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Possible effect of maternal promethazine therapy on neonatal immunologic functions
136
July 1976 TheJournalofPEDIATRICS
Possible effect of maternal promethazine therapy neonatal immunologic functions
on
The effect of the administration of promethazine in the treatment of erythroblastosis fetalis was studied in four maternal-fetal pairs. The three infants exposed for a prolonged period of time had decreased neonatal number and function of T cells, and abnormal specific humoral immune responses. The possible role of promethazine in the induction 9f fetal immunoincompetence is discussed.
Arye Rubinstein, M.D., Arthur h Eidelman,* M.D., Julian Melamed, B.Sc., M.B., B.Ch., Lawrence M. Gartner, M.D., Stephen Kandall, M.D., and Harold Schulman, M.D., Bronx, N. Y.
LARGE DOSES of promethazine, maternally adminis-
tered, have been utilized for amelioration of Rh erythroblastosis fetalis. 1 Animal studies indicate a suppressive effect on humoral and cellular immune responses. 2, ~ No data on the in vivo effects of antihistamines on the developing human fetal immunologic system have been reported. The current study reports the results of immunologic studies conducted on four Rh-positive infants born to sensitized Rh-negative mothers who were being treated with promethazine. PATIENTS
AND METHODS
Each of the newborn infants was Rh positive; their Rhnegative mothers had been sensitized to Rh antigen. Amniocentesis documented elevations of A OD 450 nm. Promethazine, 75 to 150 mg every day, had been administered to the mothers for five weeks (Patient I), six weeks (Patient II), 24 weeks (Patient III), and two weeks (Patient IV), respectively, prior to delivery. Thirteen normal term nonsensitized infants and 13 From the Departments of Cell Biology, Obstetrics, and Pediatrics, Hospital of the Albert Einstein College of Medicine, Bronx Municipal Hospital Center, Rose F. Kennedy Center for Research in Mental Retardation and Human Development, A lbert Einstein College of Medicine, Yeshiva University. Supported in part by grant A[-10702-04. *Reprint address: Hospital of the Albert Einstein College of Medicine, 1825 Eastchester Rd., Bronx, N. Y~ 10461.
Vol. 89, No. 1, pp. 136-138
adult volunteers were studied. Mothers of six of the control newborn infants received a single dose of 25 mg of promethazine as obstetrical analgesia. Serum immunoglobulins were measured by radial immunodiffusion and serum lysozyme (muramidase) by the Lyso-Plate assay. Antibodies to tetanus toxoid were measured by the hemagglutination method in Infants i, II, and III. Tetanus toxoid was administered after six weeks and boosters were given at four-week intervals. Abbreviations used EBF: erythrob!astosis fetalis PBL: peripheral blood lymphocytes SRBC: sheep red blood cells PHA: phytohemagglutinin AET: 2-amino ethylisothiouronium Neutralizing antibodies to poliomyelitis were measured in the infants after two doses of oral trivalent polio vaccine at age eight weeks and 16 weeks, respectively. Peripheral blood lymphocytes were isolated by sedimentation of heparinized cord blood in a syringe. E-rosetting for detection of T lymphocytes was performed using (1) untreated sheep red blood cellsr (2) neuraminidase-treated SRBC) and (3) sulfhydryl compound 2amino ethylisothiouronium bromide SRBC (AET). 6 The results (Table I, Fig. 1) indicate the percentage of AETrosettes, read by means of a flourescent microscope after staining in suspension with euchrysin. At age one year,
Volume 89 Number 1
Effect o f maternal prornethazine therapy
T a b l e I. Rosette formation by peripheral blood lympho-
cytes
70E
(%) I
(%)
I6
9 8 31 Range 67-80 x- 71.6 Range 31-53 x - 39
ADULT T ~ CONTROLS
~ . O
EAC:~
Patients' cord blood ll III IV Adult controls* (n = 13) Cord blood controls* (n = 13)
13 7
24 12 14 22 9-17 13.2 8-19 14
50
0 ,,', 30 10
A " "................ / z x NORMAL / .." / CORD Z."" //. BLOOD ~..."" //. ~~......" /././" ........ PATIENT T ~/"" _..,...~'/" - - - PATIENT ]Z M "'f'" ~'''''PATIENT IK o' [ 1
[ 3
I 5
1 7
AGE (Weeks)
E = Sheep erythrocytes; EACa = sheep erythrocytes coated with Forssman rabbit anti-sheeperythrocyteserum (A) and complementfrom CF~ mice. *Performed simultaneouslywith the patient's cord blood lymphocytes.
Fig. L E-Rosette formation by peripheral blood lymphocytes
PBL from Patient i was tested for E-rosetting inhibition with autologous cord blood serum. Rosetting for the quantification of B-lymphocytes with EACa (EAC1423) was performed/ using SRBC coupled with rabbit antiForssman antiserum and with complement components from C~ mouse serum. Blast-transformation of lymphocytes was measured in vitro by the incorporation of tritiated thymidine after stimulation with phytohemagglutinh~ (0.005% and 0.01%), pokweed mitogen (0.005% and 0.01%) and Concanavalin A (5-10 mg/ml).
ished at birth in all patients except Patient IV (Table I). In Patients I and III the rosettable T-lymphocytes population reached the normal adult range within six weeks. In Patient II the percentage of E-rosettes at eight weeks was still below the adult range. The percentage of EAC3 rosettes was normal in all patients, both in the cord blood and on follow-up at six weeks of age. Blast transformation of cord blood tymphocytes compared to control cord blood was reduced after stimulation; with phytohemagglutinin in Patients I, II, and Ill, with Concanavalin A in Patients II and III, and with pokeweed mitogen in Patient II. The PHA stimulation indices were 1.6 (Patient l), 3.3 (Patient II), and 4.0 (Patient III). Patient IV had a stimulation index in the range of noi:mat controls (4.1-14.4). At six weeks of age all mitogenic responses were within the normal range (PHA stimulation index ,'K = 23, range 14.0-30.7).
RESULTS All newborn infants were Coombs-positive and had hematologic evidence of hemolytic disease. Patient I developed group B streptococcal sepsis. Patients I and IV were transfused with fresh blood. In Patient I, a mild transient eosinophilia developed in the first week of life and atypical lymphocytes were noted at age three weeks. Patients II and III were transfused with irradiated (2,000 rads) blood. In Patient I cord IgM could not be detected. In Patients II, III, and IV and in all control blood sera IgM was detectable. Serum i m m u n o g l o b u l i n levels in all patients were normal at age two months. Lysozyme levels were normal. Antibodies to tetanus toxoid were detectable in the three patients following the first immunization. Repeated immunization with tetanus toxoid failed to elicit a booster effect in Patients I to III (Table I). In Patient I no neutralizing antibodies to poliomyelitis were detected after two doses of vaccine. Rosette formation of cord PBL was markedly dimin-
(% of total mononuclear cells),
DISCUSSION The mechanism by which promethazine ameliorates EBF is not yet elucidated. In experimental animal models it inhibits humoral i m m u n e responses, delayed type hypersensitivity, and macrophage activity? -~ No in vivo data on the effect of promethazine on the h u m a n fetal i m m u n e system are available. In vitro it interferes with the phagocytic capacity of normal h u m a n fetal macrophages? Consequently, its effectiveness in amelioration of EBF has been attributed to its ability to impair phagocytosis of fetal red blood cells opsonized by maternal anti-D antibodies. 8 Our studies indicate that high doses of promethazine result in a more global effect on fetal i m m u n e systems. In three newborn infants there was a marked diminution in
13 8
The Journal of Pediatrics July 1976
Rubinstein et al.
Table II. Serum antibodies to tetanus toxoid (hemagglutination test*)
I
Antibodytiters after immunization
Infant
I
Booster I
Booster II
I II III
0.16 IU 1.25 IU 0.8 IU
0.32 IU 0.65 IU 0.8 IU
0.31 IU nd nd
nd = Not done. *In 16 controls a titer above 0.5 IU was detected. Booster immunization resulted in at least four-fold titer increase. the number of circulating T cells and (Table I, Fig. 1) in PHA mitogenic responses. Spontaneous reconstitution of these perturbations required several weeks (Fig. 1). Promethazine had no immunosuppressive effect on newborn infants o f mothers who received a single dose as obstetric analgesia or in Patient IV who had only two weeks of exposure. This suggests a dose-related effect. No correlation between degree of neonatal hemolysis and T-cell suppression was noted. In fact, Patient IV with the severest degree of EBF had normal T-cell function. Alternative factors such as adrenal-mediated stress responses and excessive circulating breakdown products of erythrocytes may also cause T-cell suppression. However, serum from cord blood of Patient I failed to diminish T-cell rosettes of autologous and heterologous lymphocytes, suggesting an absence of suppressive serum factors. Hyperbilirubinemia similarly has been reported to decrease circulating T-cell count and to inhibit blast transformation of lymphocytes?. 10 This effect, however, was noted only with concentrations of indirect bilirubin in serum over 5 mg/dl, a level not reached in the cord blood of our patients. A failure to detect an adequate secondary i m m u n e response, as observed in Patients I, II, and III, is characteristic of T-cell dysfunction, H but may be related to hyperbilirubinemia. It has been reported in association with neonatal hyperbilirubinemia of more t h a n 15 m g / d1.12 In Patients I and II, concentrations of serum bilirubin exceeded this level and may have interferred with the humoral response to tetanus immunizations (Table II) beyond the neonatal period. O f additional therapeutic concern is the potential risk of graft-versus-host-reaction following transfusion of immunocompetent cells. The one patient with T-cell suppression who received nonirradiated blood transfu-
sions developed an eosinophilia and atypical lymphocytes suggestive of a mild graft-versus-host-reaction. 1~ W e r e c o m m e n d the use of irradiated blood for transfusion in promethazine-treated infants. All maternal-fetal pairs undergoing promethazine treatment should thus be carefully studied for short- and long-term evidence of immunoincompetence before final recommendations as to its role in the m a n a g e m e n t of Rh disease can be formulated. REFERENCES
1. Gudson JP, and Witherow C: Possible ameliorating effects of erythr0blastosis by promethazine hydrochloride, Am J Obstet Gynecol 117:1101, 1972. 2, De Chatelet LR, Qualliotine-Mann D, Caldwell R, McCall CE, and Gudson JP: Effects of promethazine-hydrochloride on human polymorphnuclear leukocytes, Infect Immun 7:403, 1973. 3. Myrvik QN, and Evans DG: Effects of bacillus catmette guerin on the metabolism of alveolar macrophages, Adv Exp Med Biol 1:203, 1967. 4. Lay WH, Mendes NF, Bianco C, and Nussenzweig V: Binding of sheep red blood cells to a large population of human lymphocytes, Nature 230:531, 1971. 5. Weiner MS, Bianco C, and Nussenzweig V: Enhanced binding of neuraminidase-treated sheep erythrocytes to human T lymphocytes, Blood 42:939, 1973. 6. Pellegrino MA, Ferrone S, Dierich MP, and Reisfeld RA: Enhancement of sheep red blood cell human lymphocyte rosette formation by the sulphydryl compound 2-amino ethylisothiouronium bromide, Clin Immunol Immunopathol 3:324, 1975. 7. Bianco C, Patrick R, and Nussenzweig V: A population of lymphocytes bearing a membrane recePtor for antigenantibody-complement complexes, J Exp Med 132:702, 1970. 8. Gudson JP, Iannuzzi NP, Witherow CC, and deChatelet LR: Modification of the human fetal phagocytic response by prom~ethazine hydrochloride, Am J Obstet Gynecol 119:543, 1974. 9. DeSanctis C, Malandra C, Zanetti P, Fabris L, and Ponzone A: Neonatal hyperbilirubinemia and response of lymplaocytes to phytohemagglutinin, Minerva Pediatr 20:2010, 1968. 10. Rola-Pleszcynski M, Hensen SA, Vincent MM, and Bellanti JA: Inhibitory effects of bilirubin on cellular immune responses in man, J PEDIATR86:690, 1975. 11. Mitchel, GF, Grumet FC, and McDevitt HO: Genetic control of the immune response, J Exp Med 135:126, 1972. 12. Nejedla Z: The development of immunological factors in infants with hyperbilirubinemia, Pediatrics 45:102, 1970. 13. Miller ME: Thymic dysplasia I. Graft-versus-host reaction following bone-marrow transfusion, J PEDIATR 70:730, 1967.
July 1976 TheJournalofPEDIATRICS
Possible effect of maternal promethazine therapy neonatal immunologic functions
on
The effect of the administration of promethazine in the treatment of erythroblastosis fetalis was studied in four maternal-fetal pairs. The three infants exposed for a prolonged period of time had decreased neonatal number and function of T cells, and abnormal specific humoral immune responses. The possible role of promethazine in the induction 9f fetal immunoincompetence is discussed.
Arye Rubinstein, M.D., Arthur h Eidelman,* M.D., Julian Melamed, B.Sc., M.B., B.Ch., Lawrence M. Gartner, M.D., Stephen Kandall, M.D., and Harold Schulman, M.D., Bronx, N. Y.
LARGE DOSES of promethazine, maternally adminis-
tered, have been utilized for amelioration of Rh erythroblastosis fetalis. 1 Animal studies indicate a suppressive effect on humoral and cellular immune responses. 2, ~ No data on the in vivo effects of antihistamines on the developing human fetal immunologic system have been reported. The current study reports the results of immunologic studies conducted on four Rh-positive infants born to sensitized Rh-negative mothers who were being treated with promethazine. PATIENTS
AND METHODS
Each of the newborn infants was Rh positive; their Rhnegative mothers had been sensitized to Rh antigen. Amniocentesis documented elevations of A OD 450 nm. Promethazine, 75 to 150 mg every day, had been administered to the mothers for five weeks (Patient I), six weeks (Patient II), 24 weeks (Patient III), and two weeks (Patient IV), respectively, prior to delivery. Thirteen normal term nonsensitized infants and 13 From the Departments of Cell Biology, Obstetrics, and Pediatrics, Hospital of the Albert Einstein College of Medicine, Bronx Municipal Hospital Center, Rose F. Kennedy Center for Research in Mental Retardation and Human Development, A lbert Einstein College of Medicine, Yeshiva University. Supported in part by grant A[-10702-04. *Reprint address: Hospital of the Albert Einstein College of Medicine, 1825 Eastchester Rd., Bronx, N. Y~ 10461.
Vol. 89, No. 1, pp. 136-138
adult volunteers were studied. Mothers of six of the control newborn infants received a single dose of 25 mg of promethazine as obstetrical analgesia. Serum immunoglobulins were measured by radial immunodiffusion and serum lysozyme (muramidase) by the Lyso-Plate assay. Antibodies to tetanus toxoid were measured by the hemagglutination method in Infants i, II, and III. Tetanus toxoid was administered after six weeks and boosters were given at four-week intervals. Abbreviations used EBF: erythrob!astosis fetalis PBL: peripheral blood lymphocytes SRBC: sheep red blood cells PHA: phytohemagglutinin AET: 2-amino ethylisothiouronium Neutralizing antibodies to poliomyelitis were measured in the infants after two doses of oral trivalent polio vaccine at age eight weeks and 16 weeks, respectively. Peripheral blood lymphocytes were isolated by sedimentation of heparinized cord blood in a syringe. E-rosetting for detection of T lymphocytes was performed using (1) untreated sheep red blood cellsr (2) neuraminidase-treated SRBC) and (3) sulfhydryl compound 2amino ethylisothiouronium bromide SRBC (AET). 6 The results (Table I, Fig. 1) indicate the percentage of AETrosettes, read by means of a flourescent microscope after staining in suspension with euchrysin. At age one year,
Volume 89 Number 1
Effect o f maternal prornethazine therapy
T a b l e I. Rosette formation by peripheral blood lympho-
cytes
70E
(%) I
(%)
I6
9 8 31 Range 67-80 x- 71.6 Range 31-53 x - 39
ADULT T ~ CONTROLS
~ . O
EAC:~
Patients' cord blood ll III IV Adult controls* (n = 13) Cord blood controls* (n = 13)
13 7
24 12 14 22 9-17 13.2 8-19 14
50
0 ,,', 30 10
A " "................ / z x NORMAL / .." / CORD Z."" //. BLOOD ~..."" //. ~~......" /././" ........ PATIENT T ~/"" _..,...~'/" - - - PATIENT ]Z M "'f'" ~'''''PATIENT IK o' [ 1
[ 3
I 5
1 7
AGE (Weeks)
E = Sheep erythrocytes; EACa = sheep erythrocytes coated with Forssman rabbit anti-sheeperythrocyteserum (A) and complementfrom CF~ mice. *Performed simultaneouslywith the patient's cord blood lymphocytes.
Fig. L E-Rosette formation by peripheral blood lymphocytes
PBL from Patient i was tested for E-rosetting inhibition with autologous cord blood serum. Rosetting for the quantification of B-lymphocytes with EACa (EAC1423) was performed/ using SRBC coupled with rabbit antiForssman antiserum and with complement components from C~ mouse serum. Blast-transformation of lymphocytes was measured in vitro by the incorporation of tritiated thymidine after stimulation with phytohemagglutinh~ (0.005% and 0.01%), pokweed mitogen (0.005% and 0.01%) and Concanavalin A (5-10 mg/ml).
ished at birth in all patients except Patient IV (Table I). In Patients I and III the rosettable T-lymphocytes population reached the normal adult range within six weeks. In Patient II the percentage of E-rosettes at eight weeks was still below the adult range. The percentage of EAC3 rosettes was normal in all patients, both in the cord blood and on follow-up at six weeks of age. Blast transformation of cord blood tymphocytes compared to control cord blood was reduced after stimulation; with phytohemagglutinin in Patients I, II, and Ill, with Concanavalin A in Patients II and III, and with pokeweed mitogen in Patient II. The PHA stimulation indices were 1.6 (Patient l), 3.3 (Patient II), and 4.0 (Patient III). Patient IV had a stimulation index in the range of noi:mat controls (4.1-14.4). At six weeks of age all mitogenic responses were within the normal range (PHA stimulation index ,'K = 23, range 14.0-30.7).
RESULTS All newborn infants were Coombs-positive and had hematologic evidence of hemolytic disease. Patient I developed group B streptococcal sepsis. Patients I and IV were transfused with fresh blood. In Patient I, a mild transient eosinophilia developed in the first week of life and atypical lymphocytes were noted at age three weeks. Patients II and III were transfused with irradiated (2,000 rads) blood. In Patient I cord IgM could not be detected. In Patients II, III, and IV and in all control blood sera IgM was detectable. Serum i m m u n o g l o b u l i n levels in all patients were normal at age two months. Lysozyme levels were normal. Antibodies to tetanus toxoid were detectable in the three patients following the first immunization. Repeated immunization with tetanus toxoid failed to elicit a booster effect in Patients I to III (Table I). In Patient I no neutralizing antibodies to poliomyelitis were detected after two doses of vaccine. Rosette formation of cord PBL was markedly dimin-
(% of total mononuclear cells),
DISCUSSION The mechanism by which promethazine ameliorates EBF is not yet elucidated. In experimental animal models it inhibits humoral i m m u n e responses, delayed type hypersensitivity, and macrophage activity? -~ No in vivo data on the effect of promethazine on the h u m a n fetal i m m u n e system are available. In vitro it interferes with the phagocytic capacity of normal h u m a n fetal macrophages? Consequently, its effectiveness in amelioration of EBF has been attributed to its ability to impair phagocytosis of fetal red blood cells opsonized by maternal anti-D antibodies. 8 Our studies indicate that high doses of promethazine result in a more global effect on fetal i m m u n e systems. In three newborn infants there was a marked diminution in
13 8
The Journal of Pediatrics July 1976
Rubinstein et al.
Table II. Serum antibodies to tetanus toxoid (hemagglutination test*)
I
Antibodytiters after immunization
Infant
I
Booster I
Booster II
I II III
0.16 IU 1.25 IU 0.8 IU
0.32 IU 0.65 IU 0.8 IU
0.31 IU nd nd
nd = Not done. *In 16 controls a titer above 0.5 IU was detected. Booster immunization resulted in at least four-fold titer increase. the number of circulating T cells and (Table I, Fig. 1) in PHA mitogenic responses. Spontaneous reconstitution of these perturbations required several weeks (Fig. 1). Promethazine had no immunosuppressive effect on newborn infants o f mothers who received a single dose as obstetric analgesia or in Patient IV who had only two weeks of exposure. This suggests a dose-related effect. No correlation between degree of neonatal hemolysis and T-cell suppression was noted. In fact, Patient IV with the severest degree of EBF had normal T-cell function. Alternative factors such as adrenal-mediated stress responses and excessive circulating breakdown products of erythrocytes may also cause T-cell suppression. However, serum from cord blood of Patient I failed to diminish T-cell rosettes of autologous and heterologous lymphocytes, suggesting an absence of suppressive serum factors. Hyperbilirubinemia similarly has been reported to decrease circulating T-cell count and to inhibit blast transformation of lymphocytes?. 10 This effect, however, was noted only with concentrations of indirect bilirubin in serum over 5 mg/dl, a level not reached in the cord blood of our patients. A failure to detect an adequate secondary i m m u n e response, as observed in Patients I, II, and III, is characteristic of T-cell dysfunction, H but may be related to hyperbilirubinemia. It has been reported in association with neonatal hyperbilirubinemia of more t h a n 15 m g / d1.12 In Patients I and II, concentrations of serum bilirubin exceeded this level and may have interferred with the humoral response to tetanus immunizations (Table II) beyond the neonatal period. O f additional therapeutic concern is the potential risk of graft-versus-host-reaction following transfusion of immunocompetent cells. The one patient with T-cell suppression who received nonirradiated blood transfu-
sions developed an eosinophilia and atypical lymphocytes suggestive of a mild graft-versus-host-reaction. 1~ W e r e c o m m e n d the use of irradiated blood for transfusion in promethazine-treated infants. All maternal-fetal pairs undergoing promethazine treatment should thus be carefully studied for short- and long-term evidence of immunoincompetence before final recommendations as to its role in the m a n a g e m e n t of Rh disease can be formulated. REFERENCES
1. Gudson JP, and Witherow C: Possible ameliorating effects of erythr0blastosis by promethazine hydrochloride, Am J Obstet Gynecol 117:1101, 1972. 2, De Chatelet LR, Qualliotine-Mann D, Caldwell R, McCall CE, and Gudson JP: Effects of promethazine-hydrochloride on human polymorphnuclear leukocytes, Infect Immun 7:403, 1973. 3. Myrvik QN, and Evans DG: Effects of bacillus catmette guerin on the metabolism of alveolar macrophages, Adv Exp Med Biol 1:203, 1967. 4. Lay WH, Mendes NF, Bianco C, and Nussenzweig V: Binding of sheep red blood cells to a large population of human lymphocytes, Nature 230:531, 1971. 5. Weiner MS, Bianco C, and Nussenzweig V: Enhanced binding of neuraminidase-treated sheep erythrocytes to human T lymphocytes, Blood 42:939, 1973. 6. Pellegrino MA, Ferrone S, Dierich MP, and Reisfeld RA: Enhancement of sheep red blood cell human lymphocyte rosette formation by the sulphydryl compound 2-amino ethylisothiouronium bromide, Clin Immunol Immunopathol 3:324, 1975. 7. Bianco C, Patrick R, and Nussenzweig V: A population of lymphocytes bearing a membrane recePtor for antigenantibody-complement complexes, J Exp Med 132:702, 1970. 8. Gudson JP, Iannuzzi NP, Witherow CC, and deChatelet LR: Modification of the human fetal phagocytic response by prom~ethazine hydrochloride, Am J Obstet Gynecol 119:543, 1974. 9. DeSanctis C, Malandra C, Zanetti P, Fabris L, and Ponzone A: Neonatal hyperbilirubinemia and response of lymplaocytes to phytohemagglutinin, Minerva Pediatr 20:2010, 1968. 10. Rola-Pleszcynski M, Hensen SA, Vincent MM, and Bellanti JA: Inhibitory effects of bilirubin on cellular immune responses in man, J PEDIATR86:690, 1975. 11. Mitchel, GF, Grumet FC, and McDevitt HO: Genetic control of the immune response, J Exp Med 135:126, 1972. 12. Nejedla Z: The development of immunological factors in infants with hyperbilirubinemia, Pediatrics 45:102, 1970. 13. Miller ME: Thymic dysplasia I. Graft-versus-host reaction following bone-marrow transfusion, J PEDIATR 70:730, 1967.