Depressed phytohemagglutinin and concanavalin A responses in premature infants

CLINICAL

IMMUNOLOGY

AND

Depressed

IMMUNOPATHOLOGY

19, 260-267 (1981)

Phytohemagglutinin and Concanavalin Responses in Premature Infants

A

AILA LEINO, 0. RUUSKANEN, P. KERO, J. ESKOLA, AND P. TOIVANEN Departments

of

Medical

Microbiology

and Pediatrics.

Turku

CJnir,ersity.

Turku.

Finland

Received October 1, 1980 Lymphocyte responses to phytohemagglutinin (PHA) and concanavalin A (Con A) were studied, using whole blood cultures, in 54 preterm infants weighing 800 to 2500 g, in 17 infants weighing >2500 g, and in five small-for-dates weighing 520 to 1820 g. All samples studied were collected within 12 h after the delivery. Half of the infants weighing cl000 g had abnormally low responses to PHA and Con A. The responsiveness increased with the increasing birth weight, being similar to that of the full-term newborns already in infants with a birth weight of 1251- 1500 g. Seven of the low responders were studied again at the age of 4- 13 days. All the responses were then normal, suggesting a role for immunosuppressive factors in the plasma.

INTRODUCTION

Several studies indicate that human T lymphocytes acquire immunocompetence during the fetal life, even though at the time of full-term birth their functional capacity may not be equal to that of adult lymphocytes (l-4). The criteria used include mixed lymphocyte culture, cell-mediated lympholysis, and responses to T-cell mitogens phytohemagglutinin (PHA) and concanavalin A (Con A). During the intrauterine life, PHA responsive cells appear first in the thymus at lo- 12 weeks of gestation (5-7) and Con A responsive cells a few weeks later (8). Bone marrow and liver cells of human fetuses do not usually respond to these mitogens (7-9). In the blood PHA responsive cells have first been demonstrated at 14.5 weeks of gestation (7). All these findings are based on the use of cells obtained from therapeutic abortions. No corresponding data are available regarding premature infants and, in general, there are only few reports on immune responses of premature infants. Cantell et al. (10) observed a normal capacity for interferon production in the prematures. Granberg et al. (11, 12) have described a well-developed cellmediated lympholysis by peripheral blood lymphocytes in a premature of 7 months’ gestation. A positive skin test to dinitrochlorobenzene was shown by Uhr et al. (13) in 3 of 10 prematures with an average birth weight of 1255 g. In the present work, we have studied lymphocyte responses to PHA and Con A in 76 infants including 54 prematures with a gestational age of 25-36 weeks. The number of acid a-naphtyl acetate esterase (ANAE)-positive lymphocytes was also investigated. MATERIALS

AND METHODS

Patients. The material consists of 54 premature infants with a birth weight ~2500 g (mean ~frSEM, 1610 + 70) and a gestational age of 25-36 weeks (31 + 0.4), 260 0090-1229/81/050260-08$01.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

DEPRESSED

MITOGEN

RESPONSES

IN

PREMATURES

261

of 17 infants with a birth weight >2500 g (3064 +_ 110) and a gestational age of 37-40 weeks (37 ? 0.2), and of five small-for-date infants with a birth weight of 1348 + 257 g and a gestational age of 30-37 weeks (33 2 1.4). All the infants were treated in the neonatal intensive care unit of the Department of Pediatrics at the Turku University Central Hospital. The main diagnoses of the infants are listed in Table 1; usually each individual had more than one diagnosis. The whole material includes 5 infants of diabetic and 12 infants of toxemic mothers. Twenty-five of the mothers were treated with dexamethasone (4 mg two times per day, usually for 2 days) before delivery. The blood samples (1 ml) were taken into a heparinized tube from the umbilical vein or artery immediately after a catheter had been inserted. In infants without umbilical catheter the blood samples were taken from a scalp vein. All samples were collected within 12 h after the delivery. Lymphocyte transformation assay. For mitogen stimulation of lymphocytes a technique using whole blood was applied (14). In brief, a mixture of 25 ~1 of heparinized blood and 150 ~1 of RPM1 1640 (Grand Island Biological Co., Grand Island, N.Y.) was placed into the wells of a sterile microtiter plate (IS-MRC-96, Linbro Chemical Co., New Haven, Conn.). Thereafter PHA (PHA M, Difco Laboratories, Detroit, Mich.) and Con A (Pharmacia Fine Chemicals, Uppsala, Sweden) in 25 ~1 of RPM1 1640 were added into the desired wells. The control cultures received 25 ~1 of plain medium. The final concentrations used for PHA were 25, 125, and 625 pg/rnl and for Con A 5,25, and 125 pg/ml. The cultures were pulsed 16-18 hr before harvesting with 0.25 &i of 5-[lz51]iodo-2’-deoxyuridine (lZ51UdR, sp. act. 90- 110 mCi/mg, The Radiochemical Centre, Amersham, England) which was added into the wells together with 5-fluoro-2’-deoxyuridine (Fluka, Buchs, Switzerland) (15). All cultures were made in triplicate and harvested with a multiple cell culture harvester (Skatron, Lierbyen, Norway) 72 hr after the beginning of the culture. The responses are expressed as counts per minute (cpm). Low responder is defined as one giving a response of at least 2 SD below the mean for infants with a birth weight >2500 g. Leukocyte and lymphocyte counts. The differential counts of blood leukocytes were made by using standard techniques. ANAE staining was used to demonstrate the number of T lymphocytes in blood smears according to Rankiet al. (16). Stntistics. Student’s C-test and x2 test were employed to estimate the significances. RESULTS

Lymphocyte responses to PHA and Con A in infants weighing 2500 g (Fig. 1). Responses were also significantly decreased in the IOOl- to 1250-g group except at 125 &g/ml of Con A. In infants with a birth weight of 1251-2500 g, the responses to both mitogens were similar to those of the full-term newborns. Individual responses to 125 pg/ml of PHA and to 25 pg/ml of Con A are presented in Fig. 2. Half of the infants weighing (1000 g had low PHA responses, i.e., a response at least 2 SD lower than the mean response in infants weighing >2500 g, and 5 out of 12 infants had low Con A responses. In the IOOl-

Nnre.

Figures

Total

given

to the number

of infants,

12 (1)

37 (17)

refer

1 0 0 2 (1) 3 6

9 g 4 6 4 6

2500

Tachypnoea

(4) (5) (4) (3) (1)

RDS

Birth weight (g)

to infants

7

0 0 0 3 2 2

treated

8 (4)

(4 mg two times

5

12 (2) with dexamethasone

3

5

per day).

10 (3)

0 3 (I) 3 (2)

4 (2) 1 1 0

I 0

Healthy

0 0 0

1 (1)

2 (2)

0 0

Diabetes of mother

1 (1) 5 3 0

2

Toxemia of mother

0

Sepsis

STUDIED

with a mother

11

1 2 0 0 3 5

Asphyxia

TABLE 1 THE 76 INFANTS

Hypoglycemia

those in parentheses

10 (3)

1 0 0 3 (I) 3 (2) 3

Respirator

CLINICALDIAGNOSESOF

! > r.

F;; 2 0

DEPRESSED

MITOGEN

RESPONSES IN PREMATURES

263

PHA 12

9

.--

. .

4000

-1250

2 a5E 0

6

14

Con

18

17

-2500

>2500

A

L32lO-1500 Birth

-2000 weight

(g)

FIG. 1. PI-IA and Con A responses (mean 2 SD) by peripheral blood lymphocytes of 76 infants. Column (a) represents unstimulated control cultures. Concentrations of 25 (b), 125 (c), and 625 pgirnl (d) of PI-IA and of 5 (b), 25 (c), and 125 pg/ml (d) of Con A were used. Figures at the top refer to the number of individuals tested. The asterisks show the significance in comparison to the infants weighing >2500 g: *p < 0.005; **p < 0.01; ***/I < 0.001.

to 1250-g group three low responders to PHA and two low responders to Con A were found. In infants with a birth weight of >1250 g there was only one low responder in each weight group. Details of the 14 infants with a low PHA and/or Con A response are presented in

.

.

l

3 t

::

.-

.

l .

4000

-Y.-,---

l:

-I250

----

-1500

__._

-2000

c ____

?--

l

-2500

I

>2500

. ..-______

.

3-

.

: . .

.

l

.

2-

' ~1000

-1250

-1500 Birth

-2000 welaht

I -2500

I ~2500

(g)

2. Individual PHA (125 &ml) and Con A (25 pg/ml) responses in different weight groups of 76 infants. The mean response in each group is indicated by a solid line. Values below the broken line represent low responses. i.e., at least 2 SD below the mean for infants with a birth weight >2500 g. FIG,

27 29 29 33 37

1090 1360 1540 2080 2800

u Low response if defined * 125 PgIml. c 25 &ml. d m, Mother: b, baby.

29 28

1030 1060

as being

227 1639 473 562 648

194 220

324 647 1811 129 12571 608 218

PHAb

(cpm)

the mean

206 3810 1271 2089 1176

14623 556

value

760

Ar

WITH

6789 990 14819 340 3%3 499

Con

at birth

OF INFANTS

2 SD below

responses

at least

1% 256 350 133 680

936 355

360 411 430 202 361 120 176

Unstimulated

age (weeks)

2s 29 27 27 30 27 27

Mitogen

Gestational

Elm 830 890 950 980 980 loo0

63)

Birth weight

DETAILS

a birth

388

512

284

310 143 301

6

for infants

CON

weight

responses

233

with

Mitogen

TABLE 2 PHA AND/OR

13

4

13

6 11 9

(days)

Age

Low

>2500

27286

58029

43707

44564

17309 27977 20741

PHAb

(cpm)

g.

later

A RESPONSE

41779

37138

52417

63993

23078 25711 28282

Con

A’

AT BIRTH”

m, b m m, b

m m, b

m m, b m m, b m, b

Dexamethasone”

RDS RDS RDS, hypocalcemia RDS Toxemia of mother RDS RDS, hypocalcemia, hyperbilirubinemia RDS RDS, hypocalcemia, hyperbilirubinemia RDS, hypocalcemia RDS, sepsis Hypocalcemia Hyperbilirubinemia RDS

Diagnosis

g .

t:

F 2 0

DEPRESSED

MITOGEN

RESPONSES

IN

PREMATURES

265

Table 2. In 11 infants the Con A response was higher than the PHA response; 3 had a normal Con A response and a low PHA response, whereas only 1 infant had a low Con A response and a normal PI-IA response. Seven of the low responders were studied again at the age of 4- 13 days; all the responses were then normal (Table 2). Among the 14 low responders 10 infants (71%) had a respiratory distress syndrome (RDS), whereas in the whole material 37 out of 76 (4%) had RDS; the difference was not significant (p > 0.05, x2 test). Low response was associated neither with mother’s toxemia nor diabetes. Twenty-nine percent of the normal responders to PHA and 28% of the normal responders to Con A had received dexamethasone directly and/or via the mother. The corresponding percentages in the low responders were 54 and 64; the differences were not statistically significant @ > 0.05), revealing that low responders had not been treated more often with corticosteroids than those responding normally. The T lymphocyte count in the peripheral blood of the prematures was determined by ANAE staining. The relative number of T lymphocytes in the low responders to PHA was 67 -+ 9% (n = 7), in the low responders to Con A 64 + 8% (n = 6), and in the normal responders 76 ? 9% (n = 38); the differences between the low responders and the normal responders were not significant @ > 0.05). Of the six infants who died within a few weeks after the birth two were low responders and four normal responders. All others were healthy at the age of 3-4 months. DISCUSSION

Our results indicate that half of infants weighing
266

LEINO ET AL.

mother’s lymphocyte responses may be depressed (23, 24), most probably due to factors such as glucocorticoids, estrogens, progesterone, chorionic gonadotrophins, and prostaglandins, which are also found in the fetus. We find this mechanism the most likely explanation for our findings since in each of the seven infants reexamined, the immunodepression had disappeared within a few days after the birth. This conclusion is supported by observations of Yu et al. (25). At the time of birth they observed significantly lower PHA responses with unwashed than with washed newborn lymphocytes. Seven days later this difference had disappeared, indicating a suppressive effect of the newborn plasma. Here we are dealing with the advantages and disadvantages of the whole blood culture in studies for lymphocyte reactivity (14). An advantage is that the result obtained corresponds to the in V~VO situation, a disadvantage is that lymphocyte’s functional capacities as such are not always fully expressed. In fact, all exceptional results obtained with the whole blood method should be repeated by using washed lymphocytes. However, due to the large amount of blood required, with prematures and newborns this should be considered only when a second attempt with the whole blood method also indicates a decreased or lacking response. The effect of maternally derived dexamethasone on the infant’s lymphocyte responses is unclear. Hallberg and Hallberg (26) have observed that infants born by a mother treated with prednisolone during pregnancy had a low level of SRBC-binding lymphocytes which increased during the first 3 weeks of life. In the present study, infants with low mitogen responses had not received significantly more often corticosteroids than infants with normal responses. Maternal diabetes or toxemia, neonatal asphyxia, or RDS had no clear effect on the infant’s lymphocyte responses. All small-for-date infants, even an infant with a birth weight of 520 g (gestational age 31 weeks), had also normal mitogen responses. ACKNOWLEDGMENTS This work was supported by the grants of the Turku University Foundation and Sigrid Juselius Foundation, and by a contract with the Association of Finnish Life Insurance Companies.

REFERENCES 1. Toivanen, P., Asantila, T., Granberg, C.. Leino, A., and Hirvonen, T., fmmunol. RCV. 42, 185, 1978. 2. Stiehm, E. R., Winter, H. S., and Bryson, Y. J., Pediatrics 64 (suppl.). 814. 1979. 3. Stites, D. P., and Pavia, C. S., Pediatrics 64 (suppl.), 795. 1979. 4. Granberg, C., and Hirvonen, T.. Cell. Immunol. 51. 13, 1980. 5. Papiernik, M., Blood J. Hematol. 36, 470, 1970. 6. August, C. S., Berkel, A. E.. Driscoll, S., and Merler, E.. Pediatr. Res. 5, 539, 1971. 7. Stites, D. P., Carr, M. C., and Fudenberg, H. H., Cell. Immunol. 11, 257, 1974. 8. Leino, A., Hirvonen, T., and Soppi. E.. Clin. Immunol. Immunopathol. 17, 547. 1980. 9. Stites, D. P., Carr, M. C., and Fudenberg, H. H., Proc. Nat. Acad. SC;. USA 69, 1440, 1972. 10. Cantell, K., Strander, L.. Saxen, L.. and Mayer, B., J. Immunol. 100, 1304, 1968. 11. Granberg, C.. Manninen, K., and Toivanen, P., Clin. Immunol. Immunopathol. 6, 256, 1976. 12. Toivanen, P., Granberg, C.. and Manninen, K., In “Regulatory Mechanism in Lymphocyte Activation,” Proceedings of the 11th Leukocyte Culture Conference (D. 0. Lucas, Ed.). p. 638. Academic Press, New York, 1977. 13. Uhr, J. W., Dancis. J.. and Neumann, C. G., Nature (London) 187, 1130, 1960. 14. Eskola, J., Soppi, E., Viljanen. M.. and Ruuskanen, 0.. Immrmol. Commun. 4, 297, 1975.

DEPRESSED 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

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RESPONSES IN PREMATURES

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Asantila, T., and Toivanen, P., J. Immunol. Methods 6, 73, 1974. Ranki, A., Totterman, T., and Hayry, P., Sand. J. Immunol. 5, 1129, 1976. Burgio, R., Severi, F.. and Ugazio, A., Amer. J. Dis. Child. 131, 1407, 1977. Jones, W. R., Amer. J. Obstet. Gynecol. 104, 586, 1969. Olding, L. B., and Oldstone, M. B. A., J. Immunol. 116, 682, 1976. Hayward, A. R., and Lawton, A. R., J. Immunol. 119, 1213, 1977. Miyawaki, T.. Seki, H., Kubo, M., and Taniguchi, N., J. Immunol. 123, 1092, 1979. Olding, L. B., Murgita, R. A., and Wigzell, H., J. Immunol. 119, 1109, 1977. Bjune. G., Duncan, E., Barnetson, R., and Melsom, R., Clin. Exp. Immunol. 32, 517, 1978. Gill, T. J.. III, and Repetti, C. F., Amer. J. Parhol. 95, 463, 1979. Yu, V. Y. H., Wager, C. A., MacLennan, 1. C. M., and Baum, J. D., Brit. Med. J. 1, 428, 197.5. Hallberg, A.. and Hallberg, T., Int. Arch. Allergy Appl. Immune/. 55, 102, 1977.