Int. Archs Allergy appl. Immun. 50: 14-26 (1976)
Comparative Inhibitory Effects of Serum on Lymphocyte Responses to Mitogenic Stimulation C lem ent C. S. H su Samuel J. Sackett Research Laboratories, Infectious Diseases - Hypersensitivity Section, Department of Medicine, Northwestern University Medical Center, Chicago, 111.
Abstract. The effects of sera from pregnant women, human umbilical cords and non pregnant healthy control individuals on human lymphocyte responses to phytohemagglu tinin (PHA), pokeweed mitogen (PWM) and allogeneic lymphocytes (MLR) are described. Maternal sera invariably inhibited lymphocyte responses to PHA. In MLR and PWMstimulated lymphocyte responses, however, some maternal sera had pronounced inhibitory effect while other maternal sera had none. Similar dissociation of inhibitory effects of lymphocyte responses to PHA and to PWM or in MLR was noted in some cord sera but not in the control sera we studied. The observations may reflect different serum factors impinging upon different lymphocyte subpopulations responsive to different mitogens. Preliminary attempts to relate the serum inhibitory effects to various serum components revealed that inhibition of MLR appeared to correlate with increases in a-2-globulins and /1-globulins. In contrast, inhibition of PHA responses was not similarly related to any one or two specific serum constituents.
Introduction Human serum may have a marked inhibitory or potentiating effect on cultured lymphocyte responses to various mitogens. It has been reported that in pregnancy [1-3], and in a variety of other conditions [4-17], inhibi tors) in serum suppresses lymphocyte response to phytohemagglutinin (PHA), a specific thymus-derived T-cell stimulant. There are also reports [18-22] that the serum from pregnant women inhibits mixed lymphocyte reactions (MLR), which are in vitro models for graft-versus-host reactions. It is generally thought that the serum inhibitor(s) observed in these in vitro systems, particularly MLR, inhibits lymphocyte responses in vivo and there-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 10:19:41 AM
Received: April 1, 1975.
Hsu
15
fore functions as a regulator of immune responses. It is not known, how ever, whether the serum inhibitory effect on PH A response is mediated by one or more serum constituents and whether the same inhibitor(s) is also responsible for the inhibition of MLR or lymphocyte responses to other mitogenic stimulation. Since lymphocyte responses to a given mitogen in different individuals and in the same individual on different occasions may vary, it would seem difficult to resolve this question unless the inhibitor(s) of in vitro lymphocyte responses is isolated. Based on an observation that the serum inhibitory effect of PHA response is not influenced by using lymphocytes from different donors, we investigated whether there are different inhibitors for PHA, MLR and pokeweed mitogen (PWM) responses. Sera from pregnant women, human umbilical cords and nonpregnant healthy control individuals were used for the study. The results suggest that in pregnant women’s sera there are different factors involved in inhibition of lymphocyte responses to different stimulants. Similar findings were also noted in human cord serum which, in addition, was found to have weak but definite lymphocyte-stimulating property in the absence of mitogens. Preliminary studies designed to relate the serum inhibitory effects and various serum components revealed that inhibition of MLR appeared to correlate with increases in a-2-gfobulins and /i-globulins. In contrast, inhibition of PHA response was related to changes in levels of several serum constituents.
Rationale for Design o f Work It was noted in previous studies of serum inhibitor(s) [14, 23] that a similar degree of inhibitory effect of PHA stimulation can be obtained with a given serum upon repeated experiments, regardless of the lymphocyte donors. Therefore, when groups of inhibitory and noninhibitory serum samples are tested simultaneously for their effects on mitogeninduced lymphocyte responses from a single donor, the relative degree of inhibition of the individual serum should be a fairly good indicator of relative quantity of the inhibitor(s) in the serum for that particular mitogen. When this simultaneous testing of multiple sera is used to quantitate the inhibitor(s) of two different mitogens in the same sera, two patterns of relative inhibitory effects can be obtained by the same group of sera. Then the comparison of these two inhibitory patterns should reveal the identity (or nonidentity) of the inhibitor(s) of these two mitogens by virtue of the similarity (or dissimilarity) of the relative quantities of the inhibitor(s) present in the same serum samples. For instance, inhibitory serum samples, A, B and C, may allow tritiated thymidine (;iH-TdR) uptake by PHA-stimulated lymphocytes of 1,000, 1,500 and 3,500 cpm, respectively. If the inhibitor(s) of the PHA and that of MLR are identical, the serum samples. A, B, and C, may allow 3H-TdR uptake in MLR of 200, 300 and 700 cpm, respectively (in other words, same
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 10:19:41 AM
Materials and Methods
16
Hsu
ratio of cpm between serum samples as that of the PHA experiments). If, on the other hand, the inhibitors of the two mitogenic responses are not identical, the 3H-TdR uptake in MLR influenced by the same serum samples, A, B, and C, may be of entirely different proportion from that of the PH A experiment, e.g. 500, 500 and 100 cpm, respectively, depending on the quantities of MLR inhibitors present in these samples. Using this approach, we studied the effects of inhibitor(s) in sera from pregnant women, umbilical cords and nonpregnant healthy control individuals on human lymphocyte responses stimulated by PHA, allogeneic lymphocytes and PWM.
Lymphocyte Cultures and Radioactive Analysis Lymphocyte cultures were performed as previously reported [14, 23], Peripheral blood lymphocytes obtained from healthy control individuals were washed 3 times in Roswell Park Memorial Institute (RPMI) medium 1640 before distribution into culture tubes. Each culture tube contained 0.5 x 106 lymphocytes in 0.5 ml of RPMI medium 1640 con taining 20% (v/v) of the cord, maternal or control serum as well as penicillin and strepto mycin. Cultures were set up in triplicate for each serum Mitogens added include PHA-P (Difco, 12.5 //g.'ml) and PWM (Gibco, 6.25 //g/ml). The doses used had been found to produce submaximal stimulation in our system. In MLR, 0.25 x 10“ lymphocytes each from two healthy individuals were mixed. In the second series of experiments, purified protein derivative (PPD; Parke-Davis; 2 /tg ml) was also used as stimulant. The cultures were maintained 3 days in water vapor saturated 5% COa/95% air atmosphere at 37 C for PHA-P-, and PWM-stimulated cultures and 6 days for MLR, PPD-stimulated cultures and cultures without mitogens. In the second series of experiments, PWMstimulated cultures were also maintained for 6 days. 24 h before harvesting the cells, methyl-3H-thymidine (spec. act. 1.9 Ci/msi; Schwarz/Mann) at a final concentration of 1 /iCi/mM was added to the culture. At the time of the harvest, the cells were washed twice each with normal saline and 5% trichloroacetic acid. The precipitates were dissolved in 0.5 ml of Hydroxide of Hyaminc I0X (Packard). 10 ml of Bray's solution was added, and radioactivity was counted in a Packard Scintillation Counter (Model 3375) with back ground counts subtracted automatically. The results are expressed as mean cpm of tri
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 10:19:41 AM
Serum Samples Two series of experiments were performed. In the first series, sera were obtained from 10 normal pregnant women at term, 9 newborns (umbilical cord blood) and II nonpregnant adults. Eight newborns were delivered by spinal or epidural anesthesia with procaine, and no anesthesia was used for the other newborn. Mothers of two newborns (Sos and Bla) were also included in our study. Among pregnant women, 6 (Agu, Spa, Fid, Sos, Bla and Riv) had one to three pregnancies previously and 3 (But, Ace and Rom) were primigrávidas. The record for another woman (Plu) was not available. Control sera were obtained from 6 healthy nonpregnant women and 5 healthy men who worked in the laboratory. The pregnant or nonpregnant control individuals tested had not been taking medications for at least 3 days prior to collection of sera other than multivitamins, except that two control subjects (Del and Ste) were taking oral contraceptives. In the second series, sera were obtained from 5 primigravid women between 33 and 42 weeks of gestation and 5 age-matched nonpregnant healthy women on no medications. All sera were collected aseptically and stored in aliquots at -20 C until used.
Serum Lymphocyte Inhibitory Effects
17
plicates. In the second series of experiments, for simplicity, lymphocyte responses are expressed in percentages relative to that of the cultures containing serum from control individual Ell which is arbitrarily defined as 100%. Measurement o f Serum Protein and Cortisol Levels The scrum components were measured in 10 serum samples used in the second series of experiments. For serum protein electrophoresis, a Microzone® electrophoresis system with cellulose acetate media (Beckman) was used. Serum immunoglobulins, complement C’3, a-2-macroglobulin, ceruloplasmin and transferrin were measured by the radial immunodiffusion technique using Immunoplates® (Hyland). For determination of serum cortisol levels, a radioimmunoassay was used which was described by R uder et al. [24] and kindly performed by Dr. R uder working in the Endocrinology section, Department of Medicine, Northwestern University Medical Center. Statistical Analysis o f the Results Student’s t test was used to evaluate the significance (p) of the difference in cpm be tween two groups. Correlation coefficients (r) and p values were calculated to assess the similarity of inhibitory patterns between two experiments and between the serum inhibitory effects and the levels of various serum proteins and cortisol.
First Series o f Experiments Effects of the 30 sera on PHA-P-stimulated lymphocyte responses are shown in figure 1. All maternal sera were found to inhibit PHA response to a similar degree. Among sera from control individuals, those of Del and perhaps Ste showed inhibitory effects. Among cord sera, those of Yoh and Iclc were inhibitory. There were highly significant differences (p0.9). In the MLR experiment (fig. 2), the maternal sera again showed an inhibitory effect in comparison with the control sera (p
Comparative Inhibitory Effects of Serum on Lymphocyte Responses to Mitogenic Stimulation C lem ent C. S. H su Samuel J. Sackett Research Laboratories, Infectious Diseases - Hypersensitivity Section, Department of Medicine, Northwestern University Medical Center, Chicago, 111.
Abstract. The effects of sera from pregnant women, human umbilical cords and non pregnant healthy control individuals on human lymphocyte responses to phytohemagglu tinin (PHA), pokeweed mitogen (PWM) and allogeneic lymphocytes (MLR) are described. Maternal sera invariably inhibited lymphocyte responses to PHA. In MLR and PWMstimulated lymphocyte responses, however, some maternal sera had pronounced inhibitory effect while other maternal sera had none. Similar dissociation of inhibitory effects of lymphocyte responses to PHA and to PWM or in MLR was noted in some cord sera but not in the control sera we studied. The observations may reflect different serum factors impinging upon different lymphocyte subpopulations responsive to different mitogens. Preliminary attempts to relate the serum inhibitory effects to various serum components revealed that inhibition of MLR appeared to correlate with increases in a-2-globulins and /1-globulins. In contrast, inhibition of PHA responses was not similarly related to any one or two specific serum constituents.
Introduction Human serum may have a marked inhibitory or potentiating effect on cultured lymphocyte responses to various mitogens. It has been reported that in pregnancy [1-3], and in a variety of other conditions [4-17], inhibi tors) in serum suppresses lymphocyte response to phytohemagglutinin (PHA), a specific thymus-derived T-cell stimulant. There are also reports [18-22] that the serum from pregnant women inhibits mixed lymphocyte reactions (MLR), which are in vitro models for graft-versus-host reactions. It is generally thought that the serum inhibitor(s) observed in these in vitro systems, particularly MLR, inhibits lymphocyte responses in vivo and there-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 10:19:41 AM
Received: April 1, 1975.
Hsu
15
fore functions as a regulator of immune responses. It is not known, how ever, whether the serum inhibitory effect on PH A response is mediated by one or more serum constituents and whether the same inhibitor(s) is also responsible for the inhibition of MLR or lymphocyte responses to other mitogenic stimulation. Since lymphocyte responses to a given mitogen in different individuals and in the same individual on different occasions may vary, it would seem difficult to resolve this question unless the inhibitor(s) of in vitro lymphocyte responses is isolated. Based on an observation that the serum inhibitory effect of PHA response is not influenced by using lymphocytes from different donors, we investigated whether there are different inhibitors for PHA, MLR and pokeweed mitogen (PWM) responses. Sera from pregnant women, human umbilical cords and nonpregnant healthy control individuals were used for the study. The results suggest that in pregnant women’s sera there are different factors involved in inhibition of lymphocyte responses to different stimulants. Similar findings were also noted in human cord serum which, in addition, was found to have weak but definite lymphocyte-stimulating property in the absence of mitogens. Preliminary studies designed to relate the serum inhibitory effects and various serum components revealed that inhibition of MLR appeared to correlate with increases in a-2-gfobulins and /i-globulins. In contrast, inhibition of PHA response was related to changes in levels of several serum constituents.
Rationale for Design o f Work It was noted in previous studies of serum inhibitor(s) [14, 23] that a similar degree of inhibitory effect of PHA stimulation can be obtained with a given serum upon repeated experiments, regardless of the lymphocyte donors. Therefore, when groups of inhibitory and noninhibitory serum samples are tested simultaneously for their effects on mitogeninduced lymphocyte responses from a single donor, the relative degree of inhibition of the individual serum should be a fairly good indicator of relative quantity of the inhibitor(s) in the serum for that particular mitogen. When this simultaneous testing of multiple sera is used to quantitate the inhibitor(s) of two different mitogens in the same sera, two patterns of relative inhibitory effects can be obtained by the same group of sera. Then the comparison of these two inhibitory patterns should reveal the identity (or nonidentity) of the inhibitor(s) of these two mitogens by virtue of the similarity (or dissimilarity) of the relative quantities of the inhibitor(s) present in the same serum samples. For instance, inhibitory serum samples, A, B and C, may allow tritiated thymidine (;iH-TdR) uptake by PHA-stimulated lymphocytes of 1,000, 1,500 and 3,500 cpm, respectively. If the inhibitor(s) of the PHA and that of MLR are identical, the serum samples. A, B, and C, may allow 3H-TdR uptake in MLR of 200, 300 and 700 cpm, respectively (in other words, same
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 10:19:41 AM
Materials and Methods
16
Hsu
ratio of cpm between serum samples as that of the PHA experiments). If, on the other hand, the inhibitors of the two mitogenic responses are not identical, the 3H-TdR uptake in MLR influenced by the same serum samples, A, B, and C, may be of entirely different proportion from that of the PH A experiment, e.g. 500, 500 and 100 cpm, respectively, depending on the quantities of MLR inhibitors present in these samples. Using this approach, we studied the effects of inhibitor(s) in sera from pregnant women, umbilical cords and nonpregnant healthy control individuals on human lymphocyte responses stimulated by PHA, allogeneic lymphocytes and PWM.
Lymphocyte Cultures and Radioactive Analysis Lymphocyte cultures were performed as previously reported [14, 23], Peripheral blood lymphocytes obtained from healthy control individuals were washed 3 times in Roswell Park Memorial Institute (RPMI) medium 1640 before distribution into culture tubes. Each culture tube contained 0.5 x 106 lymphocytes in 0.5 ml of RPMI medium 1640 con taining 20% (v/v) of the cord, maternal or control serum as well as penicillin and strepto mycin. Cultures were set up in triplicate for each serum Mitogens added include PHA-P (Difco, 12.5 //g.'ml) and PWM (Gibco, 6.25 //g/ml). The doses used had been found to produce submaximal stimulation in our system. In MLR, 0.25 x 10“ lymphocytes each from two healthy individuals were mixed. In the second series of experiments, purified protein derivative (PPD; Parke-Davis; 2 /tg ml) was also used as stimulant. The cultures were maintained 3 days in water vapor saturated 5% COa/95% air atmosphere at 37 C for PHA-P-, and PWM-stimulated cultures and 6 days for MLR, PPD-stimulated cultures and cultures without mitogens. In the second series of experiments, PWMstimulated cultures were also maintained for 6 days. 24 h before harvesting the cells, methyl-3H-thymidine (spec. act. 1.9 Ci/msi; Schwarz/Mann) at a final concentration of 1 /iCi/mM was added to the culture. At the time of the harvest, the cells were washed twice each with normal saline and 5% trichloroacetic acid. The precipitates were dissolved in 0.5 ml of Hydroxide of Hyaminc I0X (Packard). 10 ml of Bray's solution was added, and radioactivity was counted in a Packard Scintillation Counter (Model 3375) with back ground counts subtracted automatically. The results are expressed as mean cpm of tri
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 10:19:41 AM
Serum Samples Two series of experiments were performed. In the first series, sera were obtained from 10 normal pregnant women at term, 9 newborns (umbilical cord blood) and II nonpregnant adults. Eight newborns were delivered by spinal or epidural anesthesia with procaine, and no anesthesia was used for the other newborn. Mothers of two newborns (Sos and Bla) were also included in our study. Among pregnant women, 6 (Agu, Spa, Fid, Sos, Bla and Riv) had one to three pregnancies previously and 3 (But, Ace and Rom) were primigrávidas. The record for another woman (Plu) was not available. Control sera were obtained from 6 healthy nonpregnant women and 5 healthy men who worked in the laboratory. The pregnant or nonpregnant control individuals tested had not been taking medications for at least 3 days prior to collection of sera other than multivitamins, except that two control subjects (Del and Ste) were taking oral contraceptives. In the second series, sera were obtained from 5 primigravid women between 33 and 42 weeks of gestation and 5 age-matched nonpregnant healthy women on no medications. All sera were collected aseptically and stored in aliquots at -20 C until used.
Serum Lymphocyte Inhibitory Effects
17
plicates. In the second series of experiments, for simplicity, lymphocyte responses are expressed in percentages relative to that of the cultures containing serum from control individual Ell which is arbitrarily defined as 100%. Measurement o f Serum Protein and Cortisol Levels The scrum components were measured in 10 serum samples used in the second series of experiments. For serum protein electrophoresis, a Microzone® electrophoresis system with cellulose acetate media (Beckman) was used. Serum immunoglobulins, complement C’3, a-2-macroglobulin, ceruloplasmin and transferrin were measured by the radial immunodiffusion technique using Immunoplates® (Hyland). For determination of serum cortisol levels, a radioimmunoassay was used which was described by R uder et al. [24] and kindly performed by Dr. R uder working in the Endocrinology section, Department of Medicine, Northwestern University Medical Center. Statistical Analysis o f the Results Student’s t test was used to evaluate the significance (p) of the difference in cpm be tween two groups. Correlation coefficients (r) and p values were calculated to assess the similarity of inhibitory patterns between two experiments and between the serum inhibitory effects and the levels of various serum proteins and cortisol.
First Series o f Experiments Effects of the 30 sera on PHA-P-stimulated lymphocyte responses are shown in figure 1. All maternal sera were found to inhibit PHA response to a similar degree. Among sera from control individuals, those of Del and perhaps Ste showed inhibitory effects. Among cord sera, those of Yoh and Iclc were inhibitory. There were highly significant differences (p0.9). In the MLR experiment (fig. 2), the maternal sera again showed an inhibitory effect in comparison with the control sera (p
