CELLULAR
IMMUNOLOGY
44,
395-403 (1979)
Cellular Cooperation in Mitogen-Induced Human Leukocyte Inhibitory Factor (LIF) Synthesis LISA Institute
RASANEN,'ELIISA
of Biomedical
Sciences,
KARHUMAKI,AND
University
Received
of Tampere,
September
KAI KROHN
SF-33101
Tampere
10, Finland
12, 1978
Interactions between human T and B lymphocytes and between lymphocyte subpopulations and accessory cells in lymphokine synthesis were investigated. The cells were stimulated with leukoagglutinin (LA), concanavalin A (Con A), protein A (prot A) and anti-&-microglobulin (anti-&m). The presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose-migration method. The results indicated that monocytes augmented LIF synthesis of T cells but suppressed that of B cells. Monocyte-helper effect was mediated by both cell-cell contact and soluble factors. In addition, T lymphocytes were found to augment B-ceil LIF production. B lymphocytes enhanced Con A- but suppressed LA-induced LIF production by T cells. T-cell/B-cell collaboration was based on a direct cell-cell contact and no soluble factors were found.
INTRODUCTION Two populations of lymphocytes exist (1). T lymphocytes are responsible for the phenomena of cell-mediated immunity while B lymphocytes act by secreting antibodies. Both T and B cells are, able to produce lymphokines (2-4) and these cells may interact with each other in lymphokine synthesis. In addition, phagocytic cells are known to play a role in lymphokine production (5- 11). Thus lymphokine synthesis is the result of interactions between various cells. There are only a few reports on the role of phagocytes in T- and B-cell lymphokine synthesis (6, 9, 11). The results of these reports are contradictory especially concerning B-cell lymphokine synthesis. Therefore it was of interest to study the role of monocytes and other accessory cells in human leukocytemigration inhibitory factor (LIF) production. At present a large number of reports exist on the role of T cells in antibody synthesis and transformation. Interactions between T and B lymphocytes in lymphokine production have been studied less extensively. As a rule only the effect of T cells on B-cell function has been investigated. There are only a few reports on the effect of B cells on T-cell transformation (12,13) or lymphokine synthesis (11,14). The aim of this investigation was to analyze the interactions between T and B lymphocytes and between lymphocytes and phagocytes in lymphokine synthesis. In addition, the mechanisms by which the cells affect each other were studied. ’ Address correspondence to: Liisa Rasanen, Institute Tampere, SF-33101 Tampere 10, Finland.
of Biomedical
Sciences, University
of
395 0008-8749/79/060395-0%02.00/0
Copyright 0 1979by Academic Press, Inc. AU rights of reproduction in any form reserved.
396
RiiSiiNEN,
KARHUMiiKI,
MATERIALS
AND
KROHN
AND METHODS
Separation of cells. Mononuclear cells from healthy human individuals were obtained by Ficoll-Isopaque centrifugation of heparinized or titrated peripheral blood (15). After removal of carbonyl iron-fed monocytes T and B cells were purified by sheep red blood cell (SRBC)-rosette formation and centrifugation on Ficoll-Isopaque as described earlier (16). The purity of the cell populations was studied, as described before (16), staining nonspecific esterase activity and surface membrane immunoglobulin and using SRBC-rosette formation. Monocytes and guinea pig macrophages were purified as follows. Human mononuclear or noninduced peritoneal cells from random-bred guinea pigs were incubated for 30 min in tissue-culture flasks (growth area 25 cm2, Falcon, Oxnard, Calif.) in 10 ml of Hanks’ balanced salt solution (HBSS) supplemented with 10% fetal bovine serum (Flow Laboratories, Irvine, Scotland). To remove the nonadherent cells the flasks were shaken vigorously with HBSS, after which phosphate-buffered saline containing 0.02% EDTA was added to the flasks. After a 2-hr incubation at room temperature the adherent cells were removed by pipetting vigorously with a Pasteur pipet. The viability of the recovered monocytes and macrophages was >85%. Granulocytes were obtained by sedimentation of blood and centrifugation of the leukocyte-rich plasma on Ficoll-Isopaque. In order to obtain red blood cells the blood was centrifuged and the leukocyte-rich upper layer of the pellet discarded. Cell cultures. The cells were suspended at a concentration of 2 x lo6 cells/ml in MEM for suspension cultures (Flow Laboratories) supplemented with 10% horse serum (Microbiological Associates, Bethesda, Md.). Duplicate cultures containing 2 x lo5 lymphocytes per well were set up in U-bottomed microplates (Sterilin Ltd., Middlesex, England). The mitogens used were LA (Pharmacia Fine Chemicals, Uppsala, Sweden), Con A (Pharmacia Fine Chemicals), prot A (Pharmacia Fine Chemicals), and anti-&m (Dakopatts A/S, Copenhagen, Denmark). Unless otherwise stated the cells were pulse treated for 30 min with LA, Con A and prot A, and with medium alone (control cultures) as described before (16). Anti-&m was present during the whole culture period and after incubation the controls were reconstituted with corresponding amounts of this mitogen. All cultures were incubated at 37°C in a 5% CO, atmosphere for 3 days. Purornycin treatment of cells. In order to block the protein synthesis of monocytes and lymphocytes in cooperation experiments these cells were pulse treated with puromycin as described by Gorski et al. (17). In brief, the cells were incubated with 500 pg/ml of puromycin for 90 min, after which they were washed five times and cultured together with untreated cells. To avoid the double pulsing of the cells the mitogens were present during the whole culture period. Control cultures were reconstituted with corresponding amounts of these mitogens before being assayed. Cell cultures in Marbrook-type culture vessels. In order to study whether the monocyte-helper effect was mediated by soluble factors, T lymphocytes and monocytes were cultured in Marbrook-type culture vessels. The vessels were constructed by gluing a 0.22-pm filter with nontoxic cement (both from Millipore Co., Bedford, Mass.) on a bottomless tissue-culture tube and placing this in a bigger tube. The inner part had been sterilized with uv light. Monocytes were placed in the
CELLULAR
COOPERATION
IN LIF
397
SYNTHESIS
upper compartment and T lymphocytes in the lower one. The total volume of the culture was 0.7 ml. It is possible that although puromycin blocks the synthesis of soluble factors it does not prevent the release of preformed factors. Therefore cultures in which monocytes were both treated with puromycin and placed in Marbrook chambers were set up. No augmentation of LIF synthesis was seen in these cultures. Production of lymphocyte-culture supernatants. To study whether T lymphocytes are able to produce soluble factors which augment LIF synthesis by B lymphocytes and vice versa, lymphocytes were first stimulated with mitogens and cultured. T lymphocytes were pulse treated with 320 pg/ml of Con A and with medium alone, and B lymphocytes with 20 pg/ml of prot A and with medium alone. The culture supernatants were collected after 12, 24, or 48 hr of incubation and lymphocytes were cultured in various dilutions of these supernatants. T cells were pulse treated with 80 pg/ml of Con A, 20 &ml of prot A, and with medium alone (controls) and were cultured in B-cell supernatants. B lymphocytes were pulsed with 320 &ml of Con A, 5 pg/ml of prot A, and with medium alone and were cultured in T-cell supernatants. Leukocyte-migration inhibitory-factor assay. LIF activity was tested in the culture supernatants by the agarose-migration method as described before (16). The migration index (MI) was calculated as follows: MI =
area of migration in the presence of test supernatant area of migration in the presence of control supernatant
.
The method worked with standard deviations ranging from 0 to 18% (mean 7.2%). Generally MIS smaller than 0.85 represented significant inhibition of migration as calculated by Student’s t test. The significance of differences among MIS was calculated by Mann-Whitney’s U test. RESULTS Effect of Monocytes
on the LIF Response
of T and B Lymphocytes
The separation method used yielded T-cell populations contaminated with an average of 2.4% (range l-4%) B lymphocytes and less than 0.1% monocytes, and B-cell populations contaminated with 2.5% (l-5%) T cells and 2.9% (l-4%) monocytes. The purity of monocytes and guinea pig macrophages was on the average 95.1% (88-99%) and 96.3% (91-98%), respectively, Figure 1 shows the effect of increasing the amount of monocytes on mitogen-induced LIF synthesis of a constant number of lymphocytes. Monocytes enhanced T-cell LIF production, the optimum amount of monocytes being 5- 10%. The difference in LIF production between lymphocytes cultured in the absence and in the presence of 5% monocytes was statistically significant (P < 0.01 in the cases of Con A and the lower concentration of LA, P < 0.05 for the higher concentration of LA). The two concentrations of mitogens used had in previous experiments proved to be optimal and suboptimal (16). The augmentation of LIF synthesis was greater at suboptimal concentrations of mitogens. Monocytes by themselves did not produce LIF (data not shown). The effect of various accessory cells on LA- and Con A-stimulated T-cell LIF
398
RASANEN, ‘.‘I
KARHUMAKI,
LA
d11.25
1
2.5
AND KROHN Con
A
IO
5
1
2.5 %
FIG. 1. The effect of A- and anti-p,m-induced which were 2.5 &ml antiserum for anti-&m. and 80 &ml and 1:4,
I
5
I
10
m
8
20
40
1
40
MONOCYTES
monocytes on LA- and Con A-induced LIF synthesis of T lymphocytes and prot LIF production by B cells. (0) Denotes suboptimal concentrations of mitogens for LA, 80 &ml for Con A, 5 &ml for prot A, and a dilution of 1:8 of the Optimal concentrations (0) of LA, Con A, prot A, and anti-&m were 40, 320, respectively. Each point represents the mean k SD of six experiments.
production was also investigated. There was no difference in the augmenting capacity between autologous and allogeneic monocytes. Xenogeneic guinea pig macrophages caused a slight, statistically nonsignificant increase in LIF production while granulocytes and erythrocytes had no effect (data not shown). Figure 1 also represents the effect of additional monocytes on prot A- and anti-&m-stimulated B-cell lymphokine synthesis. LIF production diminshed with increasing amounts of monocytes both at optimal and suboptimal concentrations of mitogens. Augmentation of T-Cell LIF Response by Puromycin-Treated Monocyte-Derived Soluble Factors
Monocytes and
In order to prevent the possible elaboration of soluble factors, monocytes were pulse treated with puromycin, Treatment of lymphocytes with 500 pg./ml of puromycin inhibited LIF synthesis totally and caused an average of 86% inhibition of protein synthesis as assessed by the incorporation of r3H]leucine in leucine-free cultures. The inhibition of protein synthesis lasted at least for 3 days. Puromycin did not leak from the cells or the leakage was very small since culture supernatants of
CELLULAR
COOPERATION
IN LIF SYNTHESIS
399
puromycin-treated cells did not inhibit LIF production. Puromycin, 5 pg/ml, in the culture medium was able to completely block LIF synthesis. Figure 2A shows the effect of puromycin-treated monocytes on T-cell LIF synthesis. Puromycin-treated monocytes augmented LIF production but to a lesser degree than nontreated monocytes (Fig. 1). The optimum amount of puromycintreated monocytes was lo-20% and this gave a statistically significant difference (P < 0.05) compared to cultures containing no additional monocytes. The elaboration of soluble helper factors by monocytes was studied by culturing monocytes and T lymphocytes in double-chambered Marbrook-type culture vessels. Monocytes were able to elaborate soluble factors that enhanced T-cell LIF production (Fig. 2B). The optimum amount of monocytes was about 20% which caused in the case of Con A-migration inhibition significantly different (P < 0.05) from cultures without additional monocytes. The augmentation caused by monocyte-derived factors was smaller than that caused by nontreated or puromycin-treated monocytes. LIF Production
by T Cells, B Cells, and Their Combinations
Figure 3 shows the LIF synthesis ability of T cells, B cells, and their combinations (T + B) stimulated with mitogens. In all experiments the total lymphocyte number was kept constant. LA and Con A caused LIF synthesis in purified T cells whereas B cells responded by LIF production after stimulation with prot A or anti-&m. With Con A, prot A, and anti-/&m T- and B-cell mixtures produced more LIF than T or B cells alone. The presence of about 20% T or B cells among B or T lymphocytes caused optimum LIF production which differed significantly (P < 0.01) from the LIF synthesis level of these cells cultured alone. LA was an exception and with it LIF production diminished with increasing amounts of B lymphocytes. Autologous and allogeneic cells were also compared for their ability to affect LIF
% PUROMYCINTREATED
MONOCYTES
% MONOCYTES MARBROOK
IN CHAMBERS
FIG. 2. (A) Augmentation of the LIF response to T lymphocytes to LA and Con A by puromycin-treated monocytes. The concentrations of LA (0) and Con A (0) were 0.6 and 20 &ml, respectively, and the mitogens were present during the whole culture period. (B) The effect of monocyte-derived factors on T-cell LIF response. The cells were stimulated with LA, 2.5 CLg/ml(O), and Con A, 80 &ml(O), and cultured in the separate compartments of Marbrook-type culture vessels. The values represent the mean * SD of four experiments.
400
RASANEN,
J, % B CELLS %
9595
III 9095>95 10 5
IMMUNOLOGY
44,
395-403 (1979)
Cellular Cooperation in Mitogen-Induced Human Leukocyte Inhibitory Factor (LIF) Synthesis LISA Institute
RASANEN,'ELIISA
of Biomedical
Sciences,
KARHUMAKI,AND
University
Received
of Tampere,
September
KAI KROHN
SF-33101
Tampere
10, Finland
12, 1978
Interactions between human T and B lymphocytes and between lymphocyte subpopulations and accessory cells in lymphokine synthesis were investigated. The cells were stimulated with leukoagglutinin (LA), concanavalin A (Con A), protein A (prot A) and anti-&-microglobulin (anti-&m). The presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose-migration method. The results indicated that monocytes augmented LIF synthesis of T cells but suppressed that of B cells. Monocyte-helper effect was mediated by both cell-cell contact and soluble factors. In addition, T lymphocytes were found to augment B-ceil LIF production. B lymphocytes enhanced Con A- but suppressed LA-induced LIF production by T cells. T-cell/B-cell collaboration was based on a direct cell-cell contact and no soluble factors were found.
INTRODUCTION Two populations of lymphocytes exist (1). T lymphocytes are responsible for the phenomena of cell-mediated immunity while B lymphocytes act by secreting antibodies. Both T and B cells are, able to produce lymphokines (2-4) and these cells may interact with each other in lymphokine synthesis. In addition, phagocytic cells are known to play a role in lymphokine production (5- 11). Thus lymphokine synthesis is the result of interactions between various cells. There are only a few reports on the role of phagocytes in T- and B-cell lymphokine synthesis (6, 9, 11). The results of these reports are contradictory especially concerning B-cell lymphokine synthesis. Therefore it was of interest to study the role of monocytes and other accessory cells in human leukocytemigration inhibitory factor (LIF) production. At present a large number of reports exist on the role of T cells in antibody synthesis and transformation. Interactions between T and B lymphocytes in lymphokine production have been studied less extensively. As a rule only the effect of T cells on B-cell function has been investigated. There are only a few reports on the effect of B cells on T-cell transformation (12,13) or lymphokine synthesis (11,14). The aim of this investigation was to analyze the interactions between T and B lymphocytes and between lymphocytes and phagocytes in lymphokine synthesis. In addition, the mechanisms by which the cells affect each other were studied. ’ Address correspondence to: Liisa Rasanen, Institute Tampere, SF-33101 Tampere 10, Finland.
of Biomedical
Sciences, University
of
395 0008-8749/79/060395-0%02.00/0
Copyright 0 1979by Academic Press, Inc. AU rights of reproduction in any form reserved.
396
RiiSiiNEN,
KARHUMiiKI,
MATERIALS
AND
KROHN
AND METHODS
Separation of cells. Mononuclear cells from healthy human individuals were obtained by Ficoll-Isopaque centrifugation of heparinized or titrated peripheral blood (15). After removal of carbonyl iron-fed monocytes T and B cells were purified by sheep red blood cell (SRBC)-rosette formation and centrifugation on Ficoll-Isopaque as described earlier (16). The purity of the cell populations was studied, as described before (16), staining nonspecific esterase activity and surface membrane immunoglobulin and using SRBC-rosette formation. Monocytes and guinea pig macrophages were purified as follows. Human mononuclear or noninduced peritoneal cells from random-bred guinea pigs were incubated for 30 min in tissue-culture flasks (growth area 25 cm2, Falcon, Oxnard, Calif.) in 10 ml of Hanks’ balanced salt solution (HBSS) supplemented with 10% fetal bovine serum (Flow Laboratories, Irvine, Scotland). To remove the nonadherent cells the flasks were shaken vigorously with HBSS, after which phosphate-buffered saline containing 0.02% EDTA was added to the flasks. After a 2-hr incubation at room temperature the adherent cells were removed by pipetting vigorously with a Pasteur pipet. The viability of the recovered monocytes and macrophages was >85%. Granulocytes were obtained by sedimentation of blood and centrifugation of the leukocyte-rich plasma on Ficoll-Isopaque. In order to obtain red blood cells the blood was centrifuged and the leukocyte-rich upper layer of the pellet discarded. Cell cultures. The cells were suspended at a concentration of 2 x lo6 cells/ml in MEM for suspension cultures (Flow Laboratories) supplemented with 10% horse serum (Microbiological Associates, Bethesda, Md.). Duplicate cultures containing 2 x lo5 lymphocytes per well were set up in U-bottomed microplates (Sterilin Ltd., Middlesex, England). The mitogens used were LA (Pharmacia Fine Chemicals, Uppsala, Sweden), Con A (Pharmacia Fine Chemicals), prot A (Pharmacia Fine Chemicals), and anti-&m (Dakopatts A/S, Copenhagen, Denmark). Unless otherwise stated the cells were pulse treated for 30 min with LA, Con A and prot A, and with medium alone (control cultures) as described before (16). Anti-&m was present during the whole culture period and after incubation the controls were reconstituted with corresponding amounts of this mitogen. All cultures were incubated at 37°C in a 5% CO, atmosphere for 3 days. Purornycin treatment of cells. In order to block the protein synthesis of monocytes and lymphocytes in cooperation experiments these cells were pulse treated with puromycin as described by Gorski et al. (17). In brief, the cells were incubated with 500 pg/ml of puromycin for 90 min, after which they were washed five times and cultured together with untreated cells. To avoid the double pulsing of the cells the mitogens were present during the whole culture period. Control cultures were reconstituted with corresponding amounts of these mitogens before being assayed. Cell cultures in Marbrook-type culture vessels. In order to study whether the monocyte-helper effect was mediated by soluble factors, T lymphocytes and monocytes were cultured in Marbrook-type culture vessels. The vessels were constructed by gluing a 0.22-pm filter with nontoxic cement (both from Millipore Co., Bedford, Mass.) on a bottomless tissue-culture tube and placing this in a bigger tube. The inner part had been sterilized with uv light. Monocytes were placed in the
CELLULAR
COOPERATION
IN LIF
397
SYNTHESIS
upper compartment and T lymphocytes in the lower one. The total volume of the culture was 0.7 ml. It is possible that although puromycin blocks the synthesis of soluble factors it does not prevent the release of preformed factors. Therefore cultures in which monocytes were both treated with puromycin and placed in Marbrook chambers were set up. No augmentation of LIF synthesis was seen in these cultures. Production of lymphocyte-culture supernatants. To study whether T lymphocytes are able to produce soluble factors which augment LIF synthesis by B lymphocytes and vice versa, lymphocytes were first stimulated with mitogens and cultured. T lymphocytes were pulse treated with 320 pg/ml of Con A and with medium alone, and B lymphocytes with 20 pg/ml of prot A and with medium alone. The culture supernatants were collected after 12, 24, or 48 hr of incubation and lymphocytes were cultured in various dilutions of these supernatants. T cells were pulse treated with 80 pg/ml of Con A, 20 &ml of prot A, and with medium alone (controls) and were cultured in B-cell supernatants. B lymphocytes were pulsed with 320 &ml of Con A, 5 pg/ml of prot A, and with medium alone and were cultured in T-cell supernatants. Leukocyte-migration inhibitory-factor assay. LIF activity was tested in the culture supernatants by the agarose-migration method as described before (16). The migration index (MI) was calculated as follows: MI =
area of migration in the presence of test supernatant area of migration in the presence of control supernatant
.
The method worked with standard deviations ranging from 0 to 18% (mean 7.2%). Generally MIS smaller than 0.85 represented significant inhibition of migration as calculated by Student’s t test. The significance of differences among MIS was calculated by Mann-Whitney’s U test. RESULTS Effect of Monocytes
on the LIF Response
of T and B Lymphocytes
The separation method used yielded T-cell populations contaminated with an average of 2.4% (range l-4%) B lymphocytes and less than 0.1% monocytes, and B-cell populations contaminated with 2.5% (l-5%) T cells and 2.9% (l-4%) monocytes. The purity of monocytes and guinea pig macrophages was on the average 95.1% (88-99%) and 96.3% (91-98%), respectively, Figure 1 shows the effect of increasing the amount of monocytes on mitogen-induced LIF synthesis of a constant number of lymphocytes. Monocytes enhanced T-cell LIF production, the optimum amount of monocytes being 5- 10%. The difference in LIF production between lymphocytes cultured in the absence and in the presence of 5% monocytes was statistically significant (P < 0.01 in the cases of Con A and the lower concentration of LA, P < 0.05 for the higher concentration of LA). The two concentrations of mitogens used had in previous experiments proved to be optimal and suboptimal (16). The augmentation of LIF synthesis was greater at suboptimal concentrations of mitogens. Monocytes by themselves did not produce LIF (data not shown). The effect of various accessory cells on LA- and Con A-stimulated T-cell LIF
398
RASANEN, ‘.‘I
KARHUMAKI,
LA
d11.25
1
2.5
AND KROHN Con
A
IO
5
1
2.5 %
FIG. 1. The effect of A- and anti-p,m-induced which were 2.5 &ml antiserum for anti-&m. and 80 &ml and 1:4,
I
5
I
10
m
8
20
40
1
40
MONOCYTES
monocytes on LA- and Con A-induced LIF synthesis of T lymphocytes and prot LIF production by B cells. (0) Denotes suboptimal concentrations of mitogens for LA, 80 &ml for Con A, 5 &ml for prot A, and a dilution of 1:8 of the Optimal concentrations (0) of LA, Con A, prot A, and anti-&m were 40, 320, respectively. Each point represents the mean k SD of six experiments.
production was also investigated. There was no difference in the augmenting capacity between autologous and allogeneic monocytes. Xenogeneic guinea pig macrophages caused a slight, statistically nonsignificant increase in LIF production while granulocytes and erythrocytes had no effect (data not shown). Figure 1 also represents the effect of additional monocytes on prot A- and anti-&m-stimulated B-cell lymphokine synthesis. LIF production diminshed with increasing amounts of monocytes both at optimal and suboptimal concentrations of mitogens. Augmentation of T-Cell LIF Response by Puromycin-Treated Monocyte-Derived Soluble Factors
Monocytes and
In order to prevent the possible elaboration of soluble factors, monocytes were pulse treated with puromycin, Treatment of lymphocytes with 500 pg./ml of puromycin inhibited LIF synthesis totally and caused an average of 86% inhibition of protein synthesis as assessed by the incorporation of r3H]leucine in leucine-free cultures. The inhibition of protein synthesis lasted at least for 3 days. Puromycin did not leak from the cells or the leakage was very small since culture supernatants of
CELLULAR
COOPERATION
IN LIF SYNTHESIS
399
puromycin-treated cells did not inhibit LIF production. Puromycin, 5 pg/ml, in the culture medium was able to completely block LIF synthesis. Figure 2A shows the effect of puromycin-treated monocytes on T-cell LIF synthesis. Puromycin-treated monocytes augmented LIF production but to a lesser degree than nontreated monocytes (Fig. 1). The optimum amount of puromycintreated monocytes was lo-20% and this gave a statistically significant difference (P < 0.05) compared to cultures containing no additional monocytes. The elaboration of soluble helper factors by monocytes was studied by culturing monocytes and T lymphocytes in double-chambered Marbrook-type culture vessels. Monocytes were able to elaborate soluble factors that enhanced T-cell LIF production (Fig. 2B). The optimum amount of monocytes was about 20% which caused in the case of Con A-migration inhibition significantly different (P < 0.05) from cultures without additional monocytes. The augmentation caused by monocyte-derived factors was smaller than that caused by nontreated or puromycin-treated monocytes. LIF Production
by T Cells, B Cells, and Their Combinations
Figure 3 shows the LIF synthesis ability of T cells, B cells, and their combinations (T + B) stimulated with mitogens. In all experiments the total lymphocyte number was kept constant. LA and Con A caused LIF synthesis in purified T cells whereas B cells responded by LIF production after stimulation with prot A or anti-&m. With Con A, prot A, and anti-/&m T- and B-cell mixtures produced more LIF than T or B cells alone. The presence of about 20% T or B cells among B or T lymphocytes caused optimum LIF production which differed significantly (P < 0.01) from the LIF synthesis level of these cells cultured alone. LA was an exception and with it LIF production diminished with increasing amounts of B lymphocytes. Autologous and allogeneic cells were also compared for their ability to affect LIF
% PUROMYCINTREATED
MONOCYTES
% MONOCYTES MARBROOK
IN CHAMBERS
FIG. 2. (A) Augmentation of the LIF response to T lymphocytes to LA and Con A by puromycin-treated monocytes. The concentrations of LA (0) and Con A (0) were 0.6 and 20 &ml, respectively, and the mitogens were present during the whole culture period. (B) The effect of monocyte-derived factors on T-cell LIF response. The cells were stimulated with LA, 2.5 CLg/ml(O), and Con A, 80 &ml(O), and cultured in the separate compartments of Marbrook-type culture vessels. The values represent the mean * SD of four experiments.
400
RASANEN,
J, % B CELLS %
9595
III 9095>95 10 5
