Lymphocyte response to plant mitogens

GYNECOLOGIC

ONCOLOGY

8,

198-203 (1979)

Lymphocyte B. DAUNTER, Ph.D.,’ Department of Obstetrics Queensland, Royal

Response

to Plant Mitogens

S. K. KHOO, M.D., and Gynaecology, Brisbane Hospital,

Clinical Brisbane,

AND E. V. MACKAY, M.D. Sciences Building, University Queensland 4029, Australia

of

ReceivedJune21, 1978 Cell-mediated immuneresponse was measured by the uptake of tritiated thymidine by lymphocytes cultured in serum-free medium in the presence and absence of different doses of phytohemagglutinin-M (PHA). Four groups were studied: (i) healthy women (controls); (ii) pregnant women; (iii) patients with carcinoma of the cervix; (iv) patients with ovarian carcinoma. The cancer patients were studied before and after treatment which consisted of radiotherapy and/or chemotherapy. No significant difference in response was found in the controlsin terms of the response to the different doses of PHA or between the controls and pregnant women. When the pregnant women were grouped according to trimester dates, variability in response was observed which was dependent on the dose of PHA used. However, there was a decreased response in the second trimester relative to the first and third trimester at different doses of PHA. The lymphocyte response of the cancer patients was depressed only at 5 pg/ml of PHA. The response before treatment was decreased relative to the response after treatment. After treatment the response of the patients with ovarian carcinoma was similar to the control value.

It has been reported that the blastogenic response of lymphocytes to the plant mitogen phytohemagglutinin-M (PHA) in lymphoid malignancies [l-3] and nonlymphoid malignancies [4] is depressed. However, it has also been reported that in nonlymphoid malignancies the lymphocyte response to PHA is not depressed [5]. In addition, if the lymphocytes from cancer patients are washed their response to PHA can be enhanced [6]. A similar controversy exists as to whether the lymphocyte response to PHA during pregnancy is depressed [7-93 or not [lo, 111. In the present study the activity of washed peripheral blood lymphocytes has been investigated using different concentrations of PHA. PATIENTS AND METHODS

Subjects Studied A total of 204 women were included in the study; they were placed in one of the following three groups: (i) Healthy controls. Ninety-seven hospital staff aged between 18 and 60 years who appeared in good health. ’ To whom requests for reprints should be addressed: Department of Obstetrics and Gynaecology, Clinical Sciences Building, Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia. 198 0090-8258/79/0.50198-06$0 1.0010 Copyright All rights

@ 1979 by Academic Press, Inc. of reproduction in any form reserved.

LYMPHOCYTE

RESPONSE

TO

PLANT

MITOGENS

199

(ii) Pregnant M’umen. Sixty patients with uncomplicated pregnancies who were attending the antenatal clinic. The gestational maturity ranged from 10 to 42 weeks. (iii) Patients w*ith cancer. Thirty-one patients who had cancer of the uterine cervix, and 16 patients with ovarian carcinoma. Blood was obtained before and after primary treatment was commenced. From the information derived from clinical assessment and histological examination, these patients were considered to have either locally invasive cancer (confined to the organ of origin) or disseminated cancer (spread had occurred beyond the organ of origin). The treatment for carcinoma of the cervix was radiotherapy. Treatment of ovarian carcinoma consisted of radiotherapy and/or chemotherapy. Lymphocyte

Response

(i) Separation of lymphocytes. Lymphocytes were separated from peripheral blood by centrifugation in an Isopaque-Ficoll gradient of density 1.073 [ 121. Blood was collected in ice-cooled flasks to which phenol-free heparin (5 units/ml) had been added. The buffy coat in the mixture was removed and the cells were washed twice in Hanks’ balanced salt solution and finally resuspended in tissue culture media RPM1 1640 (Grand Island Biological Co., N. Y.) enriched with L-glutamine and buffered with N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (pH 7.2, adjusted with sodium hydroxide), without the addition of serum, antibiotics, or sodium bicarbonate. The volumes were adjusted to give 1 x 10” lymphocytes/ml and the cells dispersed in sterile polystyrene Falcon tubes in triplicate. (ii) Cultures of 1ymphocytc.s. Triplicate cultures of 1 ml each were set up without PHA, as well as with PHA, in doses of 5, 20, and 100 pg in 0.05 ml. The lymphocytes were then cultured in closed Falcon tubes at 37°C in air for 72 hr. (iii) Measurement of acti\‘ity. The response was estimated by the incorporation of tritiated thymidine by the cell cultures. To each culture tube 1 $Zi of tritiated thymidine was added 24 hr before termination of culture. At the end of culture the lymphocytes were transferred to 4-cm diamter fiberglass filter papers on Buchner funnels and washed with 10 ml each of saline, 10% trichloroacetic acid, and methanol. The dried filter papers were transferred to 10 ml of scintillation fluid and radioactivity was determined in a liquid scintillation p-counter (Nuclear, Chicago). Results were expressed as the mean counts per minute (cpm) of triplicate cultures. The unstimulated activity was defined as the mean cpm of triplicate cultures without PHA. The response index of PHA was defined as the log ratio of the mean cpm of the triplicate stimulated and unstimulated cultures. Statistical

Analysis

The t test was used to compare the groups’ Cl-31 mean log index of response to the different doses of PHA. RESULTS

There was no significant difference in the control lymphocyte response or those from the pregnant women to the different concentrations of PHA (Tables 1 and 2).

200

DAUNTER,

CONTROL PHA bdml)

AND

MACKAY

TABLE 1 TO PHYTOHEMAGGLUTININ-M

(PHA)

No. of controls

Mean log index

Log (SD)

P

97 97 97

1.45 1.40 1.40

0.36 0.24 0.30

> 0.1

5 20 100 B Comparison

RESPONSE

KHOO,

by f test.

TABLE 2 LYMPHOCYTE RESPONSE TO PHYTOHEMAGGLUTININ (PHA) CONTROLS vs PREGNANCY AND CANCER PATIENTS BEFORE TREATMENT”

Group Control Pregnant Carcinoma Carcinoma

cervix ovary

a 5 pg/ml

No. of patients

Mean log index

Log (SD)

P

97 60 31 16

1.45 1.37 0.82 0.85

0.36 0.28 0.24 0.20

> 0.1 < 0.001 < 0.001

PHA.

COMPARISON

OF THE LYMPHOCYTE

TABLE 3 RESPONSE TO PHA Mean

PHA 6%) 5 20 100

First

trimester (n=8)

0.67 2 0.54 1.28 k 0.54 1.86 k 0.32

COMPARISON

IN EACH

TRIMESTER

log index

Second trimester (n=27) p = 0.05 p < 0.05 p < 0.02

OF LYMPHOCYTE

0.33 + 0.38 0.85 2 0.52 1.40 k 0.48

TABLE RESPONSE

OF PREGNANCY

4 TO PHA

Third

p < 0.05 p < 0.01 p < 0.001

CONTROLS

trimester (n =25)

0.70 2 0.78 1.32 k 0.65 1.85 k 0.36

vs PREGNANCY

P PHA (/a) 5 20 100

First

trimester < 0.001 > 0.1 < 0.001

Second

trimester

i 0.001 < 0.001 > 0.1

Third

trimester < 0.001 > 0.1 < 0.001

LYMPHOCYTE

RESPONSE

TO

PLANT

201

MITOGENS

When the pregnant women were grouped according to trimester dates, a decrease in the lymphocyte response was found in the second trimester compared to the first and third trimesters at all concentrations of PHA investigated. However, this decrease in lymphocyte response was only really significant at the higher PHA concentrations (Table 3). Relative to the controls the lymphocyte response in the first and third trimesters was significantly decreased at 5 pg/ml PHA and significantly increased at 100 pgiml PHA; at 20 pg/ml PHA the lymphocyte response was similar to that of the controls. In the second trimester the lymphocyte response was significantly decreased at 5 and 20 pgiml PHA, but not at 100 pg/ml PHA where the response was similar to that of the controls (Table 4). A significant decrease in the lymphocyte response to S&ml PHA was found in both groups of cancer patients before treatment. After treatment there was a significant increase in the lymphocyte response to 5 pgiml PHA in both groups of cancer patients (Table 51, but in the group of patients with carcinoma of the cervix this response was still significantly depressed relative to that of the controls (Table 6). DISCUSSION

There was no statistical difference in the response of the control lymphocytes or that of the pregnant women to the different concentrations of PHA (5,20, and 100 pgiml). This suggests that lymphocyte PHA receptors were saturated at 5 pg/ml TABLE LYMPHOCYTE

RESPONSE AND

Before No. of patients cervix

31 Carcinoma 16

ovary

“ 5 &ml

After

Log

Log

(SD)

P

0.82

0.24

1.3s

0.24

< 0.001

0.85

0.20

1.41

0.30

< 0.001

TABLE RESPONSE

TREATMENT

cervix ovary PHA.

TO PHA

COMPARED

No. of patients

Group

(’ 5 &ml

treatment

Mean log index

(SD)

BEFORE

PHA

LYMPHOCYTE

Control Carcinoma Carcinoma

TO PHA

treatment

Mean log index

Carcinoma

5

IN CANCER PATIENTS AFTER TREATMENT”

97 31 16

6 IN CANCER

WITH

CONTROL

Mean log index I .45 1.35 1.41

PATIENTS

AFTER

VALUES”

Log (SD)

P

0.36 0.24 0.30

> 0.02 > 0.1

202

DAUNTER,

KHOO,

AND

MACKAY

PHA. Similarly, this applies to the lymphocytes obtained from women with carcinoma of the cervix or ovary, in which a decrease in the lymphocyte response was only found at 5 pug/ml PHA. When the pregnant women were grouped according to trimester dates and compared to the controls, there was a significant decrease in the lymphocyte response to 5 pg/ml PHA in each trimester. At 20 pg/ml PHA the lymphocyte response was only decreased significantly in the second trimester, the values in the first and third trimester being similar to the control values. The lymphocyte response to 100 pg/rnl PHA was increased to similar levels in the first and third trimester. In the second trimester the lymphocyte response to 100 ,ug/ml PHA was similar to the controls. These results demonstrate that the maternal lymphocyte response is not only dependent on the concentration of PHA but also on the stage of pregnancy. In addition the pattern of lymphocyte reactivity changes during pregnancy which may reflect the response of subpopulations of lymphocytes to changes in their milieu as pregnancy progresses. A comparison of the lymphocyte response in each trimester indicates a decreased lymphocyte response to PHA in the second trimester relative to the first and third trimesters. This difference in lymphocyte response becomes increasingly more significant (< P) with higher concentrations of PHA. This evidence supports the previous conclusion that the response of maternal lymphocytes to PHA appears to depend upon the dose of PHA used. These data may explain the controversy as to whether or not maternal lymphocyte response to PHA is suppressed or not. The lymphocyte response of the cancer patients was only depressed at 5 pg/ml PHA. In patients with carcinoma of the cervix after treatment the response to 5 pg/ml ,PHA was depressed relative to the controls, but increased relative to the response before treatment. The lymphocyte response of ovarian cancer patients after treatment was increased relative to the response before treatment and was similar to the control lymphocyte response. It has not been established whether or not the increased lymphocyte response of the cancer patients after treatment is of prognostic value. In addition, in both groups of cancer patients before treatment, the lymphocyte response at 5 ,ug/ml PHA was similar to the lymphocyte response at 20 pgiml PHA in the second trimester of pregnancy. This may suggest some similarity between the immune response of the cancer patient and that of the pregnant woman. However, these data,reflect the possibility that the PHA receptors on lymphocytes from cancer patients and healthy controls are saturated at 5 pg/ml PHA and that the lymphocyte response in pregnancy is dependent on the concentration of PHA and stage of pregnancy. Therefore, any similarity between the lymphocyte response of these groups may be conincidental. ACKNOWLEDGMENTS We wish to acknowledge Queensland Cancer Fund.

Miss J. Fox for her technical

assistance.

This study

was supported

by the

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LYMPHOCYTE

RESPONSE TO PLANT

MITOGENS

203

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