Steroid sensitivity of the PHA and PWM responses of fractionated human lymphocytes in vitro

Printed in Sweden Copyright @ 1976 by Academic Press, Inc. AI/ rights of reproduction in any form reserved

Experimental

STEROID

SENSITIVITY

FRACTIONATED

Cell Research 97 (1976) 233-240

OF THE

PHA

AND

PWM RESPONSES

HUMAN

LYMPHOCYTES

OF

IN VITRO

H. BLOMGREN’ and B. ANDERSSONZ ‘Radiumhemmet,

Karolinska Sjukhuset, Stockholm, Karolinska Institutet, Stockholm,

and 2Department S-10401 Sweden

of Tumor Biology,

SUMMARY The in vitro PHA and PWM responses of various fractions of human peripheral lymphocytes were tested for sensitivity to water-soluble prednisolone. Removal of cells having the capacity to phagocytize carbonyl iron particles or cells having a tendency to adhere to plastics increased the steroid sensitivity of both the PHA and the PWM responses. T and B cell-rich preparations were obtained by passing cell suspensions, depleted of macrophages-monocytes as described above, through anti-Ig labelled bead columns or by a rosette centrifugation technique. The mitogenic response of cell suspensions enriched for B cells were more steroid resistant than suspensions enriched for T cells.

Corticosteroids inhibit both humoral and cell-mediated immune responses [l, 2, 3, 12, 14, 15, 241). Recently it was observed that, at certain concentrations, the corticosteroids also inhibit the responses to phytohemagglutinin (PHA) and poke weed mitogen (PWM) of human lymphocytes in vitro. The PWM response was found to be more steroid resistant than the PHA response [ 19, 201. Some investigators found that cultures of human peripheral blood lymphocytes stimulated with these phytomitogens contain both T and B blast cells [I 1, 13, 17, 25, 26, 271. Since it was reported that PWMstimulated cultures contained a higher frequency of B cell blasts than PHA-stimulated cultures [ll, 131, it was speculated that blastogenesis of human B cells may be more corticosteroid resistant than that of T cells [19, 201. In a previous article we were unable to 16-751818

demonstrate that the PWM response of human lymphocytes is more resistant to water-soluble prednisolone than the PHA response [S]. On the other hand, it was observed that phagocytic cells, mainly macrophages and monocytes, release soluble factors which markedly increase the steroid resistance of both the PHA and PWM responses [S]. In the present study we have investigated the sensitivity of the PHA and PWM responses of separated subpopulations of human peripheral lymphocytes, enriched for T or B cells. It is shown that the mitogenic response of the T cells is more sensitive to corticosteroids than that of the B cells. MATERIAL

AND METHODS

suspensions. Healthy members of the laboratory staff, 25-35 years of age, served as lymphocyte donors. Venous blood was drawn in

Preparation

of leukocyte

likptl

Cell

Res 97 (1976)

234

Blomgren and Andersson

heparinized syringes and leukocytes were separated by centrifugation of the blood on a Ficoll-Isopaque gradient 1211. The leukocytes were washed once by ~entrifug&ion and suspended in Eagle’s Minimal Essential Medium with Earle’s salts (MEM). The number of nucleated cells and viability were determined in a Biirker chamber after trypan blue staining. These crude cell suspensions, containing 90-95 % of lymphocyte-like cells will hereafter be termed non-purified. Removal of phagocytic and adherent cells. Phagocytic cells were removed from the non-purified cell preparation by adding carbonyl ion powder to approx. 5 x IO’ cells suspended in MEM. After incubation at 37°C the susoensions were cleared from iron particles by a magnet. Details of this procedure have been given before [8]. The resulting cell preparations will hereafter be termed Fe-purified. In some experiments we depleted Fe-purified cell preparations from cells which stick firmly to plastics. The cells were suspended at 3.0~ lOg/ml in 5 ml of MEM containing 20% of human serum (HS). The HS was previously decomplemented bv heating at 56°C for 30 min. After I h of incubation at 3PC in horizontal plastic flasks (25 cm*, Falcon Tissue Culture Flask) the lymphocytes were poured off after vigorous agitation of the flasks, and saved for experiments or for further separation procedures. This adherence step reduced the cell number by approx. 5%. The resulting cell suspensions will hereafter be termed Fe-purified, non-adherent. Separation of T and B lymphocytes by a rosette centrifugation technique. This method has been described in detail before [22]. In short, preparations of Feouritied or Fe-ouritied, non-adherent cells were incubated with sheep erythrocytes (SRBC). During this sten. SRBC will adhere to T cells in an immunologicaliy non-specific way [IO, 211. The cells were then suspended in heat-inactivated fetal calf serum and layered on Ficoll-Isopaque. During centrifugation, the T cell-SRBC complexes will sediment due to their higher density whereas non-T cells, mainly B cells, will stay at the fluid interphase. These two cell fractions were collected and exposed to ammonium chloride to lyse SRBC. For simplicity, these cell populations will be termed T and B cell preparations. Purification of T cells by anti-Ig columns. This technique has been described in detail before [28]. The principle is that B cells, which have membrane-bound Ig molecules will stick to bead columns coated with rabbit anti-human-lg antiserum, whereas T cells, not havina membrane-associated lg. will pass through. Our columns, filled with glass beads 256pm in diameter. were 1.5 cm in diameter and 90 cm long. The experimental columns contained beads coated with a double layer of human Ig (Behringwerke) and rabbit anti-human-Ig serum and the control columns contained beads coated with human Ig and normal rabbit serum. Approx. IOncells from Fe-purified cell preparations were placed on top of the columns, passed through with a flow rate of l-2 ml/min at room temperature and collected. The experimental columns retained approx. 60% of the ceils and the control columns approx. 40%. Expf/ Cell Res 97 (1976)

Determination of frequency qf T and B cells. T cells were identified as cells binding 3 or more SRBCs as described earlier 171. Cells having membrane bound Igmolecules were- classified as B-cells. Such molecul& were detected by staining the cells with a fluoresceinconjugated goat anti-human-Ig serum followed by staining with a fluoresceinconjugated rabbit antigoat-Ig serum (Hyland Laboratories). The frequency of fluorescent cells was then determined. Details of the procedure have been given before [9]. At least a hundred cells were scored for frequency of T or B cells. Mitogens and steroid. PHA was Bacto-phytohemagglutinin M (Difco lab., Detroit, Mich.). The contents of commercially available vials of PHA were dissolved in 5.0 ml of MEM. This solution will hereafter be referred to as 100% PHA. PWM (GIBCo, Grand Island, N.Y). The contents of commercially available vials of PWM were dissolved in 5.0 ml of MEM. This solution will be referred to as 100% PWM. Water-soluble nrednisolone natrium succinate was the steroid used (Soludacortin, Merck, Darmstadt, BRD). It was dissolved in MEM and immediately used for experiments. Cell culture conditions and measurements of DNA synthesis. Two different culture techniques were employed which have been described in detail before [23]. The methods will be termed macro- and micro-culture techniques. Using the macro-technique 0.5 x IOB lymphocytes were cultured in glass tubes containing I.0 ml of MEM, supplemented with penicillin, streptomycin and 10% of heat-inactivated HS. The same medium was used for the microtechnique. In these tests the lymphocytes were cultured in the wells of micro test plates containing 0.2 ml of medium. Unless otherwise stated, I .0x lo5 cells were cultured in each well. Half of the cultures received mitogen and the others served as controls. After 4 days of incubation at 37°C in a humidified 5% CO,-air atmosphere each culture received I.0 &i of [3H]TdR (spec. act. 5 Ci/mmole; The Radiochemical Centre, Amersham). Twenty-four hours later the cultures were terminated and incorporated radioactivity determined as described previously [23]. Radioactivity, expressed as cpm, in control cultures was subtracted from the radioactivity obtained in corresponding cultures stimulated with mitogen. Mean values from duplicate cultures were calculated on an arithmetic basis.

RESULTS Steroid sensitivity of the PHA or PWM responses of column purified T cells

of lymphoid cells were depleted of phagocytic cells by the carbonyl ion powder method and then passed through control column or anti-human-Ig coated columns. The steroid sensitivity of the mitogen responses of the cells was then

Preparations

tested.

Steroid sensitivity

of human lymphocytes

Table 1. Inhibition of the PHA-response (0.75 %) by prednisolone purified and column passed lymphocyte preparations

of non-purified,

235 Fe-

Fe-purified lymphocyte suspensions were filtered through control or experimental columns. PHA stimulations are expressed as % of those obtained in control cultures without steroid. The frequency of Ig-bearing cells in the various cell preparations and the absolute stimulations, expressed as cpm, in cultures not exposed to steroids are indicated in the table. The macro culture technique was employed Prednisolone cont. (dml)

Experimental column

Non-purified

Fe-purified

Control column

(l4.0)0 100 (I2 820)* 86.8 102.5 79.2 68.4 71.2 23.9

(14.9) loo (5 590) 116.7 107.6 80.2 43.6 18.0 2.6

100 (3 870) 187.9 154.9 114.4 30.7 20.3 2.5

100 (3 490) 174.6 88.6 58.0 12.1 2.8 I.7

(17.0)” 100 (31 93O)b 109.5 117.9 77.4 31.5 IO.1 I

(16.1) 100 (I2 700) 82.3 96.2 44.9

(4.9) 100 (II 500) 113.3 109.9 43.1 6.7 I I

(0) 100 (13 300) 87. I 76.9 26.4 1.6 I I

Expt I 0

0.001 0.01 0.1 I.0 10.0 100.0

(6.9)

(1.0)

Expt II

0 0.001 0.01 0.1 1.0 10.0 100.0

:i I:0

u Frequency of Ig-bearing cells, expressed as %. * Absolute isotope uptakes, expressed as cpm.

Tables 1 and 2 show that the steroid sensitivity increased after removal of phagocytic cells. Sensitivity was further increased by passing suspensions of Fe-purified cells through control and experimental columns. However, the cells which had passed the experimental columns were more sensitive than those passed through control columns. Dose-response profiles of T and B cells to mitogens

T and B cells were separated from Fepurified cell suspensions by the rosette sedimentation method. These cell preparations, and the original preparations from which they were derived, were tested for response to various concentrations of PHA or PWM. Fig. I shows that non-purified cell preparations were more responsive to both mito-

gens than the Fe-purified, T and B preparations. The dose-response profiles of the cell populations differed somewhat. The B cell preparations required lower mitogen concentrations for optimal stimulation. Steroid sensitivity of the mitogen responses of T and B cells separated by ‘rosette-sedimentation’ Various numbers of stimulated cells. Fig. 2 shows that the steroid sensitivity of the PHA responses of all the cell preparations increased by decreasing the number of cells per culture. In all experiments it was observed that removal of phagocytic cells increased the steroid sensitivity of the lymphocyte population. Moreover, these tests showed that the PHA response of the B cell preparation was more resistant than that of Exprl Cell Res 97 (1976)

236

Blomgren and Andersson

Table 2. Experiments analogous to those presented in table 1, except that PWM (0.09 %) was used as a stimulant Prednisolone cont. (m/ml)

Non-purified

Fe-purified

Control column

Exptl column

(12SP 100(3 570) 77.0 97.2 87.0 66.0 77.4 23.3

(13.0) 100(3 260) 134.6 87.3 44.8 38.4 22.6 5.2

(13.6) 100(3 020) 89.5 83.3 39.8 17.4 6.5 3.4

100(2 950) 97.4 70.3 18.I 1.6 1.4 2.8

(19.2) 100(2 470) 102.5 113.I 90.0 69.7 60.4 12.8

(18.0) 100(3 690) 98.2 88.3 79. I 55.8 37.3 9.9

(12.7) 100(2 200) 73.4 70.8 28.3 14.2 8.3 9.4

(3.7) 100(2 240) 69.5 43.9 13.9 7.8 3.1 I

Expr I 0 0.001 0.01 0.1

I.0 10.0 100.0

(2.0)

Expr II 0

0.001 0.01 0.1 I.0 10.0 100.0

a Frequency of Ig-bearing cells, expressed as %.

the T cells. This was most evident using 100000 cells/culture. Fig. 3 depicts the results using PWM as a stimulation. These tests also show that the response of the B cell preparation was more steroid resistant than that of the T cells. Various concentrations of mitogens. The prednisolone sensitivity of the response of T and B cells to various concentrations of the mitogens was also tested. Table 3 shows that the sensitivity of the PHA responses of both T and B cells increased as the mitogen concentrations were decreased. The steroid sensitivity of the PWM response of T and B cells similarly increased as the mitogen concentration decreased (table 4). Sensitivity of cell preparations depleted of phagocytic and adherent cells. One explanation of the different steroid sensitivities of the mitogen responses of the T and B cell preparations could be that the latter cell preparations contain a higher freExptl Cell Res 97 (1976)

quency of contaminating monocytes and macrophages, which are known to increase steroid resistance of lymphocytes. To further enrich for lymphocytes, Fe-purified cell preparations were depleted of adherent

-m . .

Fig. 1. Abscissa: (left) PHA; (right) nate: cpm (X 10e3).

PWM cont.; ordi-

Stimulations of various cell preparations by different concentrations of (left) PHA and (right) PWM. The same cell donorwas used for both tests. The frequency of SBBC-binding cells in the cell preparations is indicated in the figure. O-O, Non-purified; O-O, Fe-purified: A-A, T cells; A-A, B cells.

Steroid sensitivity of human lymphocytes

237

100.000 CEL%/CUmlRE

120 1 100

60

60

40

20 \o ~ O

joaol

6

od

PO 10

0:01

loo

i@001 i b 0;01

0.1

l-o

10

100

0

0.01

Figs 2, 3. Abscissa:

prednisolone cont. (w/ml); ordinate: isotope uptake (% of control). Fig. 2. Inhibition of the PHA response of vatious cell preparations by prednisolone. Three different cell concentrations were employed. Thymidine incorporations are expressed as % of those obtained in correspond-

ing cultures without steroid. PHA was kept at a concentration of 0.37 %. The same cell donor was used for all tests. Symbols as in fig. 1. The frequency of SRBCbinding cells in the cell preparations was as follows: Non-purified, 59%; Fe-purified, 61%; T cells, 91%; B cells, 12%.

cells. B and T cells were then fractionated from these preparations of highly purified lymphocytes. Table 5 shows that there was a gradual loss of steroid resistance of the PHA response of the cells as they were depleted of first phagocytic and then adherent cells. This table also shows that the T cell preparation was more sensitive than the B cell preparation. Similar conclusions can be drawn from experiments using PWM as a stimulant (table 6).

cell donors can be largely explained by variation of the numbers of contaminating monocytes-macrophages, which seem to counteract the activity of the steroids [8]. In mice, there is evidence that B cells are more resistant to cortisone than peripheral

100

60

DISCUSSION The in vivo lymphocytolytic effect of corticosteroids varies extensively between different species. For instance, mice are extremely sensitive whereas guinea pigs and humans are relatively steroid resistant [4,5, 161. Moreover, steroid sensitivity of the PHA and PWM responses in vitro of lymphocytes from various individuals of the same species shows a large range of variation [8]. The variation between different

O

L, , . , . . 0

j oa obo1

0.1

1.0

10

loo

0

‘,

&&ol

.

.

0.1

.

.

1.0 lo

. 100

Fig. 3. Experiment analogous to that presented in fig. 2 except that PWM was used as a stimulant (0.09%). The same cell donor was used for all tests. The frequency of SRBC-binding cells in the various cell preparations was as follows: Non-purified, 59%; Fe-purified, 61%; T cells, 91%; B cells, 12%. Ekptl Cd Res 97 11976)

238

Blomgren and Andersson

T cells. It has namely been shown that the graft-vs-host responsiveness (a T cell-mediated reaction) of spleen cells of mice is reduced to a higher extent than the humoral The cells were separated by rosette sedimentation. The frequency of SRBC-binding cells in the cell prepa- antibody producing capacity (a B cell reacrations and the absolute stimulations, expressed as cpm, in cultures not exposed to steroid are shown in tion) [6]. Moreover, other investigators the table. The micro culture technique was employed have claimed that the response of human lymphocytes to PWM is more steroid rePrednisolone Final concentration of PHA cont. sistant than their response to PHA [ 19, 201. 0.19% 0.05 7% 0.75 % (i-czlml) Since B cells seem to be triggered to blast transformation to a higher extent in PWM T cells (89 % of SRBC-binding cells) 0 100(I52 72Op 100(54 430) No response than in PHA cultures of human lympho0.001 105.7 115.7 cytes [I I, 131it was speculated that blasto0.01 92.9 77.8 0.1 67.0 12.7 genesis of B cells is more steroid resistant I.0 58.6 1.9 than that of T cells [l9, 201. 10.0 39.3 I.6 100.0 17.0 I In this investigation we have enriched human T and B lymphocytes and tested the B cells (IO % of SRBC-binding cells) steroid sensitivity of the mitogen responses 0 100(I I9 150) 100(74 480) 100(3 170) 0.001 91.9 III.6 90.8 of these cell fractions. The results have 0.01 83.2 80.5 19.4 shown that preparations of T lymphocytes 0.1 74.6 69.4 I I.0 73.6 65.7 I obtained by passing lymphoid cells through 10.0 68.2 62.5 I anti-Ig-coated column beads were more 100.0 43.4 38. I I sensitive to steroids than cells passed through control columns. The increment of Table 3. Steroid sensitivity of the responses of T and B cell preparations to various concentrations of PHA

Table 4. Experiment analogous to that presented in table 3 except that PWM was used as a stimulant Prednisolone cont. (kdml)

Final concentration of PWM 0.37%

0.09%

0.02%

0.005 %

cells) 100 (24 74Op 102.5 84.9

100(21 450) 100.3 79.3

100(7 040) 115.7 72.3

100(I 210) 141.8 22.8

T cells (90 % of SRBC-binding

0 ’ 0.001 0.01 0.1 1.0 10.0 100.0

58.3 46.6 42.6 19.8

B cells (13 % of SRBC-binding cells) 0 100(31 310)

0.001 0.01 0.1 I.0 10.0 100.0

109.5 92.3 83.5 71.0 76.3 70.7

47.3 30.5 21.8 7.7

43.1 9.4 2.5 I

100(34 020) 106.7 93.4 71.1 53.1 46.1 36.8

100(36 3 IO) 80.7 71.5 40.3 25.1 18.I 10.3

a Absolute isotope uptakes, expressed as cpm. Exprl Cell Res 97 (1976)

I I I 100(I8 750) 114.4 60.7 28.8 4.2 1.4 I

Steroid sensitivity of human lymphocytes

239

Table 5. Steroid sensitivity of the response of various cell preparations to PHA (0.37%) The T and B cell fractions were separated from preparations of Fe-purified, non-adherent cell suspensions by rosette sedimentation. The frequency of SRBC-binding cells in the cell preparations and the absolute stimulations, expressed as cpm, in cultures not exposed to steroid are shown in the table. The micro culture technique was employed Frequency of SRBC binding cells, expressed as % Prednisolone cont. (dml) 0 EY 0:1 1.0 10.0 100.0

Non-purified (61%)

Fe-purified (65 %)

Fe-purified, non-adherent (65 %)

T cells (94 %)

B cells (16%)

100 (144 700)” 80.4 94.2

100(230 000) 100.3 103.5

100(173 900) 76.2 91.4

100(172 200) 96.6 95.6

100(165 600) 112.3 96.2

91.8 92.6 78.0 35.0

101.4 84.2 60.2 30.1

62.2 23.6 12.7 2.3

71.5 26.5 15.3 1.3

108.3 89.8 91.8 47. I

,J Absolute isotope uptakes, expressed as cpm.

sensitivity after passage through control columns, which was observed in all experiments, can be explained by the fact that adherent cells and also B cells are retained in the columns. The mitogen responses of T and B cell preparations separated by “rosette sedimentation”, were found to exhibit different sensitivities to prednisolone; the former being more sensitive. Although the cell suspensions used for “buoyant density” centrifugation were depleted of phagocytic cells it is possible that monocytes-macrophages with a low phagocytic

activity remained in the suspensions. These few contaminant cells may concentrate in the fluid interphase, the B cell fraction, after buoyant density centrifugation. This could thus explain why the mitotic responses of the B cell preparation were more steroid resistant than the T cells. In an attempt to exclude this possibility we removed adherent cells from the Fe-purified cell suspensions and then fractionated out T and B cells. These cell preparations, thus highly depleted of monocytes and macrophages, still showed a B cell fraction with a higher

Table 6. Experiment analogous to that presented in table 5 except that PWM (0.09 %) was used as a stimulant Frequency of SRBC binding cells, expressed as cpm Prednisolone cont. OLdml)

Non-purified (61%)

Fe-purified (64 %)

Fe-purified, non-adherent (66%)

T cells (95 %)

B cells (14%)

0 0.001 0.01 0.1 1.0 10.0 100.0

100 (22 340)” 116.2 107.2 122.9 121.3 117.9 127.8

100 (17 640) 122.I 106.0 94.7 69.0 65.0 53.5

100(7 190) 132.5 107.8 92.6 39. I 26.8 10.7

100(8 200) 96.0 51.5 20.9 28.5 I 1

100(IO 280) 84.8 77.1 55.6 42.1 33.5 25.1

a Absolute isotope uptakes, expressed as cpm. Exprl Cell Res 97 (1976)

240

Blomgren and Andersson

steroid resistance than that of their T cell fraction. Our B cell preparations were constantly contaminated by approx. 15% of T cells. The B cell preparations can easily be further enriched by a second “rosette sedimentation”. However, B cell preparations with a very low contamination of T cells (approx. 5% or less) were not possible to test for their steroid sensitivity since they exhibit practically no increment of DNA synthesis upon exposure to PHA or PWM. A prerequisite for blastogenesis of B cells in cultures seems to be the presence of phytomitogen responsive T cells. It has been suggested by some investigators that B cells do not respond to soluble phytomitogens but are triggered by mitogenic factors released by phytomitogen activated T cells [25, 261. We can thus only conclude that blastogenesis of B cell rich population is more steroid resistant than that of T cells. We cannot state that blastogenesis of T and B cells differ in their steroid sensitivity. As a byproduct of the present investigation we observed that steroid sensitivity of the PHA and PWM responses increase as the cell concentration or mitogen concentration of the cultures were decreased. The reason for the enhancement of sensitivity by decreasing the cell concentration can be explained by the fact that the concentration of monocytes-macrophages, known to produce steroid protective factors, was also decreased. However, the fact that sensitivity increased by decreasing the concentration of phytomitogen seems to indicate that a lymphocyte which receives many “signals” to undergo blast transformation is more resistant to steroids than a cell which only receives a few “signals”. In conclusion, this investigation strongly indicates that blastogenesis of B cells in cultures of human lymphocytes are more corExprl Cell Res 97 (1976)

ticosteroid resistant than that of T cells. Such a difference also seems to exist between human T and B cells with regard to sensitivity to ionizing radiation [ 181. The authors wish to thank Miss Marja excellent technical assistance. This work was supported by grants Society in Stockholm and the Swedish

Rikkinen

for her

from the Cancer Cancer Society.

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June

IO. 1975