The inhibition of polymorphonuclear and lymphocyte-mediated fetal red blood cell lysis by promethazine in vitro JOHN
P. GUSDON,
GLENN RODNEY Winston-Salem,
A.
HERBST,
M.A.
MARRIOTT, North
M.D.
JR.,
B.A.
Carolina
in vitro method for the measurement of fetal red blood cell destWtiOn by Fc receptor-bearing poiymorphonuclear leukocytes and lymphocytes has been developed. Cord blood was collected at the time of delivery from infants who were Rh-D positive and compatible with their mother, as well as infants who were Rh-D positive and incompatible with their Rh-D-sensitized mothers. Red blood cells, lymphocytes, and PdyrnWphonUclear preparations were individually separated from each cord Mood sample. Rh-D-positive cord red blood cells from compatible and incompatible pregnancies complicated by erythroblastosis were coated with anti-Rh-D. All target red blood cells, opsonized and not opsonized, were labeled with chromium-51 and cultured with autologws lymphocytes or polymorphonuclear leukocytes at an 6ffector:red blood ceil ratio of 10: 1. Cultures containing either no promethazine hydrochloride or varying amounts of the drug were studied. Antibody alone did not cause red cell lysis. Both lymphocytes and polyrnorphonuclear leukocytes were effective in causing the lysis of red blood ceils bearing antibody. Lysis of red blood calls by polyrnorphonuclear leukocytes or lymphocytes was inhibited by promethazine hydrochloride. Excessive amounts of prom&ha&e hydmchkxide alone cause lysis of the red cells. At the low doses utilized for inhiiitkm in this experiment, the action of promethazine hydrochloride on the lymphocytes was found to be reversible. Promethazine is believed to have exhibited two different types of actii in this study: (1) It inhibited antibody-dependent, cell-mediated lysis of autologous red blood c&s coated with antibody; (2) it stabilized the red blood cell membrane so that even spontaneous lysis of red blood cells was decreased 50 per cent. (AM. J. OBSTET. GYNECOL. 130: 391, 1978.)
An
MECHANISMS of fetal red blood cell destruction in vivo in erythroblastosis are not absolutely known. In vitro studies done in our laboratory showed that specific antibody to the Rh-D antigen and complement does not result in significant fetal red blood cell lysis.’ In both that and a later study, we demonstrated that fetal and neonatally obtained peritoneal macrophages
THE
From the Dgatiment of Obstetlics Bowman Gray School of Medicine IJniverrity.
and Gynecology, of Wakt Forest
Supported in part by Wyeth Labs., Philadelphia, Pennsylvania. Presented at the Twenty-jouvth Annual Meeting of the Society for Gynecologic investigation, Tucson, Ariwna, March 23-25, 1977. Reprint requests: Dr. John P. Gus&n, Jr., Department of Obstetrics and Gynecology, Bowvn~n Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103. 0002-9378/78/04130-0391$00.70/O
@ 1978
The
C. V. Mosby
Co.
were capable of binding fetal Rh-D positive red blood cells which were opsonized with anti-D.2 These data suggested that fetal red blood cell destruction could be mediated in vivo by fetal macrophages. In neither of these studies, however, did we actually document red blood cell phagocytosis or destruction. Jandl and Tomlinson,3 however, had shown earlier that destruction of red cells which were opsonized by nonhemolytic antibody is accomplished by cells of the reticuloendothelial system, particularly those of the spleen and liver. Rosenau,* using time-lapse cinematography, demonstrated that lymphoid cells were able to lyse in vitro, without complement-specific cells against which the mice had been sensitized in vivo. Chicken erythrocytes had been shown to be an excellent target for lymphoid cells from donors sensitized to that antigem Based on that earlier work, Holm and Hammarstroms labeled human red blood cells with “‘Cr to study the lysis of 391
392
Gusdon, Herb+ and Marriott
February 15; 1978 Am. j. Obstet. Gynecoi.
Table I. Protocol used in determining the effect of fetal effector cells (polymorphonuclear leukocytes or lymphocytes) on opsonized red blood cell destruction =Cr-FRBC* ( + I - opxmization) 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18.
+ + + + +
+
Fetal effector cells
+ +
+ + + -
5 + + -
*%r-treated
Promethuzine hydrochloride (pglml.)
5 5 5 5
10 10 10 10 0 (distilled water) 0 (distilled water) fetal red blood cells.
antibody-coated red cells by macrophages and leukocytes.s However, although those cells were capable of destroying the opsonized red blood cells, peripheral lymphocytes were not. In our earlier studies of fetal macrophages, we were able to demonstrate resetting of the opsonized red blood cell-bound fetal macrophages. However, seemingly in accord with the data of Holm and Hammarstrom, we were not able to demonstrate rosetting by fetal peripheral lymphocyte preparations.‘, 2 In this study, we documented effective opsonized red blood cell destruction by fetal peripheral lymphocytes and polymorphonuclear leukocytes. It is clear that erythroblastosis fetalis is an immunologically mediated disease process. Unless a maternal antibody to a fetal red blood cell antigen has been shown to have developed, the disease does not occur. Yet, because in vitro studies with the use of anti-D aIone did not result in red blood cell destruction, it seemed that the actual de$truction must be cellmediated.’ These studies, therefore, have been undertaken in an effort to document the role of antibody and some of the types of cells which might be involved. In addition, it was thought that the possible protective and ameliorating role of promethazine hydrochloride in the treatment of affected patients might be more effectively elucidated. During the course of this study, data that documented the presence of Fc receptors on polymorphonuclear cells were published.’ We believed, therefore, that it might be possible for the fetal polymorphonuclear leukocytes to bind and to destroy op-
sonized done.
red blood
cells, and appropriate
studies were
Material and methods Fifty to one hundred milliliters of cord blood was collected at the time of delivery and anti-coagulated with 2 U. of sodium heparin per milliliter. Aliquots of 25 ml. (or one half of the amount collected) of blood were used for lymphocyte separation, and 25 ml. was used for the polymorphonuclear leukocyte isolation. Lymphocyte separation was accomplished by methods previously described,s with only slight modification. Preparation of lymphocytes. Five hundred milligrams of carbonyl iron powder was added to 25 ml. of heparinized cord blood that was then incubated in a 37” C. water bath for 30 minutes with swirling at 10 minute intervals. At the end of the incubation, 15 ml. of Roswell Park Memorial Institute (RPMI) 164OP (Gibco) was added and the suspension was placed on a large magnet. After the iron-laden phagocytes were withdrawn from the suspension, the blood was poured into four test tubes, underlayered with 5 ml. of Ficol Hypaque (specific gravity 1.076), and then centrifuged at 300 g for 20 minutes. Lymphocytes were collected from the Ficol Hypaque-serum interface, washed three times in RPM1 1640f media, counted, and resuspended at an appropriate concentration for use in the experiment. Polymorphonuclear leukocyte preparation. The other 25 ml. aliquot of cord blood was used for isolation of the polymorphonuclear leukocytes. Eight milliliters of plasma gel+ was added to 25 ml. of blood, and the suspension was mixed, poured into a conical centrifuge tube, and then allowed to settle for one hour. The straw-colored supernatant solution was removed and centrifuged at 500 g for 10 minutes. After one washing with RPM1 1640f, the cells were resuspended in media to a 12 C.C.volume, and 4 ml. was poured into each of three test tubes. The tubes were then underlayered with 3 ml. of Ficol Hypaque and centrifuged for 30 minutes at 350 g. Lymphocyte-free granulocytes were found in a pellet at the bottom of the test tube along with contaminating red blood cells. After careful removal of the pellet, it was washed once with phosphate-buffered saline and the red cells were lysed by resuspending the pellet in 6 ml. of distilled water for 20 seconds followed by the immediate addition of 2 ml. of 3.5 per cent sodium *RF’MI 1640f is RPM1 1640 media enriched with 20 per cent fetal calf serum and penicillin and streptomycin. tRoger BeIIon Laboratories, Neulley, France. Distributed by the HTI Corporation, Buffalo, New York.
Volume Number
Inhibition
130 4
chloride. After one more wash in phosphate-buffered saline, the cells were counted and resuspended in RPM1 164Of. Differential counts were performed on the final lymphocyte and polymorphonuclear leukocyte preparations with the use of cytocentrifuge smears, stained with Wright’s stain. Lymphocyte preparations were found to contain approximately 95 per cent lymphocytes. Polymorphonuclear leukocytes were usually 95 with approximately 5 per cent per cent pure, eosinophil contamination. The cell preparations used were more than 9.5 per cent viable as determined by trypan blue dye exclusion. Red blood cell preparations. Red blood cells were obtained from the pellet below the lymphocytes in the lymphocyte-extraction procedure. The red blood cells were removed, washed three times in RPM1 1640f, and counted. The washed cells were opsonized and labeled with %r* simultaneously as follows: To two tubes each containing 4 x lo6 fetal red blood cells, 200 &i of Wr was added. To tube No. 1, 0.2 ml. of anti-Rh-Df was added. In specific instances, twice as much antibody was used to opsonize the fetal red blood cells. In some instances, when the fetal red blood cells were already opsonized by maternal antibody as a result of erythroblastosis, no additional antibody was added; in others, more antibody was added. Cytotoxicity assay. Lymphocytes or polymorphonuclear leukocytes and red blood cells were placed into sterile, screw-cap, glass, disposable test tubes. A ratio of 10 effector cells to one target red blood cell was used in all experiments. Promethazine hydrochIoride$ was placed into solution containing RPM1 1640f. In those experiments in which promethazine was used, it was added directly to the effector cell-red blood cell suspension. A volume of 2 c. c. was maintained in all tubes. The tubes were incubated at 37°C. in an atmosphere of air with 5 per cent carbon dioxide for 18 to 24 hours. A typical protocol is outlined in Table I. Fig. 1 is a composite of the different components of these experiments. At the end of the incubation all tubes were centrifuged at 100 g for 10 minutes; the supernatant solutions were removed, and the pellet and supernatant solutions were counted separately. There was variability in the number of lymphocytes and polymorphonuclear leukocytes we were able to harvest from each delivery. Although some deliveries *Amersham/Searle Corp., tBlood type reagent, Ortho Jersey. $Phenergan was supplied Pennsylvania, as a crystalline, 39311, B7040, and B5610).
Arlington Heights, Illinois. Diagnostics, Inc., Raritan,
New
by Wyeth Labs., Philadelphia, pure powder in 5 Cm. lots (Lots
Fig.
of fetal
1. A standard
red blood
number
cell lysis by promethazine
of S’Cr-labeletl
red
blood
present in every tube. Experiments were performed use of anti-Rh-D
in half
of the tubes
to opsonize
393
cells is
with the the $‘C:r-
labeled cells. The effector cells were either t&al lymphocytes or polymorphonuclear leukocytes, and either no promethazine hydrochloride or varying amctunts of promethazine hydrochloride were used in cliff&-em experiments. The starred cells indicate the “‘O-labeled t>psonized red blood cells.
yielded a large amount of-cord blood, others yielded far less. Therefore, there was variability in the number of effector cells used in individual experiments. This fact, coupled with the differences in the uptake of %r by the fetal red blood cells in different experiments, prevents the comparison of absolute valrles obtained in individual experiments. However, since the effectorto-target ratio of 10: 1 was kept constant in all experiments and control studies were maintained within each experiment, results were calculated 01i a percentage basis. Maximum “‘Cr release for each red blood cell preparation was determined by distilled water Ivsis. Spontaneous “‘0 release was deterfnined by the
394
Gusdon, Herb&, and Marriott
Table
II. In vitro protection
7
of opsonized
red blood cells from destruction Inhibition
Fetal
No. of experiments 17
February 1.5, 1978 Am. J. Obstet. Gynecol.
ORBC* destruction without pomethazine hydrochloride (70)
effector cell
lymphocyte Polymorphonuclear
34.6 2 12 32.1 t 6
I pgtlml.
30 213 22.2 t 12
by fetal effector ofORLK*
cells
Destruction
(%) (G.E.M.)
5 ~gtl??d.
10 PgLgtlml.
36 2 10.1 32.4 -+ 8.2
42.3 2 8.2 36 tf- 10.2
*Opsonized red blood cell. tOf promethazine hydrochloride. Table blood
III. In vitro protection of opsonized of patients with erythroblastosis
red blood
cells from destruction
by effector
Inhibition Patient
seuerity of disease
Trpe of effectvr cell
ORBC destruction without prometharine hydrochloride (70)
L. A. B.
Very mild Moderate
L L
34.2 29.0
K.
*Of promethazine
cells from cord
of ORBC
destruction
1 pg*lml.
5 pg*lml.
30.8
32.1 58.6
(%) 10 pg*lml.
42.3 72.8
hydrochloride.
amount of 51Cr released by labeled red blood cells to which no fetal effector cells had been added. In addition, the effect of promethazine hydrochloride at concentrations of 1, 5, and 10 +g per milliliter was studied. Therefore, specific lysis was calculated according to the formula: % Specific lysis = fetal effector cell-mediated %r release spontaneous %r release maximum “‘Cr release - spontaneous %r release
x 100.
The amount of spontaneous release varied between 6 and 12 per cent in all experiments. The inhibition by promethazine hydrochloride of opsonized red blood cell destruction (per cent protection) by fetal effector cells was determined in the following manner: The destruction (per cent release) by the effector cells was measured by using either no promethazine hydrochloride or different concentrations of the drug within each individual experiment. The per cent of specific lysis obtained in the presence of promethazine hydrochloride was subtracted from the per cent of specific lysis obtained in duplicate tubes in which promethazine hydrochloride was not present. This difference was then divided by the total specific lysis obtained in the absence of the drug and multiplied by 100. The reversibility of the effect of promethazine hydrochloride was studied in experiments by adding promethazine hydrochloride (10 wg per milliliter) to cultures and then washing it out after two hours of incubation and replacing it with normal RPM1 1640f. R@NJJtS
The degree of specific lysis varied between experiments. We have done several preliminary experiments
in which the red blood cells were opsonized with varying amounts of antibody. These data indicate that there is a marked increase in the destruction of the more heavily opsonized fetal red blood cells. One case of particular significance was in a very mildly affected erythroblastotic pregnancy. In this case the child was minimally affected and the indirect Coombs test was only 1+ at the time of delivery at 38 weeks. These cells, which were utilized without any additional antibody, yielded only 8 per cent specific destruction. The same red blood cells that were opsonized by adding a standard amount of antibody yielded 44 per cent specific destruction by the same number of fetal effector cells. In other cases similar results were attained. An interesting finding was that the use of anti-D resulted in two to three times more absorption of %r by the fetal red blood cells than by the red cells to which no anitbody was added. However, in each situation, the amount of spontaneous lysis during the 18 hour course of incubation was the same. Red blood cells to which anti-D was added spontaneously lysed 10.2 2 2.1 per cent (S.E.M.). Red blood cells from the same cord blood, to which an equal amount of %r had been added but which were not opsonized, lysed 9.3 c 3.1 per cent (S.E.M.) during the incubation period. In 17 experiments with lymphocytes, spontaneous lysis varied between 6 and 19.5 per cent. The mean spontaneous release without promethazine hydrochloride was 10.2 per cent, decreasing to 5.1 per cent with promethazine hydrochloride, which suggested a stabilization of membranes. In Table II data are presented that deal with the in vitro protection of opsonized red blood cells from destruction by fetal effector cells with the use of different concentrations of promethazine hydrochloride. The
Volume
130
Number
I
Inhibition
of fetal red blood
cell lysis
by promethazine
395
numbers of lymphocyte experiments exceed those done with polymorphonuclear leukocytes. However, significant destruction of opsonized red blood cells has been obtained with both types of fetal effector cells in vitro. Promethazine hydrochloride at these concentrations has inhibited the amount of opsonized red blood cell destruction. We found, however, that by increasing the concentration of promethazine hydrochloride to 100 pg per milliliter we obtained almost complete red blood cell destruction. As presented in Table III, the same degree of opsonized red blood cell destruction and its inhibition is seen in patients with pre-existing erythroblastosis. In experiments done to determine the reversibility of the effects of promethazine hydrochloride on lymphocytes, we found that the degree of opsonized red blood cell destruction was the same after removal of the promethazine hydrochloride as that in the control, which was never exposed to the drug. In order to verify visually the presence of Fc receptors on polymorphonuclear leukocytes, a fetal polymorphonuclear leukocyte and opsonized red blood cell preparation was made in the same manner as if a T-lymphocyte rosette test were being done.’ Fig. 2 represents photographic evidence of a fetal polymorphonuclear leukocyte bearing Fc receptors that bind the fetal red blood cells.
Comment Antibody-dependent, cell-mediated cytotoxicity represents an in vitro model of the effector arm of cellmediated immunity. This cytotoxicity required immunoglobulin G antibodies directed against membrane antigens of the target cells and is expressed only if the antibodies have intact Fc regions which react with Fc receptors on the effector cell surface. In most of the studies on animal and human systems, the antibodydependent effector cell has been identified as a lymphoid cell, which is distinct from B and T cells and which has been tentatively called the K ce11.9-12 Paraskevas and associatesr3 were able to show that approximately 25 per cent of the T cells in the spleen were capable of taking up cytophilic complexes whether these complexes were formed in vivo, i.e., from serum six hours after immunization, or in vitro. The same number of T cells in the spleen were shown to become immunoglobulin positive within six hours after immunization.‘“. I5 Their data also pointed out that the Fc fragment was of significant importance for the uptake of complexes in vitro. Other workersi6-*’ have documented the presence of Fc receptors on the subpopulation of normal nonactivated T cells by a variety of techniques. This kind of information is of par-
Fig. 2. Shows an isolated polymorphonuc-leak. opsonized red blood cells resetting around
Iwkocvte
with
ir
titular importance in documenting that K. T, and K lymphocytes have Fc receptors and call be involved in cell-mediated immune lysis. Because of the recent reports dealing with the presence of Fc receptors on polymorphonuclear leukocytes, we began to isolate polymorphonuclear leukocytes from the cord blood as well as the lymphocytes. In this study, both of these cell types were used and have been shown to cause a significant amount of antibody dependent, cellmediated destruction of the opsonized red blood cells by these fetal effector cells in vitro. At levels of 1,5. and 10 pg per milliliter of promethazine hydrochloride, significant inhibition of this lysis was seen. This effect was reversible and, therefore, not due to permanent cellular impairment. It is at these low levels that the clinical application of prometha/ine hydrochloride might be considered. In some of our earlier studies, we used concentrations of promethazine hvdrochloride of up to 150 to 200 pg per milliliter. However. we found that fetal red blood cells are lysed b!; (irug levels in excess of 100 pg per milliliter.
396
Gusdon, Herbst, and Marriott
February 15, 1978 Am. J. Obstet. Gynecol.
It has been shown previously that the phenothiazines protect erythrocytes from hypotonic hemolysiszO and retard the rate of hemolysis.21-23 Seem&n and Weinsteinz4 extended these observations with a number of tranquilizers and were able to show ihat in the presence of low concentrations of many of the phenothiazines human erythrocytes are protected or stabilized against hypotonic and mechanical hemolysis. They showed that erythrocyte stabilization is long lasting but that the drug may be removed from the membranes by washing the red cells, which then returned to their former state of fragility. They suggest that this decrease in osmotic fragility might be explained by an expansion of the cell membranes. The data that have been developed in these experiments, depending upon the release of %r, suggest that indeed the spontaneous destruction of red blood cells that would occur during an 18 hour incubation period is decreased by approximately 50 per cent in the presence of promethazine hydrochloride. A suggestion as to another potential use of this membrane-stabilizing property of promethazine hydrochloride was recently made.25T 26 It was suggested, since lysis of fetal red blood cells was a significant problem during early fetoscopy, that the use of promethazine hydrochloride in the mother prior to the attempt to obtain fetal red blood cells by fetoscopy might stabilize the fetal red blood cell membranes sufficiently to ameliorate this problem.
The present study, therefore, suggests that one of the mechanisms of destruction of fetal red blood cells in vivo may be a function of fetal lymphocytes and polymorphonuclear leukocytes and Fc receptors. The data suggest that these effects may be inhibited by promethazine hydrochloride. The clinical use of this compound during pregnancy for the purpose of ameliorating the effects of erythroblastosis was the subject of an earlier preliminary report.27 We have continued to use this compound for that purpose, and our clinical experience with these affected patients will be the subject of a later report. Clinical studies are also underway at other institutions. Although promethazine hydrochloride is not a panacea, if administration is begun early and it is given in adequate amounts, we have found that frequently we able to avoid the use of intrauterine transfusions and allow more time in utero for maturation of the baby prior to delivery. Some of the basic reasons for this clinical effectiveness have been described in this paper. A most interesting recent publication@ also described a significant increase in the production of glucuronyl transferase production induced by promethazine hydrochloride. This, too, may play a role in the amelioration of the disease process after the birth of the affected
REFERENCES
1.
Gusdon, J. P., Jr., Iannuzzi, N. P., Witherow, C. C., and DeChatelet, L. R.: Modification of the human fetal phagocytic response by promethazine hydrochloride, AM. J. OBSTET.
2.
GYNECOL.
543,
GYNECOL.
125: 224,
1976.
Jandl, J. H., and Tomlinson, A. S.: The destruction of red cells by antibodies in man. II. Psycogenic, leukocytic and dermal responses to immune hemolysis, J. Clin. Invest. 37: 1202,
9.
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Gusdon, J. P., Caudle, M. R., Herbst, G. A., and Iannuzzi, N. P.: Phagocytosis and erythroblastosis. I. Modification of the neonatal response by promethazine hydrochloride, AM. J. OBSTET.
3.
119:
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Rosenau, W.: Interaction of lymphoid cells with target cells in tissue culture, in Amos, B., and Koprowski, H., editors: Cell-bound Antibodies, Philadelphia, 1963, The Wistar Institute of Anatomy and Biology, pp. 75-80. 5. Perlmann, P., and Helm, G.: Cytotoxic effects of Iymphoid cells in vitro, Adv. Immuiol. 11: 117, 1969. ’
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6. Holm. G.. and Hammarstrom. S.: Haemolvtic activitv of human blood monocytes. Lyiis of human erythrocytes treated with anti-A serum, Clin. Exp. Immunol. 13: 29, 1973. 7. Rabellino, E. M., and Metcalf, D.: Receptors for C3 and IgG on macrophage neutrophil and eosinophil colony cells grown in vitro, J. Immunol. 115: 688, 1975. 8. Aiuti, F., Cerottini, J. E., et al.: International Union of Immunological Societies Report, July, 1974. Identifica-
10.
child.
tion, enumeration and isolation of B and T lymphocytes from peripheral blood, Clin. Immunol. Immunopathol. 3: 584, 1975. Perhnann, P., Perlmann, H., and Wigzell, H.: Lymphocyte mediated cytotoxicity in vitro. Induction and inhibition by humoral antibody and nature of effector cells, Transplant. Rev. 13: 91, i972. Trinchieri. G.. DeMarchi. M.. Mavr. W.. Savi. M.. and I
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Ceppelini, R.: Lymphocyte aniibody 1ymphocystoly;ic interaction (LALI) with special emphasis on HL-A, Transplant. Pro;. 5: 1631, 19^73. * 11. Harding. B.. Pudifin. D. 1.. Got& F.. and MacLennan. I. C. M.:“Cytbtoxic lymp&cytes from rats depleted ‘of thymus processed cells, Nature (New Biol.) 232: 80, 197 1. 12. Van Boxel, J. A., Stobo, J. D., Paul, W. E., and Green, I.: Antibody-dependent lymphoid cell-mediated cytotoxicitv: No requirement for thvmus-derived lymphocytes, I. I Stence 175: 194, 1972. ’ 13. Paraskevas. F.. Lee. S. T.. and Orr. K. B.: The function of T cells car&&g re&pto& for co&plexes of Ig and antigens, Immunol. Commun. 5: 501, 1976. 14. Paraskevas, F., Orr, K. B., and Lee, S. T.: Cell surfaceassociated gamma globulins in lymphocytes. III. Changes of y globulin-carrying lymphocytes during primary response, J. Immunol. 109: 1254, 1972. 15. Lee, S. T., and Paraskevas, F. J.: Cell surface-associated gamma globulins in lymphocytes. III. Lack of detection of surface y globulin on B-cells and acquisition of surface
Volume Number
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21.
Inhibition of fetal red blood cell lysis by promethazine
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y G globulin by T cells during primary response, J. Immu&l. 109: 1262, 1972. - _ Soteriades-Vlachos, G.. Gvonavossv. M. I. C.. and Playfair, J. H. L.: Rosette form:tion by mouse lymphocytes. III. Receptors for immunoglobulin on normal and activated T cells, Clin. Exp. Immunol. 18: 187, 1974. Stout, R. D.. and Herzenhere. L. A.: The Fc receutor on thymus-derived lymphocyte: I. Detection of a subpopulation of murine T lymphocytes bearing the Fc receptor, J. Exp. Med. 142: 611, 1975. Basten, A., Miller, J. F. A. P., Warner, N. L., Abraham, R., Chia, E., and Gamble, J.: A subpopulation of T cells bearing Fc receptors, J. Immunol. 115: 1159, 1975. Anderson, C. L., and Grey, H. M.: Receptors for aggregated IgG on mouse lymphocytes. Their presence on thymocytes, thymus-derived, and bone marrow derived lymphocytes, J. Exp. Med. 139: 1175, 1974. Chaplin, H., Jr., Crawford, H., Cutbush, M., and Mollison, P. L. The effects of phenothiazine derivative (RP. 3300) on red cell preservation, J. Clin. Pathol. 5: 91, 1952. Freeman, A. R.. and Spirtes, M. A.: Effect of some phenothiazine derivatives on the hemolysis of red blood cells in vitro, Biochem. Pharmacol. 11: 161, 1962.
22. Freeman,
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27.
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A. R., and Spines. M. A.: Effects of chlorpromazine on biological membranes. II. Chlorpromazine-induced changes in human erythrocytes, B&hem. Pharmacol. 12: 47, 1963. Freeman, A. R., and Spirtes, M. A.: Further effects of chlorpromazine on the human erythrocyte membrane, Biochem. Pharmacol. 12: 1235, 1962. Seeman, P.. and Weinstein, J. L.: Erythrocyte membrane stabilization by tranquilizers and antihistamines, Biothem. Pharmacol. 15: 1737, 1966. Naftolin, F.: Personal communication. Gusdon, J. P., Jr., and Herbst, G. A.: ‘The inhibition of PMN and lymphocyte-mediated lysis by promethazine in an in vitro erythroblastosis model, Gynrcol. Invest, S: 9, 1977. Gusdon, J. P., Jr., and Witherow. C. C.: Possible ameliorating effects of erythroblastosis by promethazine hydrochloride, AM. J. OBSTET. GYNFCOL. 117: 1101, 1973. Vaisman, S. I., Lee, K. S., and Gardner, L. M.: The effect of promethazine hydrochloride on bilirubin metabolism in the rat, Pediatr. Res. 10: 788, 1976.
IntonnrRtasr for authors Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following statement, signed by each author: “The undersigned author(s) transfers all copyright ownership of the manuscript entitled (title of article) to The C. V. Mosby Company in the event the work is published. The author(s) warrants that the article is original, is not under consideration by another journal, and has not been previously published.” Authors will be consulted, when possible, regarding republication of their material.
P. GUSDON,
GLENN RODNEY Winston-Salem,
A.
HERBST,
M.A.
MARRIOTT, North
M.D.
JR.,
B.A.
Carolina
in vitro method for the measurement of fetal red blood cell destWtiOn by Fc receptor-bearing poiymorphonuclear leukocytes and lymphocytes has been developed. Cord blood was collected at the time of delivery from infants who were Rh-D positive and compatible with their mother, as well as infants who were Rh-D positive and incompatible with their Rh-D-sensitized mothers. Red blood cells, lymphocytes, and PdyrnWphonUclear preparations were individually separated from each cord Mood sample. Rh-D-positive cord red blood cells from compatible and incompatible pregnancies complicated by erythroblastosis were coated with anti-Rh-D. All target red blood cells, opsonized and not opsonized, were labeled with chromium-51 and cultured with autologws lymphocytes or polymorphonuclear leukocytes at an 6ffector:red blood ceil ratio of 10: 1. Cultures containing either no promethazine hydrochloride or varying amounts of the drug were studied. Antibody alone did not cause red cell lysis. Both lymphocytes and polyrnorphonuclear leukocytes were effective in causing the lysis of red blood ceils bearing antibody. Lysis of red blood calls by polyrnorphonuclear leukocytes or lymphocytes was inhibited by promethazine hydrochloride. Excessive amounts of prom&ha&e hydmchkxide alone cause lysis of the red cells. At the low doses utilized for inhiiitkm in this experiment, the action of promethazine hydrochloride on the lymphocytes was found to be reversible. Promethazine is believed to have exhibited two different types of actii in this study: (1) It inhibited antibody-dependent, cell-mediated lysis of autologous red blood c&s coated with antibody; (2) it stabilized the red blood cell membrane so that even spontaneous lysis of red blood cells was decreased 50 per cent. (AM. J. OBSTET. GYNECOL. 130: 391, 1978.)
An
MECHANISMS of fetal red blood cell destruction in vivo in erythroblastosis are not absolutely known. In vitro studies done in our laboratory showed that specific antibody to the Rh-D antigen and complement does not result in significant fetal red blood cell lysis.’ In both that and a later study, we demonstrated that fetal and neonatally obtained peritoneal macrophages
THE
From the Dgatiment of Obstetlics Bowman Gray School of Medicine IJniverrity.
and Gynecology, of Wakt Forest
Supported in part by Wyeth Labs., Philadelphia, Pennsylvania. Presented at the Twenty-jouvth Annual Meeting of the Society for Gynecologic investigation, Tucson, Ariwna, March 23-25, 1977. Reprint requests: Dr. John P. Gus&n, Jr., Department of Obstetrics and Gynecology, Bowvn~n Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103. 0002-9378/78/04130-0391$00.70/O
@ 1978
The
C. V. Mosby
Co.
were capable of binding fetal Rh-D positive red blood cells which were opsonized with anti-D.2 These data suggested that fetal red blood cell destruction could be mediated in vivo by fetal macrophages. In neither of these studies, however, did we actually document red blood cell phagocytosis or destruction. Jandl and Tomlinson,3 however, had shown earlier that destruction of red cells which were opsonized by nonhemolytic antibody is accomplished by cells of the reticuloendothelial system, particularly those of the spleen and liver. Rosenau,* using time-lapse cinematography, demonstrated that lymphoid cells were able to lyse in vitro, without complement-specific cells against which the mice had been sensitized in vivo. Chicken erythrocytes had been shown to be an excellent target for lymphoid cells from donors sensitized to that antigem Based on that earlier work, Holm and Hammarstroms labeled human red blood cells with “‘Cr to study the lysis of 391
392
Gusdon, Herb+ and Marriott
February 15; 1978 Am. j. Obstet. Gynecoi.
Table I. Protocol used in determining the effect of fetal effector cells (polymorphonuclear leukocytes or lymphocytes) on opsonized red blood cell destruction =Cr-FRBC* ( + I - opxmization) 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18.
+ + + + +
+
Fetal effector cells
+ +
+ + + -
5 + + -
*%r-treated
Promethuzine hydrochloride (pglml.)
5 5 5 5
10 10 10 10 0 (distilled water) 0 (distilled water) fetal red blood cells.
antibody-coated red cells by macrophages and leukocytes.s However, although those cells were capable of destroying the opsonized red blood cells, peripheral lymphocytes were not. In our earlier studies of fetal macrophages, we were able to demonstrate resetting of the opsonized red blood cell-bound fetal macrophages. However, seemingly in accord with the data of Holm and Hammarstrom, we were not able to demonstrate rosetting by fetal peripheral lymphocyte preparations.‘, 2 In this study, we documented effective opsonized red blood cell destruction by fetal peripheral lymphocytes and polymorphonuclear leukocytes. It is clear that erythroblastosis fetalis is an immunologically mediated disease process. Unless a maternal antibody to a fetal red blood cell antigen has been shown to have developed, the disease does not occur. Yet, because in vitro studies with the use of anti-D aIone did not result in red blood cell destruction, it seemed that the actual de$truction must be cellmediated.’ These studies, therefore, have been undertaken in an effort to document the role of antibody and some of the types of cells which might be involved. In addition, it was thought that the possible protective and ameliorating role of promethazine hydrochloride in the treatment of affected patients might be more effectively elucidated. During the course of this study, data that documented the presence of Fc receptors on polymorphonuclear cells were published.’ We believed, therefore, that it might be possible for the fetal polymorphonuclear leukocytes to bind and to destroy op-
sonized done.
red blood
cells, and appropriate
studies were
Material and methods Fifty to one hundred milliliters of cord blood was collected at the time of delivery and anti-coagulated with 2 U. of sodium heparin per milliliter. Aliquots of 25 ml. (or one half of the amount collected) of blood were used for lymphocyte separation, and 25 ml. was used for the polymorphonuclear leukocyte isolation. Lymphocyte separation was accomplished by methods previously described,s with only slight modification. Preparation of lymphocytes. Five hundred milligrams of carbonyl iron powder was added to 25 ml. of heparinized cord blood that was then incubated in a 37” C. water bath for 30 minutes with swirling at 10 minute intervals. At the end of the incubation, 15 ml. of Roswell Park Memorial Institute (RPMI) 164OP (Gibco) was added and the suspension was placed on a large magnet. After the iron-laden phagocytes were withdrawn from the suspension, the blood was poured into four test tubes, underlayered with 5 ml. of Ficol Hypaque (specific gravity 1.076), and then centrifuged at 300 g for 20 minutes. Lymphocytes were collected from the Ficol Hypaque-serum interface, washed three times in RPM1 1640f media, counted, and resuspended at an appropriate concentration for use in the experiment. Polymorphonuclear leukocyte preparation. The other 25 ml. aliquot of cord blood was used for isolation of the polymorphonuclear leukocytes. Eight milliliters of plasma gel+ was added to 25 ml. of blood, and the suspension was mixed, poured into a conical centrifuge tube, and then allowed to settle for one hour. The straw-colored supernatant solution was removed and centrifuged at 500 g for 10 minutes. After one washing with RPM1 1640f, the cells were resuspended in media to a 12 C.C.volume, and 4 ml. was poured into each of three test tubes. The tubes were then underlayered with 3 ml. of Ficol Hypaque and centrifuged for 30 minutes at 350 g. Lymphocyte-free granulocytes were found in a pellet at the bottom of the test tube along with contaminating red blood cells. After careful removal of the pellet, it was washed once with phosphate-buffered saline and the red cells were lysed by resuspending the pellet in 6 ml. of distilled water for 20 seconds followed by the immediate addition of 2 ml. of 3.5 per cent sodium *RF’MI 1640f is RPM1 1640 media enriched with 20 per cent fetal calf serum and penicillin and streptomycin. tRoger BeIIon Laboratories, Neulley, France. Distributed by the HTI Corporation, Buffalo, New York.
Volume Number
Inhibition
130 4
chloride. After one more wash in phosphate-buffered saline, the cells were counted and resuspended in RPM1 164Of. Differential counts were performed on the final lymphocyte and polymorphonuclear leukocyte preparations with the use of cytocentrifuge smears, stained with Wright’s stain. Lymphocyte preparations were found to contain approximately 95 per cent lymphocytes. Polymorphonuclear leukocytes were usually 95 with approximately 5 per cent per cent pure, eosinophil contamination. The cell preparations used were more than 9.5 per cent viable as determined by trypan blue dye exclusion. Red blood cell preparations. Red blood cells were obtained from the pellet below the lymphocytes in the lymphocyte-extraction procedure. The red blood cells were removed, washed three times in RPM1 1640f, and counted. The washed cells were opsonized and labeled with %r* simultaneously as follows: To two tubes each containing 4 x lo6 fetal red blood cells, 200 &i of Wr was added. To tube No. 1, 0.2 ml. of anti-Rh-Df was added. In specific instances, twice as much antibody was used to opsonize the fetal red blood cells. In some instances, when the fetal red blood cells were already opsonized by maternal antibody as a result of erythroblastosis, no additional antibody was added; in others, more antibody was added. Cytotoxicity assay. Lymphocytes or polymorphonuclear leukocytes and red blood cells were placed into sterile, screw-cap, glass, disposable test tubes. A ratio of 10 effector cells to one target red blood cell was used in all experiments. Promethazine hydrochIoride$ was placed into solution containing RPM1 1640f. In those experiments in which promethazine was used, it was added directly to the effector cell-red blood cell suspension. A volume of 2 c. c. was maintained in all tubes. The tubes were incubated at 37°C. in an atmosphere of air with 5 per cent carbon dioxide for 18 to 24 hours. A typical protocol is outlined in Table I. Fig. 1 is a composite of the different components of these experiments. At the end of the incubation all tubes were centrifuged at 100 g for 10 minutes; the supernatant solutions were removed, and the pellet and supernatant solutions were counted separately. There was variability in the number of lymphocytes and polymorphonuclear leukocytes we were able to harvest from each delivery. Although some deliveries *Amersham/Searle Corp., tBlood type reagent, Ortho Jersey. $Phenergan was supplied Pennsylvania, as a crystalline, 39311, B7040, and B5610).
Arlington Heights, Illinois. Diagnostics, Inc., Raritan,
New
by Wyeth Labs., Philadelphia, pure powder in 5 Cm. lots (Lots
Fig.
of fetal
1. A standard
red blood
number
cell lysis by promethazine
of S’Cr-labeletl
red
blood
present in every tube. Experiments were performed use of anti-Rh-D
in half
of the tubes
to opsonize
393
cells is
with the the $‘C:r-
labeled cells. The effector cells were either t&al lymphocytes or polymorphonuclear leukocytes, and either no promethazine hydrochloride or varying amctunts of promethazine hydrochloride were used in cliff&-em experiments. The starred cells indicate the “‘O-labeled t>psonized red blood cells.
yielded a large amount of-cord blood, others yielded far less. Therefore, there was variability in the number of effector cells used in individual experiments. This fact, coupled with the differences in the uptake of %r by the fetal red blood cells in different experiments, prevents the comparison of absolute valrles obtained in individual experiments. However, since the effectorto-target ratio of 10: 1 was kept constant in all experiments and control studies were maintained within each experiment, results were calculated 01i a percentage basis. Maximum “‘Cr release for each red blood cell preparation was determined by distilled water Ivsis. Spontaneous “‘0 release was deterfnined by the
394
Gusdon, Herb&, and Marriott
Table
II. In vitro protection
7
of opsonized
red blood cells from destruction Inhibition
Fetal
No. of experiments 17
February 1.5, 1978 Am. J. Obstet. Gynecol.
ORBC* destruction without pomethazine hydrochloride (70)
effector cell
lymphocyte Polymorphonuclear
34.6 2 12 32.1 t 6
I pgtlml.
30 213 22.2 t 12
by fetal effector ofORLK*
cells
Destruction
(%) (G.E.M.)
5 ~gtl??d.
10 PgLgtlml.
36 2 10.1 32.4 -+ 8.2
42.3 2 8.2 36 tf- 10.2
*Opsonized red blood cell. tOf promethazine hydrochloride. Table blood
III. In vitro protection of opsonized of patients with erythroblastosis
red blood
cells from destruction
by effector
Inhibition Patient
seuerity of disease
Trpe of effectvr cell
ORBC destruction without prometharine hydrochloride (70)
L. A. B.
Very mild Moderate
L L
34.2 29.0
K.
*Of promethazine
cells from cord
of ORBC
destruction
1 pg*lml.
5 pg*lml.
30.8
32.1 58.6
(%) 10 pg*lml.
42.3 72.8
hydrochloride.
amount of 51Cr released by labeled red blood cells to which no fetal effector cells had been added. In addition, the effect of promethazine hydrochloride at concentrations of 1, 5, and 10 +g per milliliter was studied. Therefore, specific lysis was calculated according to the formula: % Specific lysis = fetal effector cell-mediated %r release spontaneous %r release maximum “‘Cr release - spontaneous %r release
x 100.
The amount of spontaneous release varied between 6 and 12 per cent in all experiments. The inhibition by promethazine hydrochloride of opsonized red blood cell destruction (per cent protection) by fetal effector cells was determined in the following manner: The destruction (per cent release) by the effector cells was measured by using either no promethazine hydrochloride or different concentrations of the drug within each individual experiment. The per cent of specific lysis obtained in the presence of promethazine hydrochloride was subtracted from the per cent of specific lysis obtained in duplicate tubes in which promethazine hydrochloride was not present. This difference was then divided by the total specific lysis obtained in the absence of the drug and multiplied by 100. The reversibility of the effect of promethazine hydrochloride was studied in experiments by adding promethazine hydrochloride (10 wg per milliliter) to cultures and then washing it out after two hours of incubation and replacing it with normal RPM1 1640f. R@NJJtS
The degree of specific lysis varied between experiments. We have done several preliminary experiments
in which the red blood cells were opsonized with varying amounts of antibody. These data indicate that there is a marked increase in the destruction of the more heavily opsonized fetal red blood cells. One case of particular significance was in a very mildly affected erythroblastotic pregnancy. In this case the child was minimally affected and the indirect Coombs test was only 1+ at the time of delivery at 38 weeks. These cells, which were utilized without any additional antibody, yielded only 8 per cent specific destruction. The same red blood cells that were opsonized by adding a standard amount of antibody yielded 44 per cent specific destruction by the same number of fetal effector cells. In other cases similar results were attained. An interesting finding was that the use of anti-D resulted in two to three times more absorption of %r by the fetal red blood cells than by the red cells to which no anitbody was added. However, in each situation, the amount of spontaneous lysis during the 18 hour course of incubation was the same. Red blood cells to which anti-D was added spontaneously lysed 10.2 2 2.1 per cent (S.E.M.). Red blood cells from the same cord blood, to which an equal amount of %r had been added but which were not opsonized, lysed 9.3 c 3.1 per cent (S.E.M.) during the incubation period. In 17 experiments with lymphocytes, spontaneous lysis varied between 6 and 19.5 per cent. The mean spontaneous release without promethazine hydrochloride was 10.2 per cent, decreasing to 5.1 per cent with promethazine hydrochloride, which suggested a stabilization of membranes. In Table II data are presented that deal with the in vitro protection of opsonized red blood cells from destruction by fetal effector cells with the use of different concentrations of promethazine hydrochloride. The
Volume
130
Number
I
Inhibition
of fetal red blood
cell lysis
by promethazine
395
numbers of lymphocyte experiments exceed those done with polymorphonuclear leukocytes. However, significant destruction of opsonized red blood cells has been obtained with both types of fetal effector cells in vitro. Promethazine hydrochloride at these concentrations has inhibited the amount of opsonized red blood cell destruction. We found, however, that by increasing the concentration of promethazine hydrochloride to 100 pg per milliliter we obtained almost complete red blood cell destruction. As presented in Table III, the same degree of opsonized red blood cell destruction and its inhibition is seen in patients with pre-existing erythroblastosis. In experiments done to determine the reversibility of the effects of promethazine hydrochloride on lymphocytes, we found that the degree of opsonized red blood cell destruction was the same after removal of the promethazine hydrochloride as that in the control, which was never exposed to the drug. In order to verify visually the presence of Fc receptors on polymorphonuclear leukocytes, a fetal polymorphonuclear leukocyte and opsonized red blood cell preparation was made in the same manner as if a T-lymphocyte rosette test were being done.’ Fig. 2 represents photographic evidence of a fetal polymorphonuclear leukocyte bearing Fc receptors that bind the fetal red blood cells.
Comment Antibody-dependent, cell-mediated cytotoxicity represents an in vitro model of the effector arm of cellmediated immunity. This cytotoxicity required immunoglobulin G antibodies directed against membrane antigens of the target cells and is expressed only if the antibodies have intact Fc regions which react with Fc receptors on the effector cell surface. In most of the studies on animal and human systems, the antibodydependent effector cell has been identified as a lymphoid cell, which is distinct from B and T cells and which has been tentatively called the K ce11.9-12 Paraskevas and associatesr3 were able to show that approximately 25 per cent of the T cells in the spleen were capable of taking up cytophilic complexes whether these complexes were formed in vivo, i.e., from serum six hours after immunization, or in vitro. The same number of T cells in the spleen were shown to become immunoglobulin positive within six hours after immunization.‘“. I5 Their data also pointed out that the Fc fragment was of significant importance for the uptake of complexes in vitro. Other workersi6-*’ have documented the presence of Fc receptors on the subpopulation of normal nonactivated T cells by a variety of techniques. This kind of information is of par-
Fig. 2. Shows an isolated polymorphonuc-leak. opsonized red blood cells resetting around
Iwkocvte
with
ir
titular importance in documenting that K. T, and K lymphocytes have Fc receptors and call be involved in cell-mediated immune lysis. Because of the recent reports dealing with the presence of Fc receptors on polymorphonuclear leukocytes, we began to isolate polymorphonuclear leukocytes from the cord blood as well as the lymphocytes. In this study, both of these cell types were used and have been shown to cause a significant amount of antibody dependent, cellmediated destruction of the opsonized red blood cells by these fetal effector cells in vitro. At levels of 1,5. and 10 pg per milliliter of promethazine hydrochloride, significant inhibition of this lysis was seen. This effect was reversible and, therefore, not due to permanent cellular impairment. It is at these low levels that the clinical application of prometha/ine hydrochloride might be considered. In some of our earlier studies, we used concentrations of promethazine hvdrochloride of up to 150 to 200 pg per milliliter. However. we found that fetal red blood cells are lysed b!; (irug levels in excess of 100 pg per milliliter.
396
Gusdon, Herbst, and Marriott
February 15, 1978 Am. J. Obstet. Gynecol.
It has been shown previously that the phenothiazines protect erythrocytes from hypotonic hemolysiszO and retard the rate of hemolysis.21-23 Seem&n and Weinsteinz4 extended these observations with a number of tranquilizers and were able to show ihat in the presence of low concentrations of many of the phenothiazines human erythrocytes are protected or stabilized against hypotonic and mechanical hemolysis. They showed that erythrocyte stabilization is long lasting but that the drug may be removed from the membranes by washing the red cells, which then returned to their former state of fragility. They suggest that this decrease in osmotic fragility might be explained by an expansion of the cell membranes. The data that have been developed in these experiments, depending upon the release of %r, suggest that indeed the spontaneous destruction of red blood cells that would occur during an 18 hour incubation period is decreased by approximately 50 per cent in the presence of promethazine hydrochloride. A suggestion as to another potential use of this membrane-stabilizing property of promethazine hydrochloride was recently made.25T 26 It was suggested, since lysis of fetal red blood cells was a significant problem during early fetoscopy, that the use of promethazine hydrochloride in the mother prior to the attempt to obtain fetal red blood cells by fetoscopy might stabilize the fetal red blood cell membranes sufficiently to ameliorate this problem.
The present study, therefore, suggests that one of the mechanisms of destruction of fetal red blood cells in vivo may be a function of fetal lymphocytes and polymorphonuclear leukocytes and Fc receptors. The data suggest that these effects may be inhibited by promethazine hydrochloride. The clinical use of this compound during pregnancy for the purpose of ameliorating the effects of erythroblastosis was the subject of an earlier preliminary report.27 We have continued to use this compound for that purpose, and our clinical experience with these affected patients will be the subject of a later report. Clinical studies are also underway at other institutions. Although promethazine hydrochloride is not a panacea, if administration is begun early and it is given in adequate amounts, we have found that frequently we able to avoid the use of intrauterine transfusions and allow more time in utero for maturation of the baby prior to delivery. Some of the basic reasons for this clinical effectiveness have been described in this paper. A most interesting recent publication@ also described a significant increase in the production of glucuronyl transferase production induced by promethazine hydrochloride. This, too, may play a role in the amelioration of the disease process after the birth of the affected
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Gusdon, J. P., Jr., Iannuzzi, N. P., Witherow, C. C., and DeChatelet, L. R.: Modification of the human fetal phagocytic response by promethazine hydrochloride, AM. J. OBSTET.
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Jandl, J. H., and Tomlinson, A. S.: The destruction of red cells by antibodies in man. II. Psycogenic, leukocytic and dermal responses to immune hemolysis, J. Clin. Invest. 37: 1202,
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Gusdon, J. P., Caudle, M. R., Herbst, G. A., and Iannuzzi, N. P.: Phagocytosis and erythroblastosis. I. Modification of the neonatal response by promethazine hydrochloride, AM. J. OBSTET.
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Rosenau, W.: Interaction of lymphoid cells with target cells in tissue culture, in Amos, B., and Koprowski, H., editors: Cell-bound Antibodies, Philadelphia, 1963, The Wistar Institute of Anatomy and Biology, pp. 75-80. 5. Perlmann, P., and Helm, G.: Cytotoxic effects of Iymphoid cells in vitro, Adv. Immuiol. 11: 117, 1969. ’
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6. Holm. G.. and Hammarstrom. S.: Haemolvtic activitv of human blood monocytes. Lyiis of human erythrocytes treated with anti-A serum, Clin. Exp. Immunol. 13: 29, 1973. 7. Rabellino, E. M., and Metcalf, D.: Receptors for C3 and IgG on macrophage neutrophil and eosinophil colony cells grown in vitro, J. Immunol. 115: 688, 1975. 8. Aiuti, F., Cerottini, J. E., et al.: International Union of Immunological Societies Report, July, 1974. Identifica-
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tion, enumeration and isolation of B and T lymphocytes from peripheral blood, Clin. Immunol. Immunopathol. 3: 584, 1975. Perhnann, P., Perlmann, H., and Wigzell, H.: Lymphocyte mediated cytotoxicity in vitro. Induction and inhibition by humoral antibody and nature of effector cells, Transplant. Rev. 13: 91, i972. Trinchieri. G.. DeMarchi. M.. Mavr. W.. Savi. M.. and I
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Ceppelini, R.: Lymphocyte aniibody 1ymphocystoly;ic interaction (LALI) with special emphasis on HL-A, Transplant. Pro;. 5: 1631, 19^73. * 11. Harding. B.. Pudifin. D. 1.. Got& F.. and MacLennan. I. C. M.:“Cytbtoxic lymp&cytes from rats depleted ‘of thymus processed cells, Nature (New Biol.) 232: 80, 197 1. 12. Van Boxel, J. A., Stobo, J. D., Paul, W. E., and Green, I.: Antibody-dependent lymphoid cell-mediated cytotoxicitv: No requirement for thvmus-derived lymphocytes, I. I Stence 175: 194, 1972. ’ 13. Paraskevas. F.. Lee. S. T.. and Orr. K. B.: The function of T cells car&&g re&pto& for co&plexes of Ig and antigens, Immunol. Commun. 5: 501, 1976. 14. Paraskevas, F., Orr, K. B., and Lee, S. T.: Cell surfaceassociated gamma globulins in lymphocytes. III. Changes of y globulin-carrying lymphocytes during primary response, J. Immunol. 109: 1254, 1972. 15. Lee, S. T., and Paraskevas, F. J.: Cell surface-associated gamma globulins in lymphocytes. III. Lack of detection of surface y globulin on B-cells and acquisition of surface
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A. R., and Spines. M. A.: Effects of chlorpromazine on biological membranes. II. Chlorpromazine-induced changes in human erythrocytes, B&hem. Pharmacol. 12: 47, 1963. Freeman, A. R., and Spirtes, M. A.: Further effects of chlorpromazine on the human erythrocyte membrane, Biochem. Pharmacol. 12: 1235, 1962. Seeman, P.. and Weinstein, J. L.: Erythrocyte membrane stabilization by tranquilizers and antihistamines, Biothem. Pharmacol. 15: 1737, 1966. Naftolin, F.: Personal communication. Gusdon, J. P., Jr., and Herbst, G. A.: ‘The inhibition of PMN and lymphocyte-mediated lysis by promethazine in an in vitro erythroblastosis model, Gynrcol. Invest, S: 9, 1977. Gusdon, J. P., Jr., and Witherow. C. C.: Possible ameliorating effects of erythroblastosis by promethazine hydrochloride, AM. J. OBSTET. GYNFCOL. 117: 1101, 1973. Vaisman, S. I., Lee, K. S., and Gardner, L. M.: The effect of promethazine hydrochloride on bilirubin metabolism in the rat, Pediatr. Res. 10: 788, 1976.
IntonnrRtasr for authors Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following statement, signed by each author: “The undersigned author(s) transfers all copyright ownership of the manuscript entitled (title of article) to The C. V. Mosby Company in the event the work is published. The author(s) warrants that the article is original, is not under consideration by another journal, and has not been previously published.” Authors will be consulted, when possible, regarding republication of their material.
