American Journal of Hematology 6: 135-141 (1979)

Acquired Aplastic Anemia: Antibody-Mediated Hematopoietic Failure Melvin H. Freedman, Erwin W. Gelfand, and E. Fred Saunders Departments of Pediatrics and Immunology, University o f Toronto; and the Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada

The hematopoietic failure in aplastic anemia was studied in six patients by assessing marrow erythroid stem cell (CFU-E) and granulocytic stem cell (CFU-C) colony growth in vitro. CFU-E and CIW-C were absent or reduced in five patients, and their serum did not inhibit CFU-E or CFU-C growth from control marrows. In contrast, marrow from one patient yielded 80 CFU-E/105 and 16 CFU-C/lOS (control CFU-E: 1 97/105 cells plated, range 110-387; control CFU-C: 48/105, range 15-106). His serum decreased CFU-E (1 2 2 t o 9/105) and CFU-C (71 t o 32/105) from control marrow, and CFU-E from his own marrow (80 t o 37/105 ). His peripheral blood lymphocytes cocultured with autologous or control marrow similarly decreased CFU-C. The inhibition was more marked in cocultures using a B-cell enriched lymphocyte fraction obtained by albumin density gradient centrifugation. IgG separated from the patient’s serum demonstrated a dose-responsive suppression of CFU-E ( 1 0 2 t o 8/105), and of CFU-C (58 t o 28/105) from control marrow. We conclude that of the six patients, one demonstrated an antibody-mediated hematopoietic failure. It is recommended that all patients with acquired aplastic anemia be studied for this, since in vitro testing can detect those in whom irnmunosuppression and plasmapheresis may be more appropriate therapy than marrow transplantation. Key words: aplastic anemia, stem cells, antibody-mediated

INTRODUCTION

Aplastic anemia is defined as peripheral blood pancytopenia due t o bone marrow failure. Regardless of cause, the severe acquired forms of this disorder have been associated with a poor outlook for survival, with a mortality rate of about 70% [ l ] . Traditional management of these patients has included transfusional therapy, antibiotic coverage, and androgens, but the long-term results from this approach have been disappointing. Recent data have indicated that bone marrow transplantation using histocompatible siblings as donors is effective in about 70% of patients [ 2 ] and is probably the most useful therapy available.

Received for publication October 5 , 1978; accepted January 31, 1979. Address reprint requests to Dr. Melvin H. Freedman, Division of Hematology, Hospital f o r Sick Children, 555 University Avenue, Toronto, Ontario, MSG 1x8 Canada.

0361-8609/79/0602-0135 $01.70 0 1979 Alan R. Liss, Inc.

136

Freedman, Gelfand, and Saunders

Perhaps not all patients with severe acquired aplastic anemia are suitable candidates for transplantation. If the defect is due to reduced or defective hematopoietic stem cells, transplantation seems justified; if the problem is secondary to a “hostile microenvironment” such as the presence of cellular inhibitors of hematopoiesis [3-51, then other forms of therapy may be more suitable. Studies on the mechanism of the disorder may therefore provide a basis for rational patient selection. In vitro assays for human marrow granulocytic [6] and erythroid stem cells [7] permit investigation of early hematopoietic events and yield insight into mechanisms of marrow failure. Using these tissue culture techniques, we studied six patients with acquired aplastic anemia and found that in one case the marrow failure was associated with a B-cell and IgG-mediated inhibition of hematopoiesis. SUBJECTS

Marrows from six patients with idiopathic acquired aplastic anemia were studied. There were five males and one female ranging in age from 2% t o 1 9 years. Their hematologic data are shown in Table I. All had varying degrees of anemia, neutropenia, and thrombocytopenia. Fragments from bone marrow aspirates in all were hypocellular, and marrow smears showed a symmetrical reduction of the three major hematopoietic cell lines. To confirm the diagnosis of aplastic anemia, bone marrow biopsies were performed on all except patient 5. None were taking corticosteroids, and only Patient 3 was on androgen. Prior to study, Patient 4 had not received transfusions of blood products, and Patient 1 had only received one transfusion of packed red cells. All others were transfused on numerous occasions. None were bleeding or had sepsis when studied. Patient 1 subsequently died of an acute encephalitic disorder unrelated t o marrow failure; Patients 2, 3, and 6 died from complications of aplastic anemia; patient 5 underwent successful bone marrow transplantation; and Patient 4 has shown partial marrow recovery. Marrows from controls were obtained from patients without hematologic disease or infection who had aspirates performed as part of the medical investigation of other disorders. This study was approved by the Human Experimentation Committee of the hospital. METHODS

Marrow tissue cultures were performed as previously described for CFU-E erythroid stem cells [8] and for CFU-C granulocytic stem cells [9], except that blood group AB serum from a hematologically normal volunteer was substituted for fetal calf serum. For

TABLE I. Hematological Data at the Time of Study of Six Patients With Idiopathic Acquired Aplastic Anemia

Pt .

Hb (gm/dl)

1 2 3 4 5 6

10.9 8.9 8.8 7.5 10.9 7.8

WBC (109/1)

Polys

(%I

Lymphs (%)

Monos (%)

3.6 2.6 3.1 1.9 5.6 2.1

29 10 4 60 1 26

55 84 96 38 96 70

7 6 0 2 3 4

Platelets (109/1) 17.0 7 .O 14.0 30.0 4.0 6.0

Antibody-Mediated Aplastic Anemia

137

CFU-E, marrow cells suspended in nutrient medium were exposed t o 1.O unitlml human erythropoietin and then immobilized in plasma clots (total volume 0.1 ml). CFU-C granulocytic colonies were assayed in a semi-soldi methylcellulose culture system using for growth stimulation medium “conditioned” with colony-stimulating activity (CSA) from the peripheral blood leukocytes of one normal volunteer. After suitable incubation periods, the cultures were examined microscopically, the colonies counted, and the results expressed as CFU-E or CFU-C per 10’ nucleated cells plated. In the inhibitor studies, patients’ serum was substituted for control AB serum in the erythroid and granulocytic cultures at final concentrations of 30%and 20%, respectively. The IgG fractions from Patient 1 and from a control were isolated from serum by elution from DEAE-cellulose equilibrated in 0.007 M phosphate buffer, pH 6.3, as previously described [ l o ] . The purity of the preparations was confirmed by immunoelectrophoresis. The final concentrations of IgG in both the patient’s and the control samples were approximately 10 mg/100 ml of phosphate buffer. The IgG was added to the cultures in concentrations of 0.1 t o 0.3 mg/ml. Freshly obtained peripheral blood lymphocytes from Patients 1 and 5 , and from a control, were separated from heparinized whole blood by Ficoll-Hypaque density gradient centrifugation [ 1 1 ] . The mononuclear cells were further fractionated on discontinuous bovine serum albumin gradients, which at 23-27% albumin enriched for T-cells and at 27-31% albumin enriched for B-cells [12]. With E-rosettes at 4°C used as a T-cell marker and EAC rosettes at 37°C used as a B-cell marker, an enrichment of approximately 60% was confirmed, although an admixture of T- and B-cells still existed in both fractions. In the coculture studies, 1 X lo’ of the lymphocyte preparations of Patient 1, Patient 5 , and a control were mixed with 1 X lo’ control marrow cells prior t o plating. RESULTS

CFU-E and CFU-C were completely absent in marrow cultures from Patients 2-6, with the exception of marrow from Patient 5 , which yielded 8 CFU-C/lO’ . Control values in our laboratory are 197 CFU-E/lO’ (range 110-387) and 48 CFU-C/10’ (range 15-106). In contrast, the marrow from Patient 1 yielded 80 CFU-E/lO’ and 1 6 CFU-C/105. When serum from Patient 1 was substituted for control AB serum in the autologous marrow cultures, there was a pronounced decline of CFU-E numbers from 80/10’ t o 37/10’. Since this assay system is very reproducible [8] ,this degree of change in colony numbers is highly significant statistically (P < 0.005). Autologous CFU-C studies could not be completed before the patient’s death. Table I1 shows the effect on growth of CFU-E and CFU-C from control marrow when patients’ sera were substituted for normal serum in the cultures. Serum from Patient 1 significantly inhibited both erythroid and granulocytic colony growth (P < 0.005). The effect on CFU-E was profound and almost completely abolished colony formation. In contrast, no inhibition was seen when sera from Patients 2-6 were tested in CFU-E and CFU-C assay. With the exception of serum from Patient 4, which caused a statistically insignificant (P > 0.05) decline in CFU-C numbers, stimulation of control marrow colony growth was demonstrated. Table I11 shows the results of the marrow-peripheral blood lymphocyte coculture studies. Compared to cultures without added lymphocytes, normal lymphocytes produced a modest increase in CFU-E numbers from control and from Patient 1 marrow. Inhibition of CFU-E growth from control marrow and from Patient 1 marrow was seen when co-

Freedman, Gelfand, and Saunders

138

TABLE 11. Effect of Normal and Patient Sera on CFU-E and CFU-C Growth From Control Marrows CFU-C/lOS cells plated

CFU-E/105 cells plated Patient

1 2 3 4

5 6

Normal serum

Patient’s serum

122 105 105 78 260 105

9 131 126 83 27 7 121

% Change

Normal serum

- 93 + 24 + 20 +6 +6 + 15

%

Patient’s serum

Change

32 69 87 61 150 85

- 55 -2 + 13 - 12 + 94 + 20

71 71 17 71 17 71

TABLE 111. Marrow-Peripheral Blood Lymphocyte Coculture Studies CFU-E/105 marrow cells plated Control marrow

Patient 1 marrow

247 27 8 198 258 155 267 213

80 93 33

No added lymphocytes Normal lymphocytes Patient 1 lymphocytes Normal B-cells Patient 1 B-cells Normal T-cells Patient 1 T-cells

a

PATIENT 1 I g G NORMAL I g G

- 50

’O0I

CFU-C/lOs

CFU-€/lo’

-

25

- 0

31.3

Fig. 1. The effect of adding IgG from a normal and from Patient 1 on control marrow CFU-E and CFUC.

Antibody-Mediated Aplastic Anemia

139

cultured with Patient 1 lymphocytes. The same effect was demonstrated when Patient 1 B-cell- or T-cell-enriched fractions were cocultured with control marrow, but the inhibition was more pronounced with B-cells. Compared t o normal, peripheral blood lymphocytes from Patient 5 had no effect on CFU-E growth from control marrow (1 68 vs 178 CFU-E/ 105). The effect of adding purified IgG from Patient 1 or a normal on CFU-E and CFU-C growth from control marrow is shown in Figure 1. No difference in CFU-E colony numbers was seen on adding three different concentrations of normal IgG. IgG from Patient 1 produced a dose-responsive inhibition of CFU-E growth. When 0.3 mg/ml of Patient 1 IgG was added to the cultures, colony growth was almost abrogated. Similar inhibition of CFU-C was seen when one concentration was tested. DISCUSSION

With the exception of Patient 5, whose marrow had reduced numbers of granulocytic precursors, no committed hematopoietic stem cells could be demonstrated in marrows of Patients 2-6. This finding is consistent with the concept that in acquired aplastic anemia there is an absolute reduction or impaired proliferation of pluripotent hematopoietic stem cells, or of committed stem cells that give rise to the in vitro colonies, or both. An alternate explanation is that the marrow specimens were diluted with stem cell-poor peripheral blood, and the low marrow colony yield was due t o dilution. This seems unlikely, since marrow cultures with increased cell numbers and stem cell-enriched fractions also failed t o yield colonies. Our results are consistent with those of others who have noted a diminished granulocytic stem cell compartment in aplastic anemia [ 13, 141. In some patients who finally attained hematologic “remission,” the decreased number of marrow granulocyte colony-forming cells persisted [14] , indicating a severe, possibly permanent injury t o the stem cell pool. The marrow culture characteristics of Patient 1 were different from the others. Despite pancytopenia and a hypocellular aspirate, his marrow yielded a moderate number of erythroid colonies and numbers of granulocyte colonies that fell within our lower control range. These findings support the idea that the mechanism of marrow failure in this case was not due either t o stem cell lack or t o an inability of the precursors t o proliferate and differentiate. Another distinguishing characteristic of Patient 1 was the effect of his serum on colony growth from control marrow. Whereas no significant reduction in either CFU-E or CFU-C was seen when sera from the other patients were tested, serum from patient 1 markedly impaired the colony growth from control marrow. This inhibition was produced by the IgG fraction of his serum. The suppressive effect on CFU-E and CFU-C growth was extremely potent and could be demonstrated in vitro with an IgG concentration as low as 0.1 mg/ml. The cell-mediated suppression of CFU-E growth seen in the lymphocyte-marrow cocultures was probably due to the presence of antibody-secreting B-cells, since the inhibition was most marked using lymphocyte populations enriched for B-cells. The T-cellenriched fractions also inhibited CFU-E growth, but the degree of suppression was less. Since the fractionation of T- and B-cells was not complete, the inhibition seen with the predominantly T-cell population was presumably due t o the presence of the 25% contaminating B-cells. The question arises whether the serum inhibitor of hematopoiesis from Patient 1 is an autoimmune antibody or an alloantibody. It is conceivable that, after exposure to blood products, sensitization t o donor antigens could result in alloantibody formation. However,

140

Freedman, Gelfand, and Saunders

we were not able to demonstrate serum inhibitors in the other patients who received multiple transfusions, nor were we able t o find inhibitors in chronically transfused patients that we [8] and others [ 151 have studied previously. Therefore, the incidence of alloantibody formation with characteristics similar to that of Patient 1 is probably very low. It should also be emphasized that Patient 1 received only one transfusion of packed red cells prior to study. Because the exposure t o blood products was minimal, the chance of sensitization was small. The strongest and most convincing argument that Patient 1 had an autoantibody is that his serum, as well as his peripheral blood lymphocytes, suppressed colony growth from his own marrow. Such an autologous system practically eliminates the possibility that the inhibitor was an alloantibody. Cellular inhibition of CFU-E from control marrow by aplastic anemia peripheral blood lymphocytes has been documented by Hoffman and associates [3]. Probably, these were T-cells since the serum from these patients did not have an inhibitory action on erythroid colony growth. Evidence for a population of marrow cells from aplastic anemia patients that is capable of suppressing granulocyte colony growth in autologous and in control marrow has also been published [4,5]. These reports suggest that some forms of acquired aplastic anemia are immunologic in origin and are cell-mediated. Patient 1 appears t o be another example of immune suppression of hematopoiesis, but the pathogenesis of his marrow failure seemed likely to be due t o antibody inhibition which could be shown to act against two hematopoietic cell lines. The in vitro assays of hematopoietic stem cells were valuable in the study of our patients with acquired aplastic anemia. We were able to recognize that the pathophysiology of marrow failure in Patient 1 was different from the others. The identification of such a patient is important since alternative forms of therapy, other than bone marrow transplantation, should be considered, such as immunosuppression and/or plasmapheresis. Patient 1 died before such therapy could be started. We strongly recommend that all patients with acquired aplastic anemia be tested for an immune mechanism. This will permit a more rational selection of candidates for marrow transplant at ion. ACKNOWLEDGMENTS

We thank Mrs. Wilma Vanek and Mr. Tom Grunberger for expert technical work. Human urinary erythropoietin was generously supplied by the Division of Blood Diseases and Resources of the National Heart, Lung and Blood Institute, Bethesda, Maryland. This study was supported by grants MA-4982 and MT-4875 from the Medical Research Council of Canada. Dr. Gelfand is a recipient of a Queen Elizabeth I1 Scientist Award.

REFERENCES 1. Li FP, Alter BP, Nathan DG: The mortality of acquired aplastic anemia in children. Blood 40:153162,1972. 2. Storb R, Thomas ED, Buckner CD, Fefer A, Goodell B, Neiman P, Sanders J , Singer J, Weiden P: Progress in marrow transplantation for severe aplastic anemia. Blood 52(Suppl 1):91, 1978. 3. Hoffman R, Zanjani ED, Lutton JD, Zalusky R, Wasserman LR: Suppression of erythroid-colony formation by lymphocytes from patients with aplastic anemia. N Engl J Med 296:lO-13, 1977. 4. Ascensao J, Kagan W, Moore M, Pahwa R, Hansen J, Good R : Aplastic anemia: Evidence for an immunological mechanism. Lancet 1:669-671,1976.

Antibody-Mediated Aplastic Anemia

141

5. Kagan WA, Ascensao, JA, Pahwa RN, Hansen JA, Goldstein G , Valera EB, Incefy GS, Moore MAS, Good RA: Aplastic anemia: Presence in human bone marrow of cells that suppress myelopoiesis. Proc Natl Acad Sci USA 73:2890-2894,1976. 6. Robinson WA, Pike BL: Colony growth of human bone marrow cells in vitro. In Stohlman F Jr (ed): “Hemopoietic Cellular Proliferation.” New York: Grune & Stratton, 1970, pp 249-259. 7. Tepperman AD, Curtis JE, McCulloch EA: Erythropoietic colonies in cultures of human marrow. Blood 44:659-669, 1974. 8. Freedman MH, Amato D, Saunders EF: Erythroid colony growth in congenital hypoplastic anemia. J Clin Invest 57:673-677,1976. 9. Amato D, Freedman MH, Saunders EF: Granulopoiesis in severe congenital neutropenia. Blood 47~531-538,1976. 10. Abramson N, Gelfand EW, Jandl JH, Rosen FS: The interaction between human monocytes and red cells. Specificity for IgG subclasses and IgG fragments. J Exp Med 132:1207-1215, 1970. 11. Boyum A: Isolation of mononuclear cells and granulocytes from human blood. Scand J Lab Invest 97~77-89,1968. 12. Pyke KW, Gelfand EW: Detection of T-Precursor cells in human bone marrow and foetal liver. Differentiation 5:189-191, 1976. 13. Ragab AH, Gilkerson E, Crist WM, Phelan E: Granulopoiesis in childhood aplastic anemia. J Pediatr 88:790-794, 1976. 14. Kern P, Heimpel H, Heit W, Kubanek B: Granulocytic progenitor cells in aplastic anemia. Br J Haematol 35:613-623,1977. 15. Hoffman R, Zanjani ED, Vila J, Zalusky R, Lutton JD, Wasserman, LR: Diamond-Blackfan syndrome: Lymphocyte-mediated suppression of erythropoiesis. Science 193: 899-900, 1976.

Acquired aplastic anemia: antibody-mediated hematopoietic failure.

American Journal of Hematology 6: 135-141 (1979) Acquired Aplastic Anemia: Antibody-Mediated Hematopoietic Failure Melvin H. Freedman, Erwin W. Gelfa...
425KB Sizes 0 Downloads 0 Views