct lymph separated into a major and a minor peak, the major peak occurring between the second and third peaks of splenic leukocytes. Erythrocytes were located in one peak (cavities 35-60) regardless of source. The viability of splenic leukocytes after CCD separation is shown in Table 1. Damaged cells were mainly recovered in the first peak. Cells excluding trypan blue
TABLE Viability
1
of Splenic Leukocytes
Cavities
pooled
After
Mean percentage viable cells f SEb
04 5-9 10-14 15-19 20-24 25-29 30-34 35-39 4044 45-49 SO-59 a Viability was determined polymers. b Mean of three separations
by trypan
CCD Separationa
blue exclusion
in a 5.0% dextran/3.9%
64.6 72.0 74.3 76.0 69.0 87.0 91.6 85.0 87.0 86.5 91.6
f f f f f f f f f f f
3.9 7.6 5.4 7.1 6.3 3.7 2.2 6.4 6.5 2.5 5.2
after
washing
to remove
PEG phase system.
phase system
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8
CAVITY
ET
AL.
NUMBER
FIG. 2. Countercurrent distribution of spleen cells carrying different surface markers as defined by immunofluorescence. Percentage per cavity of total white blood cells recovered (-). Percentage of cells in each fraction stained with anti-rat IgG, (direct immunofluorescence) (~---a ). Percentage of cells in each fraction stained with antiserum to rat brain, (indirect immunofluorescence) (0-O). Average of two experiments in a 5.0/3.7 phase system.
in peaks II and III averaged 90%. The average recovery of viable cells from a CCD separation was 71% of loaded, viable cells (range, 55-100, n = 15). Imvnunofluorescence studies. In the unseparated spleen cell population, 49% of the cells had surface immunoglobulin (SIg+ cells ; range, 44-56%). When CCDseparated cells were assayed for surface immunoglobulin by direct immunofluorescence, almost 50% of the cells with the lowest partition coefficient (the first peak) were SIgf (Fig. 2). The percentage of SIg+ cells constantly declined along the CCD train until fewer than 2% of the cells with the highest partition coefficient (third peak) expressed SIg. As is apparent from the figure there was a selective loss of SIg+ cells, an average of 35% (range, 25-40) of the SIg+ cells placed into the CCD apparatus were lost during the separation. The proportion of thymus-derived lymphocytes was estimated by indirect immunofluorescence using an antiserum to rat brain-associated antigens (RBAA) (12, 13). The unseparated spleen cell population contained 38% stained cells
CAVITY
NUMBER
FIG. 3. Distribution of SIg+ and SIg- spleen cells. SIgS cells were removed from one spleen cell suspension by affinity chromatography prior to CCD separation. A. Distribution of spleen cells (O-O) and spleen cells depleted of SIg+ cells (O---O) in a 5.0/3.9 phase system. B. Comparison of the percentages of SIg+ cells in the different fractions from the whole spleen cell population (O--O) and spleen cells depleted of SIgS cells by affinity chromatography (O- - -0).
CCD
SEPARATION
OF RAT
LEUKOCYTES
41.5
FIG. 4. Distribution of spleen cells responding to mitogens. Pooled fractions of cells from the CCD train were stimulated with PHA at l/100 dilution (top) and Con A at 2.5 pg ml-’ (bottom), cultivated for 72 hr, and labeled with [nzetJryZ-3H]thymidine 24 hr prior to harvest. Results of mitogen stimulation are expressed as percentage stimulation compared to unseparated control spleen cell cultures. The figures include the results of five different experiments on cells separated in a 5.0/3.7 phase system. Filled symbols represent tumor-immunized rats and unfilled symbols represent untreated rats.
(range, 3142%). F rom Fig. 2 it can be seen that RBAA+ cells account for 68--74% of the cells with a high partition coefficient (peak III) and 41-45% of the cells with a low partition coefficient (peak I). The fractions at the right end of the CCD train were thus virtually free of SIg+ cells, but no fraction was completely devoid of RBAA+ lymphocytes. No selective loss of RBAA+ lymphocytes was observed. Partition of spleen cells after removal of SIgf cells. To delineate further the distribution of SIg+ and SIg- spleen cells, SIg+ cells were removed from the spleen cell suspension prior to CCD separation (10). Figure 3A compares the distributions of spleen cells and spleen cells depleted of SIg+ cells in a 5.0/3.9-phase system. Figure 3B compares the percentage of SIg+ cells recovered in the pooled fractions. The removal of SIg+ cells decreased the percentage of leukocytes re-
416
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ET
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covered in the first peak. The 2% SIg+ cells remaining after affinity chromatography were all recovered in the first fractions. Lymphocytes with optimal response to T-cell PHA and Con A resjonsiweness. mitogens were concentrated in cavities 32 to 42, corresponding to the right half of the second peak of splenic leukocytes. A peak of stimulation was most distinct for PHA, while the response to Con A showed a somewhat broader profile (Fig. 4). Cells on either side of these cavities showed stimulation that was lower than that of unseparated control cells. These differences cannot be accounted for by a lower number of RBAA+ lymphocytes in these fractions (compare with Fig. 2). Spleen cells from both normal control rats and from rats immunized to colon carcinomas were tested and no difference in reactivity to PHA and Con A was seen. Histochenzical demonstration of intracellular esterase. Populations of rodent lymphocytes have been identified by cytochemical staining of intracellular esterases. Chloroacetate esterase is present in granulocytes but not in cells of the monocyte or lymphocyte series (17). a-Naphthyl esterase has been reported in granulocytes, monocytes, and a subpopulation of thymus-derived lymphocytes (17, 18). The distribution of these cells from rat spleen was evaluated by staining smears of cells from pooled fractions for both enzyme activities (19). The size, morphology, and number of cells with deep blue granules (chloroacetate esterase) or dark red granules (a-naphthyl esterase) was determined for at least 300 cells in each preparation. Small mononuclear cells with one to three red granules were not considered positive in this study (18). In two separations of spleen cells, small mononuclear cells heavily stained for a-naphthyl esterase accounted for fewer than 10% of the cells in peak I and 10 to 20% of the cells in the left half of peak II and rose to 45 to 5070 of the cells in the right half of peak II and peak III. As SIg+ cells are reported to lack this enzyme, we also investigated the enzyme activity of separated SIg- cells (Fig. 3). As shown in Table 2, removal of SIg+ cells increased the percentage of esterase-positive small mononuclear cells in peak I and the left half of peak II, indicating that esterase-positive cells were not retained on the anti-Ig TABLE
2
Distribution of Spleen Cells with ol-Naphthyl Chloroacetate Spleen cell peak
Cells from cavities
Percentage
Neither enzyme
I I I II II III
5-9 10-14 15-19 23-25 29-31 4549
Acetate
Esterase or
Esterase Activity
93 72 IO 66 67 47
of leukocytes immunoglobulin
with no surface with
c+Naphthylacetate esterase mononuclear cells Small
Large
2 23 21 30 31 48
3 0 2 2 3 5
Chloroacetate esterase polynuclear cells
2 4 7 2 0 0
(LSpleen cells depleted of SIg+ cells were separated in a 5.0/3.9 phase system (Fig. 3). Smears of cells from pooled fractions were stained using a combined method for both enzymes (19).
CCD
SEPARATION
OF RAT
417
LEUKOCYTES
I
, 1
mm 6. 5. 4 3. 2. 1.
8 5 4 3 2 1 0
10
20
30
CAVITY
40
50
60
NUMBER
5. Effect of changes in phase system composition on the distribution of rat spleen leucoFIG. cytes (-) and erythrocytes (---). Phase systems used were 5.0% dextran T-500 and 3.7% polyethylene glycol 6000 (5.0/3.7) or 5.0% dextran T-500 and 3.9% polyethylene glycol 6000 (5.0/3.9). Both phase systems contained 0.094 M sodium phosphate buffer, pH 7.4, and were supplemented with 5% (w/w) fetal calf serum.
affinity columns. Esterase-positive cells again accounted for 48% of the leukocytes in peak III. The distributions of large mononuclear cells with a-naphthyl esterase and polynuclear cells with chloroacetate esterase were similar in the three separations. The latter were recovered in the first peak (Table 2). a-Naphthyl esterase positive large mononuclear cells were found in all three peaks. Comparison of two diflerent phase systewzs. In this study we compared two phase systems both containing 5.0% dextran but with different polyethylene glycol (PEG)
CAVITY
NUMBER
6. Distribution of rat spleen leucocytes with surface immunoglobulin, SIg, in two different phase systems, 5.0/3.7 ( 0-0 ) and 5.0/3.9 (O---O). A. SIg+ cells in each cavity expressed as a percentage of total white blood cells recovered. B. Percentage of SIg+ cells in each fraction. FIG.
418
MALMSTRCM
ET
AL.
concentrations: 3.7 or 3.9%. The difference in the distribution of spleen cells separated in the two phase systems, 5.0/3.7 and 5.0/3.9, is shown in Fig. 5. A great part of the leukocytes was moved to the left when the concentration of PEG was increased. The distribution of red cells was essentially unaffected. The distribution of SIg+ cells was compared in both phase systems. Increase in the concentration of PEG shifted the SIgf cells to the left end of the CCD train along with the whole cell population (Fig. 6A). However, the relative percentage of the cells in the different peaks was virtually unchanged (Fig. 6B). A small fraction of the SIgf cells seemed not to be affected by this change in the phase system, as the distribution curves of these cells in both phase systems from cavities 25 through 40 were almost identical. DISCUSSION The present investigation demonstrates the feasibility of using countercurrent distribution in aqueous two-phase systems for separation of rat lymphoid cells. Lymphoid cells from different organs give distinct, highly reproducible distribution patterns. The average recovery of 70% of the cells subjected to separation is quite satisfactory when compared to other techniques and in view of the length of the procedure and the numerous tube transfers involved. The viability of the recovered cells also compares favorably with other separation methods. Leukocytes from the spleen were separated into three peaks, Lymphocytes expressing SIg were found mainly in the first peak. Lymphocytes expressing rat brain-associated antigens (RBAA), the majority of the thymus-derived lymphocytes in the spleen (20), distributed all across the CCD train and constituted most of the cells of peak II and III. Lymphocytes giving a maximum response after PHA stimulation were recovered in the right half of the second peak. The maximum response to Con A was found in the same region, but the distribution of the responding cells was somewhat broader. Small mononuclear cells with intracellular esterase were mainly recovered in peaks II and III. These cells reportedly represent a subpopulation of thymus-derived lymphocytes (18). Granulocytes as defined morphologically and histochemically were recovered in the first peak. The wide distribution of thymus-derived lymphocytes indicates great variety in surface characteristics of these cells. This might reflect the existence of several subsets of thymus-derived lymphocytes in the spleen or, alternatively, cells at various stages of maturation. We realize that the enumeration of thymus-derived lymphocytes by indirect immunofluorescence carries the risk of nonspecific staining and overestimation of the number of positively stained cells. All percentages of stained cells were corrected for nonspecifically stained cells. The frequency of the latter was determined by incubation of cells with BSA followed by the anti-rabbit Ig serum. No other attempt was made in the present investigation to rule out staining of cells with Fc receptors by, e.g., use of F(ab)z fragments of the antisera. However, our recorded frequencies of SIg+ cells and thymus-derived (RBAA+) cells in the rat spleen correspond closely to those reported by others (20, 21). In view of the wide distribution of RBAAf cells, the narrow peak of cells responding to T-cell mitogens is intriguing. The peak stimulation seen in cavities 37 to 42 cannot be accounted for by a higher number of thymus-derived lymphocytes in these fractions, as they vary from 42 to 78% along the CCD train. However, the mitogen stimulation assays are also affected by the presence or absence of other
CCD
SEPARATION
OF RAT
LEUKOCYTES
419
cell pop&tions. For example, an optimal response to mitogens requires accessory cells (22). In the lymphocyte culture system used, it has been shown that both adherent cells (removable by Sephadex G-10 columns) and normal rat serum are necessary for an optimal response (P. Malmstriim, unpublished results). However. no apparent concentration of large mononuclear cells could be detected in the region of optima] mitogen responsiveness. Large mononuclear cells with a-naphthyl esterase thus distributed all along the CCD train. It has been reported that an admixture of erythrocytes may increase the response to PHA in human systems (23) but does not affect the response to Con A. In the cavities where optimal lymphocyte stimulation is found, the ratio of erythrocytes to lymphocyes varies from 5 :l to 15 : 1. Control experiments with an admixture of rat erythrocytes from peripheral blood to lymphocytes in proportions from 1: 1 to 20 : 1 have shown slight inhibitory effects on the response to PHA and Con A except at the highest dose. 20 : 1, where a slight enhancement was seen. (P. Malmstrom, unpublished results). Moreover, the peak of erythrocytes in the 5.0/3.7 phase system was found in cavity 48. Suppressive effects on lymphocyte activation by macrophages (24) and by cooperation between macrophages and a subpopulation of thymus-derived lymphocytes (25,26) have been found to influence profoundly the response to mitogens. The existence of possible suppressor populations in this system has not been investigated. There appeared to be a selective loss of about 35% of the SIg+ cells during the separation. A reason for this might be that some SIg+ cells of the unseparated cell population, which have passively acquired Ig by their Fc receptors, lose this Ig during the separation procedure. Another possibility is that S1g-t cells are lost during the separation because of a greater tendency to adhere to plastic surfaces (27). Although many other physical separation methods also yield viable, functional cells, a distinct advantage of CCD separation is that the cell fractions are not exposed to differences in pH, osmolarity, or ion concentration during the separation procedure. Although surface charge-associated properties form one basis of cell separation in two-phase systems, the distribution of SIg+ cells and thymus-derived lymphocytes in our system differs from that reported using cell electrophoresis. With free-flow electrophoresis, SIg+ cells and thymus-derived cells in mice are separated into two distinct peaks (2). The overlap of populations in our system may simply reflect differences in the original cell composition, as other investigators removed adherent and phagocytic cells prior to separation (2). Unfortunately, cells other than monocytes and macrophages are also lost during these procedures (3). Alternatively, two-phase systems may efficiently separate subpopulations of the two main groups of lymphoid cells by virtue of ce]] characteristics other than the surface charge such as hydrophobic and other nonionic interactions between the cells and the phases. Similar subpopulations have hiterto been separated only by use of density gradient centrifugation or density gradient electrophoresis (1, 28). The phase systems used in this study were chosen on the basis of cell partition experiments in single tubes, with systematic changes in phase composition (for a discussion see Refs. 4 and 5). Systems with about 5070 leukocytes in the top phase were considered suitable. The decrease in partition coefficient of the majority of the leukocytes, when the PEG concentration is raised from 3.7 to 3.9$%, is probably due
420
MALMSTRiiM
ET
AL,
to the increase in interfacial tension (29). The reason that the leukocytes in the current experiments are shifted to a greater extent than the erythrocytes is a consequence of the very high affinity of the latter for the top phase. The polymer concentration would have to be increased appreciably to effect a decrease in the partition coefficient of rat erythrocytes. Thus, changes in phase composition do not affect the partition coefficients of different cell types to the same extent. This may be used to design phase systems for particular purposes with the different factors, electrical charge, interfacial tension, hydrophobicity, etc. interacting in a desired manner. CCD separation can thus be considered a reliable and reproducible method for the separation of rat leukocyte populations. It is also possible to make affinity phase systems specific for certain cell surface receptors. Using specific ligands coupled to one of the polymers, it has been possible to shift the partitioning of proteins (30), cell membrane fragments (31, 32), and cells (33, 34). This technique may be further developed for affinity partition of intact lymphoid cells. Also, a combination of CCD separation with other separation methods based on defined cell surface receptors may yield a finer discrimination of lymphocyte subpopulations. ACKNOWLEDGMENTS We thank Ms. Adine Karlsson, Ms. Ingar Nilsson, Ms. Mariana Nilsson, Ms. Ingela Stadenberg, and Ms. Lena Thiman for skillful technical assistance.
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CCD
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SEPARATION
OF RAT
LEUKOCYTES
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