Serum Inhibitors of Hematopoiesis in a Patient with Aplastic Anemia and Systemic Lupus Erythematosus Recovery after Exchange Plasmapheresis
JOHN J. FITCHEN, MARTIN J. CLINK ANDREW SAXON, DAVID W. GOLDE,
M.D. M.D. M.D. M.D.
Los Angeles, Coliforniu
From the Division of Hematology-Oncology and the Division of Clinical Immunology and Allergy, Department of Medicine, IJniversity of California School of Medicine, Los Angeles. California. This study was supported by Grants CA 15619. CA 15688. CA 12806 and RR 00865 from the 1J.S. Public Health Service. It was presented in part at the XXth Annual Mcoting of the American Society of Hematology, San Diego. Dccemher 3-i’. 1977. Requests for reprints should bc addressed to Dr. John H. Fitchen. Department of Medicine, IJCLA Center for the Health Sciences, Los Angeles, California 90024. Manuscript acccpted August 10. 1978.
A 36 year old black woman with serologic evidence of systemic lupus erytbematosus bad aplastic anemia that failed to respond to corticosteroid or androgen therapy. Bone marrow culture studies were performed to determine if the marrow aplasia was immunologically mediated. A potent serum inhibitor of in vitro myeloid and erythroid colony formation was demonstrated. Incubation of normal bone marrow cells with the patient’s serum and a source of complement caused a 95 per cent reduction in myeloid colony formation. Under these conditions, bone marrow from 35 normal volunteer subjects formed a mean of 5 f I colonies per z x 105 inarrow cells plated (control = 95 f 8 colo&s). The addition of the patient’s serum plus complement to normal marrow cultured in methylcellulose also caused a 98 per cent reduction in erythroid colony numbers. Assay of serum immunoglobulin fractions obtained by gel filtration demonstrated that the inhibitory activity was contained in the immunoglobulin G (IgG) fraction. In association with a series of plasma exchanges, the titer of inhibitor fell, and marrow cellula&y and peripheral blood counts returned to normal. Additional studies indicated that the inhibitor of allogeneic marrow was not directed against HLA or “B-cell” antigens. Incubation of the patient’s recovery marrow with stored preplasmapheresis serum and complement did not produce a reduction in myeloid colony formation. It s&emsmost likely that the inhibitor represents alloandbody directed against heretofore unrecognized antigens widely distributed on normal human hematopoietic progenitor cells. The observation of autologous recovery from aplastic anemia following immunosuppressive conditioning for bone marrow transplantation with antilymphocyte globulin [1] or high dose cyclophosphamide (2-51 has prompted the suggestion that some cases of aplastic anemia may be immunologically mediated. Humoral immune suppression of hematopoictic stem cells committed to a single line of differentiation has been documented. Krantz [S] has shown that pure red cell aplasia may be mediated by complement-dependent cytotoxic antibodies directed against red cell precursors. Cline et al. (71 have recently described immune suppression of myeloid precursor cells (CFU-C) in a patient with episodic panleukopenia. We now describe a patient with aplastic anemia and serologic evidence of systemic 111puserythematosus (SLE) whose serum contained complement-dependent immunoglobulin G (IgG) antibody that in-
March 1979
The American Journal of Medicine
Volume 66
537
SERUM INHIBITORS IN APLASTIC ANEMIA-FITCHEN
2
ET AL.
oRX
~, , ,
2h4
PLASMAPHERESIS
isl UCLA
;
JAN
5%
b,
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A’“‘TwfT”“al OXYHETHALONE
MAR
APR
MAY
I
JUN
I
JUL
I
AUG
I
1
SEP
1976
I
OCT
1 I
NOV
PLASMAPHERESIS
1 I
1
DEC
{
JAN
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I
I
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MAY I
I
1977
Figure 1. Clinical course of the patient. Red blood cell transfusions are indicated by small arrows at the top of the figure. The administration of corticosteroids and androgens was without sustained benefit. A series of plasma exchanges (large arrows, bottom of figure) was associated with a clear and persistent hematologic response.
hibited both granulocytic and erythroid colony formation by normal human bone marrow in vitro. A series of plasma exchanges was associated with recovery from profound pancytopenia. Although the patient’s serum inhibited myeloid stem cells from 35 normal donors, autologous inhibition (preplasmapheresis serum versus patient’s recovery marrow] was not demonstrable in vitro. CASE
REPORT
The clinical course of the patient is summarized in Figure 1. A 36 year old black woman underwent a left radical mastectomy for infiltrating ductal carcinoma in October 1975. All 12 axillary nodes were negative for cancer. In January 1976, she suffered fatigue and dyspnea on exertion and entered the hospital. A complete blood count showed a hemoglobin level of 3.5 g/dl, a white blood cell count of 3,499/m1 (segmental neutrophils 50 per cent, band forms 3 per cent, lymphocytes.49 per cent, monocytes 7 per cent) and a platelet count of 122,99O/ml. Repeated reticulocyte counts were less than 9.5 per cent. The antinuclear antibody test [ANA] was positive, as were 2 of 4 blood lupus erythematosus cell preparations. A sugar water test was negative. Bone marrow biopsy showed a profound decrease in hematopoietic elements. The patients’ blood was type A2B, Rh positive, and her serum had anti-I, antiLewisa and b, and anti-A, antibodies. She was given a transfusion of packed red blood cells and was treated with 80 mg of prednisone daily. Over the ensuing five months, she re-
538
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quired frequent transfusion. After a transient increase, a progressive decrease in the white blood cell count and platelet count was also noted over this time period (Figure 11. The patient was referred to UCLA in July 1976. Blood pressure was 110/70 mm Hg, pulse 100/min at rest and temperature 37%. The palms and mucous membranes were pale and without purpura. The liver and spleen were not palpable, and there was no lymphadenopathy. The right breast was without masses. A complete blood count showed profound pancytopenia (Figure 1, July 1976). The reticulocyte count was less than 0.1 per cent. Broad spectrum Coombs’ test was positive. A bone marrow biopsy specimen showed a hypocellular marrow. No metastatic cells were seen. An lllindium-transferrin bone marrow scan showed a marked decrease in uptake. Liverspleen scan, bone scan and right mammogram were unremarkable. ANA was positive at a titer of 1:160. Antisinglestranded DNA binding was 13 per cent. Latex fixation was negative. Total complement, and the third (C3) and fourth (C41 components of complement were normal, and elevated levels of circulating immune complexes were not found [a]. Androgen therapy and a brief trial with high-dose methyl-prednisolone were unsuccessful. Preliminary in vitro marrow culture studies indicated that the patient’s serum contained an inhibitor of granulocytemacrophage colony formation. Based on this finding, plasmapheresis was initiated in November 1976 in an attempt to remove the inhibitor. Two weeks later, the reticulocyte count had increased from 0.1 per cent to 6.6 per cent. With subsequent plasma exchanges, there was a progressive increase in the hemoglobin level, white blood cell count and platelet
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count, Monthly plasma exchanges were discontinued in February 1977. after a total of four exchanges. Repeat bone marrow examination showed normal cellularity, and I%dium-transferrin bone marrow scan showed normal uptake. Current blood counts are normal: hemoglobin 13.6 g/dl. white blood cell count 5,300 (segmented neutrophils 44 per cent, band forms 5 per cent, eosinophils 1 per cent, lymphocytes 30 per cent, monocytes 13 per cent] and platelet count 245,000/ Wl.
METHODS Inhibitor
Assays. CFU-C: Heparinized bone marrow cell suspensions were obtained from informed and consenting normal subjects. A single cell suspension at 2 X 105/ml was plated in 0.3 per cent agar over feeder*layers of peripheral blood leukocytes as previously described [9,10]. After incubation at 37’C in an atmosphere of 7.5 per cent carbon dioxide in air for 10 to 14 days, granulocyte-macrophage colonies of greater than 40 cells were enumerated. Complement-dependent antibodies active against CFU-C were assayed by the method of Terasaki and McClelland [ll] with modifications [7]. Briefly, the patient’s serum or normal AB serum (control] was added to a suspension of normal marrow cells and incubated at room temperature for 30 minutes. Rabbit serum (NRS) was added and the incubation continued for an additional 60 minutes. The NRS served as a source of complement and was not in itself inhibitory to human CFU-C. At the completion of incubation, the cell suspension was plated in duplicate for the CFU-C assay. CFU-E: CFU-E were assayed by the methylcellulose technic as previously described [12] using 1.0 U of human urinary erythropoietin (provided by the Blood Research Division, Heart and Lung Institute, National Institutes of Health]. Erythropoietin, marrow cells, 0.1 ml of serum f 0.1 ml NRS [complement source) were added to 0.8 per cent methylcellulose and plated immediately in duplicate at a final cell concentration of 2 X 105/ml. After incubation at 37’C in 7.5 per cent carbon dioxide in air for eight days, colonies containing 8 or more hemoglobinized cells were counted. Serum fracSerum Fractionation and Immunoabsorption. tionation by gel filtration and the absorption technics for establishing IgG or immunoglobulin M (IgM] specificity of antibody have been described in detail previously [7]. Tests of serum cytotoxicity against lymLymphocytotoxins. phocytes were kindly performed by Dr. Paul Terasaki. Serums were assayed against a panel of lymphocytes from 84 normal volunteer subjects (selected to insure representation of all known HLA types) according to a standard complementdependent microcytotoxicity assay [l3]. Results are expressed as per cent positive (100 X number of wells with greater than 20 per cent killing/total number of wells] and strength index (strength index = 100 X number of strong positives [greater than 80 per cent killing]/number of positives]. Absorptions. To remove anti-HLA alloantibodies, serum was incubated for 90 minutes at room temperature with an equal volume of pooled platelets from 25 random donors. The patient’s serum was also absorbed with adult and umbilical cord red blood cells (120 minutes at room temperature]. Absorbed samples were then assessed for marrow inhibitory activity using the CFU-C inhibitor assay.
Figure 2. Effect of patient’s serum f complement (C) on granulocytic colony formation (mean f standard error of 35 experiments).
Blocking Antibodies. To explore the possibility that recovery was mediated by the development of blocking antibodies, normal marrow cells were incubated with the patient’s recovery serum (November 1977) for 60 minutes at 37% and then preplasmapheresis serum was added and the incubation continued as in the CFU-C inhibitor assay. Autologous Studies. Repeated attempts to aspirate marrow prior to plasmapheresis were unsuccessful. Autologous inhibiton assays (preplasmapheresis serum versus recovery marrow) were possible after marrow recovery. The effect on the patient’s marrow of a rabbit antiserum reactive with normal human CFU-C and B lymphocytes [14] was also assessed using the CFU-C inhibitor assay. Plasma Exchange. Plasma exchanges were by continuous flow centrifugation employing an Aminco Celltrifuge@ (American Instrument Company, Silver Spring, Maryland). At monthly intervals, 5 to 6 liters of plasma were exchanged isovolumetrically with type AB fresh-frozen plasma. RESULTS Inhibition of CFU-C and CFU-E. The effect of the patient’s serum on normal human CFU-C is depicted in Figure 2. Thirty-five different normal marrows were used as sources of CFU-C. In every case, there was marked inhibition of colony formation. Extensive washing of the cells after incubation and prior to plating had no effect on inhibition, indicating that the marked reduction in colony numbers was due to toxicity to CFU-C rather than to destruction of mature cells in developing colonies. This was corroborated by the
March 1979
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100 A
100
i
80
U
13 d ” VI 2 * cy \
~ u
80
60
40
A Y u
20
/
I I NO
NORM. NORM. PT.
kM
PT. L_._.. ~_~~~
C
Figure 3. Effect of patient’s serum f complement (C) on erythroid colony formation (mean f standard error of three experiments).
failure to observe abortive colonies. Inhibitory activity was consistently complement dependent and persisted at a 1:16 dilution of the patient’s serum. Thirty normal serums were also tested, and none showed inhibitory activity. Erythroid colony formation, tested in three different normal marrows, was also markedly inhibited by the patient’s serum in the presence of complement (Figure
31. Characterization
March 1979
The American Journal of Medicine
C’
16
I&
W
+
+
C’
C’
Figure 4. Effect of immunoglobulin fractions on granulocytic colony formation (mean f standard error of two experiments). Fractions consist of immunoglobulins M (IgM) and G (IgG) with and without complement (C), as well as the IgG fraction and complement after removal of IgG antibodies.
Absorptions.
of Inhibitor. Assay of serum immunoglobulin fractions obtained by gel filtration demonstrated that the inhibitory activity was contained in the IgG fraction (Figure 4). Specific removal of IgG from the IgG fraction with Cowan I Staphylococcus aureus (protein A from this strain binds the Fc portion of IgG molecules [x]) and rabbit anti-IgG removed 77 per cent of the inhibitory activity (Figure 4), whereas treatment to remove IgM did not affect the inhibitory activity of the IgG fraction. Effect of Plasma Exchange. As shown in Figure 1, improvement occurred in association with piasma exchange with return of hemoglobin, white blood cell count and platelet count to normal levels. Samples obtained at the beginning of each plasma exchange were assayed for inhibitor with the CFU-C assay. Over the four-month period of plasmapheresis, a Is-fold decrease in the titer of inhibitory activity was noted (Table I). Total hemolytic complement and quantitative immunoglobulins remained in the normal range during and following the period of plasmapheresis. The Coombs’ test became negative, but the ANA titer was unchanged.
540
ABSORBED IgM
+
SERUM SERUM SERUM SERUM SERUM + +
C
1
Preliminary studies in our laboratory have indicated that human CFU-C probably express HLA antigens. To explore the possibility that the patient’s inhibitor simply represented anti-HLA alloantibody, we absorbed the serum with random donor platelets. The effect of this absorption is depicted in Table II. Platelet absorption effectively removed lymphocytotoxic (anti-HLA) but not anti-CFU-C activity from the patient’s &rum. Absorption of the patient’s serum with adult and cord blood red cells was also without effect on myeloid inhibitory activity. Preincubation of normal human Blocking Antibodies. marrow with the patient’s recovery serum did not alter the potent inhibitory effect of preplasmapheresis serum on normal colony formation. Autologous Studies. The patient’s stored preplasmapheresis serum [November 1976) was assayed against her own recovery marrow on four occasions between January and November 1977. At no time was inhibitory activity found. A representative experiment is depicted in Table III. Loss of activity due to prolonged storage was excluded by the demonstration of persistent potent activity against normal human CFU-C. As a positive control, the patient’s recovery marrow was also treated with an antiserum reactive with normal human CFU-C and B lymphocytes [14]. This antiserum produced marked inhibition of colony formation by the patient’s recovery marrow (Table III).
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TABLE I
TABLE III
Effect of Plasmapheresis on Circulating Inhibitor Level > 50% Inhibition of SampleTested
Plasma from Exchange #l (11176) Exchange #2 ( 12176) Exchange #3 (1777) March 31, 1977 October 10, 1977
1:16 1:8 1:2 Undiluted No inhibition
NOTE: SI = strength index. Mean f SEM of six experiments l
Lymphocytotoxins % t/s1 o/o 95171 2/o
Colonies/ 2 X ifIs Cells* 60 f 4 3fl 6fl
ET AL.
Effect of Preplasmapheresis Serum November 1976 and of Anti-B-Cell Serum on in Vitro Colony Formation by Patient’s Recovery Marrow (November 1977)
AB serum + complement Patient’s serum (November 1976) icomplement Anti-B-cell serum + complement
Effect of Random-Donor Platelet Absorption on Inhibitory Activity
AB serum + complement Patient’s serum + complement Patient’s serum (platelet-absorbed) + complement
ANEMIA-FITCHEN
Marrow Incubated with
We have described a patient with aplastic anemia and serologic evidence of SLE who had a complementdependent IgG inhibitor(s) of colony formation by both granulocytic and erythroid I’:ogenitor cells in vitro. Inhibitory activity was distinct from the patient’s erythrocyte antibodies and froim HLA antibodies, as indicated by the failure of red blood cell and random donor platelet absorption to remove the inhibitory activity against normal myeloid progenitors. The inhibitor was also not directed against the recently described B cell antigens present on CFU-C [14]. Repeated plasma exchange was associated with a progressive fall in the circulating level of inhibitor, repopulation of the patient’s marrow with hematopoietic cells and a return to normal of her hematologic status. The improvement could not be attributed to removal of complement. The dramatic hematologic response and the decrease in inhibitor titer that followed plasmapheresis led us to believe that the aplastic anemia was mediated by autoantibodies directed against antigens on hematopoietic stem cells. However, failure to demonstrate inhibitory activity of preplasmapheresis serum on autologous recovery marrow cast doubt on this interpretation. Such an interpretation is tenable only if one postulates antigenic modulation of the patient’s stem cells coincident with plasmapheresis and hematologic recovery. It seems more likely that the inhibitor represents transfusion-induced alloantibody directed against antigens widely distributed on human CFU-C but distinct
Normal Marrow Incubatedwith
IN APLASTIC
Colony Formation at a Tiler of
COMMENTS
TABLE II
INHIBITORS
Colonies/? X lo5 Marrow Cells 63 62 2
from antigens known to be expressed on these cells (“B cell,” HLAJ. If the patient’s inhibitor was in fact alloantibody, then plasmapheresis was only fortuitously related to hematologic recovery. Alternatively, plasmapheresis may have stimulated hematopoiesis by removal of some factor not detectable in our in vitro system. Duckham et al. [16] found a factor in the lipoprotein fraction of 43 per cent of serums from patients with SLE that inhibited colony formation by murine bone marrow, but the relevance of this xenogenic model to human disease is uncertain. Plasmapheresis has been employed successfully in various immune disorders, In patients with Goodpasture’s syndrome, Lockwood and colleagues [li’] were able to decrease antiglomerular basement membrane antibody levels, control pulmonary hemorrhage and improve renal function by use of plasmapheresis in conjunction with immunosuppression. This regimen was also successfplly applied to the treatment of immune complex crescentic nephritis [18]. Verrier Jones et al. ([19] treated eight patients with SLE with plasmapheresis and found clinical and immunochemical improvement in all four who had high levels of circulating immune complexes. The concurrent administration of immunosuppressive agents makes it difficult to interpret the importance of plasmapheresis in these regimens. However, Fraser et al. [ZO] succeeded in reducing antibody titers and preventing intrauterine or neonatal death in 62 per cent of patients with Rhesus isoimmunization by use of plasmapheresis alone. Despite these encouraging results and the apparently beneficial effects of plasma exchange in our patient with aplastic anemia, widespread use of plasmapheresis is probably not warranted [2l] in bone marrow failure disorders unless appropriate studies suggest that abnormal immunoglobins may play a role in their pathogenesis. ACKNOWLEDGMENT We are indebted to Dr. Frederich M. Rarick for referral of the patient and to Drs. Jacob Zighelboim, Gary Siegel and Winston Ho for their suggestions regarding therapy. Carol Le Fevre and Noelle Bersch provided outstanding technical assistance.
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REFERENCES 1. 2. 3.
4.
5.
6.
Speck B. Cluckman E, Haak HL, et al.: Treatment of aplastic anemia by antilymphocyte globulin with and without allogeneic bone-marrow infusions. Lancet 1:1145, 1977. Thomas ED, Storb R. Giblett ER. et al.: Recovery from aulastic anemia following attempted marrow transplantation. Exp Hematol4: 97, 1976. Speck B, Cornu P. Jeannet M, et al.: Autologous marrow recovery following allogeneic marrow transplantation in a patient with severe aplastic anemia. Exp Hematol4: 131. 1976. Territo MC: Autologous bone marrow repo ulation following high dose cyclophosphamide and al Pogeneic marrow transplantation in aplastic anemia. Br J Haematol36: 305. 1977. Sensenbrenner LL. Steele AA, Santos GW: Recovery of hcmatologic competence without engraftment following attempted bone marrow transplantation for aplastic anemia. Report of a case with diffusion chamber studies. Exp Hemat01 5181. 1977. Krantz SB: Pure red-cell aplasia. N Engl ] Med 291: 345, 1974.
7. 8. 9. 10. 11.
542
Chne Ml. Ooelz G. Saxon A. et al.: Autoimmune oardeukopenia.‘hl Engl J Med 295: 1489,1976. Barnett E. Chia D: Ouantification of immunozlobulin G in serum and immune complexes isolated in polyethylene glycol. Annals Rheum Dis 36 (suppl): 26.1977. Pike BL. Robinson A: Human bone marrow colony growth in agar-gel. ] Cell Physiol76: 77, 1970. Golde DW. Cline MJ: Identification of the colony-stimulating cell in human peripheral blood. J Clin Invest 51: 2981, 1972. Terasaki PI, McClelland JD: Microdroplet assay of human
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cytotoxins. Nature 204: 998.1964. 12. Golde DW, Bersch N, Cline MJ: Potentiation of erythropoiesis in vitro bv dexamethasone. I Clin Invest 57: 67.1976. 13. Mittal KK.“Mickey MR, Singal DP. et al.: Serotyping for homo-transplatation. XVIII. Rcfinemeht of microdroplet cytotoxicity test. Transplantation 6: 913, 1968. 14. Cline Ml. Billinv R: Antigens exoressed bv human B Ivmphocytes and”myeloid item cells. ] Exp- Med 146: li43. 1977. 15. Kessler SW: Ra id isolation of antigchs from cells with a staphylococca P protein A-antibody absorbent. Parameters of the interaction of antibody-antigen complexes with protein A. ] ImmunoJ 115: 1617, 1975. 16. Duckham DJ, Rhync RL Jr, Smith RE, et al.: Retardation of colony growth of in vitro bone marrow culture usin sera from patients with F&y’s syndrome, disseminated Pupus erythematosus (SLE). rheumatoid arthritis and other disease states. Arthritis Rheum 18: 323. 1975. 17. Lockwood CM, Pearson TA. Rees AJ. et al.: Immunosuppression and plasma-exchange in the treatment of Goodpasture’s syndrome Lancet 1: 711. 1976. 18. Lockwood CM, et al.: Plasma-exchange and immunosdppression in the treatment of fulminating immune-complex crescentic nephritis. Lancet 1: 63, 1977. 19. Verrier Jones J. Bucknall RC, Cumming RH, et al.: Plasmanheresis in the management of acute mstemic IUDUS erythematosus? Lancet 7: 709.1976. . 20. Fraser ID, Bennett MO, Bothamlcv JE, et al.: Intensive antenatal plasmaphercsis in severekhesus isoimmunizatlon. Lancet 1: 6. 1976. 21. Editorial: Plasmapheresis and immunosuppression. Lancct 1: 113, 1976.
Volume 66
JOHN J. FITCHEN, MARTIN J. CLINK ANDREW SAXON, DAVID W. GOLDE,
M.D. M.D. M.D. M.D.
Los Angeles, Coliforniu
From the Division of Hematology-Oncology and the Division of Clinical Immunology and Allergy, Department of Medicine, IJniversity of California School of Medicine, Los Angeles. California. This study was supported by Grants CA 15619. CA 15688. CA 12806 and RR 00865 from the 1J.S. Public Health Service. It was presented in part at the XXth Annual Mcoting of the American Society of Hematology, San Diego. Dccemher 3-i’. 1977. Requests for reprints should bc addressed to Dr. John H. Fitchen. Department of Medicine, IJCLA Center for the Health Sciences, Los Angeles, California 90024. Manuscript acccpted August 10. 1978.
A 36 year old black woman with serologic evidence of systemic lupus erytbematosus bad aplastic anemia that failed to respond to corticosteroid or androgen therapy. Bone marrow culture studies were performed to determine if the marrow aplasia was immunologically mediated. A potent serum inhibitor of in vitro myeloid and erythroid colony formation was demonstrated. Incubation of normal bone marrow cells with the patient’s serum and a source of complement caused a 95 per cent reduction in myeloid colony formation. Under these conditions, bone marrow from 35 normal volunteer subjects formed a mean of 5 f I colonies per z x 105 inarrow cells plated (control = 95 f 8 colo&s). The addition of the patient’s serum plus complement to normal marrow cultured in methylcellulose also caused a 98 per cent reduction in erythroid colony numbers. Assay of serum immunoglobulin fractions obtained by gel filtration demonstrated that the inhibitory activity was contained in the immunoglobulin G (IgG) fraction. In association with a series of plasma exchanges, the titer of inhibitor fell, and marrow cellula&y and peripheral blood counts returned to normal. Additional studies indicated that the inhibitor of allogeneic marrow was not directed against HLA or “B-cell” antigens. Incubation of the patient’s recovery marrow with stored preplasmapheresis serum and complement did not produce a reduction in myeloid colony formation. It s&emsmost likely that the inhibitor represents alloandbody directed against heretofore unrecognized antigens widely distributed on normal human hematopoietic progenitor cells. The observation of autologous recovery from aplastic anemia following immunosuppressive conditioning for bone marrow transplantation with antilymphocyte globulin [1] or high dose cyclophosphamide (2-51 has prompted the suggestion that some cases of aplastic anemia may be immunologically mediated. Humoral immune suppression of hematopoictic stem cells committed to a single line of differentiation has been documented. Krantz [S] has shown that pure red cell aplasia may be mediated by complement-dependent cytotoxic antibodies directed against red cell precursors. Cline et al. (71 have recently described immune suppression of myeloid precursor cells (CFU-C) in a patient with episodic panleukopenia. We now describe a patient with aplastic anemia and serologic evidence of systemic 111puserythematosus (SLE) whose serum contained complement-dependent immunoglobulin G (IgG) antibody that in-
March 1979
The American Journal of Medicine
Volume 66
537
SERUM INHIBITORS IN APLASTIC ANEMIA-FITCHEN
2
ET AL.
oRX
~, , ,
2h4
PLASMAPHERESIS
isl UCLA
;
JAN
5%
b,
FE6
A’“‘TwfT”“al OXYHETHALONE
MAR
APR
MAY
I
JUN
I
JUL
I
AUG
I
1
SEP
1976
I
OCT
1 I
NOV
PLASMAPHERESIS
1 I
1
DEC
{
JAN
MI\R I
I
I
FE6
BPR ,
MAY I
I
1977
Figure 1. Clinical course of the patient. Red blood cell transfusions are indicated by small arrows at the top of the figure. The administration of corticosteroids and androgens was without sustained benefit. A series of plasma exchanges (large arrows, bottom of figure) was associated with a clear and persistent hematologic response.
hibited both granulocytic and erythroid colony formation by normal human bone marrow in vitro. A series of plasma exchanges was associated with recovery from profound pancytopenia. Although the patient’s serum inhibited myeloid stem cells from 35 normal donors, autologous inhibition (preplasmapheresis serum versus patient’s recovery marrow] was not demonstrable in vitro. CASE
REPORT
The clinical course of the patient is summarized in Figure 1. A 36 year old black woman underwent a left radical mastectomy for infiltrating ductal carcinoma in October 1975. All 12 axillary nodes were negative for cancer. In January 1976, she suffered fatigue and dyspnea on exertion and entered the hospital. A complete blood count showed a hemoglobin level of 3.5 g/dl, a white blood cell count of 3,499/m1 (segmental neutrophils 50 per cent, band forms 3 per cent, lymphocytes.49 per cent, monocytes 7 per cent) and a platelet count of 122,99O/ml. Repeated reticulocyte counts were less than 9.5 per cent. The antinuclear antibody test [ANA] was positive, as were 2 of 4 blood lupus erythematosus cell preparations. A sugar water test was negative. Bone marrow biopsy showed a profound decrease in hematopoietic elements. The patients’ blood was type A2B, Rh positive, and her serum had anti-I, antiLewisa and b, and anti-A, antibodies. She was given a transfusion of packed red blood cells and was treated with 80 mg of prednisone daily. Over the ensuing five months, she re-
538
March
197s
The American Journal of Medicine
quired frequent transfusion. After a transient increase, a progressive decrease in the white blood cell count and platelet count was also noted over this time period (Figure 11. The patient was referred to UCLA in July 1976. Blood pressure was 110/70 mm Hg, pulse 100/min at rest and temperature 37%. The palms and mucous membranes were pale and without purpura. The liver and spleen were not palpable, and there was no lymphadenopathy. The right breast was without masses. A complete blood count showed profound pancytopenia (Figure 1, July 1976). The reticulocyte count was less than 0.1 per cent. Broad spectrum Coombs’ test was positive. A bone marrow biopsy specimen showed a hypocellular marrow. No metastatic cells were seen. An lllindium-transferrin bone marrow scan showed a marked decrease in uptake. Liverspleen scan, bone scan and right mammogram were unremarkable. ANA was positive at a titer of 1:160. Antisinglestranded DNA binding was 13 per cent. Latex fixation was negative. Total complement, and the third (C3) and fourth (C41 components of complement were normal, and elevated levels of circulating immune complexes were not found [a]. Androgen therapy and a brief trial with high-dose methyl-prednisolone were unsuccessful. Preliminary in vitro marrow culture studies indicated that the patient’s serum contained an inhibitor of granulocytemacrophage colony formation. Based on this finding, plasmapheresis was initiated in November 1976 in an attempt to remove the inhibitor. Two weeks later, the reticulocyte count had increased from 0.1 per cent to 6.6 per cent. With subsequent plasma exchanges, there was a progressive increase in the hemoglobin level, white blood cell count and platelet
Volume 66
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count, Monthly plasma exchanges were discontinued in February 1977. after a total of four exchanges. Repeat bone marrow examination showed normal cellularity, and I%dium-transferrin bone marrow scan showed normal uptake. Current blood counts are normal: hemoglobin 13.6 g/dl. white blood cell count 5,300 (segmented neutrophils 44 per cent, band forms 5 per cent, eosinophils 1 per cent, lymphocytes 30 per cent, monocytes 13 per cent] and platelet count 245,000/ Wl.
METHODS Inhibitor
Assays. CFU-C: Heparinized bone marrow cell suspensions were obtained from informed and consenting normal subjects. A single cell suspension at 2 X 105/ml was plated in 0.3 per cent agar over feeder*layers of peripheral blood leukocytes as previously described [9,10]. After incubation at 37’C in an atmosphere of 7.5 per cent carbon dioxide in air for 10 to 14 days, granulocyte-macrophage colonies of greater than 40 cells were enumerated. Complement-dependent antibodies active against CFU-C were assayed by the method of Terasaki and McClelland [ll] with modifications [7]. Briefly, the patient’s serum or normal AB serum (control] was added to a suspension of normal marrow cells and incubated at room temperature for 30 minutes. Rabbit serum (NRS) was added and the incubation continued for an additional 60 minutes. The NRS served as a source of complement and was not in itself inhibitory to human CFU-C. At the completion of incubation, the cell suspension was plated in duplicate for the CFU-C assay. CFU-E: CFU-E were assayed by the methylcellulose technic as previously described [12] using 1.0 U of human urinary erythropoietin (provided by the Blood Research Division, Heart and Lung Institute, National Institutes of Health]. Erythropoietin, marrow cells, 0.1 ml of serum f 0.1 ml NRS [complement source) were added to 0.8 per cent methylcellulose and plated immediately in duplicate at a final cell concentration of 2 X 105/ml. After incubation at 37’C in 7.5 per cent carbon dioxide in air for eight days, colonies containing 8 or more hemoglobinized cells were counted. Serum fracSerum Fractionation and Immunoabsorption. tionation by gel filtration and the absorption technics for establishing IgG or immunoglobulin M (IgM] specificity of antibody have been described in detail previously [7]. Tests of serum cytotoxicity against lymLymphocytotoxins. phocytes were kindly performed by Dr. Paul Terasaki. Serums were assayed against a panel of lymphocytes from 84 normal volunteer subjects (selected to insure representation of all known HLA types) according to a standard complementdependent microcytotoxicity assay [l3]. Results are expressed as per cent positive (100 X number of wells with greater than 20 per cent killing/total number of wells] and strength index (strength index = 100 X number of strong positives [greater than 80 per cent killing]/number of positives]. Absorptions. To remove anti-HLA alloantibodies, serum was incubated for 90 minutes at room temperature with an equal volume of pooled platelets from 25 random donors. The patient’s serum was also absorbed with adult and umbilical cord red blood cells (120 minutes at room temperature]. Absorbed samples were then assessed for marrow inhibitory activity using the CFU-C inhibitor assay.
Figure 2. Effect of patient’s serum f complement (C) on granulocytic colony formation (mean f standard error of 35 experiments).
Blocking Antibodies. To explore the possibility that recovery was mediated by the development of blocking antibodies, normal marrow cells were incubated with the patient’s recovery serum (November 1977) for 60 minutes at 37% and then preplasmapheresis serum was added and the incubation continued as in the CFU-C inhibitor assay. Autologous Studies. Repeated attempts to aspirate marrow prior to plasmapheresis were unsuccessful. Autologous inhibiton assays (preplasmapheresis serum versus recovery marrow) were possible after marrow recovery. The effect on the patient’s marrow of a rabbit antiserum reactive with normal human CFU-C and B lymphocytes [14] was also assessed using the CFU-C inhibitor assay. Plasma Exchange. Plasma exchanges were by continuous flow centrifugation employing an Aminco Celltrifuge@ (American Instrument Company, Silver Spring, Maryland). At monthly intervals, 5 to 6 liters of plasma were exchanged isovolumetrically with type AB fresh-frozen plasma. RESULTS Inhibition of CFU-C and CFU-E. The effect of the patient’s serum on normal human CFU-C is depicted in Figure 2. Thirty-five different normal marrows were used as sources of CFU-C. In every case, there was marked inhibition of colony formation. Extensive washing of the cells after incubation and prior to plating had no effect on inhibition, indicating that the marked reduction in colony numbers was due to toxicity to CFU-C rather than to destruction of mature cells in developing colonies. This was corroborated by the
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100 A
100
i
80
U
13 d ” VI 2 * cy \
~ u
80
60
40
A Y u
20
/
I I NO
NORM. NORM. PT.
kM
PT. L_._.. ~_~~~
C
Figure 3. Effect of patient’s serum f complement (C) on erythroid colony formation (mean f standard error of three experiments).
failure to observe abortive colonies. Inhibitory activity was consistently complement dependent and persisted at a 1:16 dilution of the patient’s serum. Thirty normal serums were also tested, and none showed inhibitory activity. Erythroid colony formation, tested in three different normal marrows, was also markedly inhibited by the patient’s serum in the presence of complement (Figure
31. Characterization
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C’
16
I&
W
+
+
C’
C’
Figure 4. Effect of immunoglobulin fractions on granulocytic colony formation (mean f standard error of two experiments). Fractions consist of immunoglobulins M (IgM) and G (IgG) with and without complement (C), as well as the IgG fraction and complement after removal of IgG antibodies.
Absorptions.
of Inhibitor. Assay of serum immunoglobulin fractions obtained by gel filtration demonstrated that the inhibitory activity was contained in the IgG fraction (Figure 4). Specific removal of IgG from the IgG fraction with Cowan I Staphylococcus aureus (protein A from this strain binds the Fc portion of IgG molecules [x]) and rabbit anti-IgG removed 77 per cent of the inhibitory activity (Figure 4), whereas treatment to remove IgM did not affect the inhibitory activity of the IgG fraction. Effect of Plasma Exchange. As shown in Figure 1, improvement occurred in association with piasma exchange with return of hemoglobin, white blood cell count and platelet count to normal levels. Samples obtained at the beginning of each plasma exchange were assayed for inhibitor with the CFU-C assay. Over the four-month period of plasmapheresis, a Is-fold decrease in the titer of inhibitory activity was noted (Table I). Total hemolytic complement and quantitative immunoglobulins remained in the normal range during and following the period of plasmapheresis. The Coombs’ test became negative, but the ANA titer was unchanged.
540
ABSORBED IgM
+
SERUM SERUM SERUM SERUM SERUM + +
C
1
Preliminary studies in our laboratory have indicated that human CFU-C probably express HLA antigens. To explore the possibility that the patient’s inhibitor simply represented anti-HLA alloantibody, we absorbed the serum with random donor platelets. The effect of this absorption is depicted in Table II. Platelet absorption effectively removed lymphocytotoxic (anti-HLA) but not anti-CFU-C activity from the patient’s &rum. Absorption of the patient’s serum with adult and cord blood red cells was also without effect on myeloid inhibitory activity. Preincubation of normal human Blocking Antibodies. marrow with the patient’s recovery serum did not alter the potent inhibitory effect of preplasmapheresis serum on normal colony formation. Autologous Studies. The patient’s stored preplasmapheresis serum [November 1976) was assayed against her own recovery marrow on four occasions between January and November 1977. At no time was inhibitory activity found. A representative experiment is depicted in Table III. Loss of activity due to prolonged storage was excluded by the demonstration of persistent potent activity against normal human CFU-C. As a positive control, the patient’s recovery marrow was also treated with an antiserum reactive with normal human CFU-C and B lymphocytes [14]. This antiserum produced marked inhibition of colony formation by the patient’s recovery marrow (Table III).
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TABLE I
TABLE III
Effect of Plasmapheresis on Circulating Inhibitor Level > 50% Inhibition of SampleTested
Plasma from Exchange #l (11176) Exchange #2 ( 12176) Exchange #3 (1777) March 31, 1977 October 10, 1977
1:16 1:8 1:2 Undiluted No inhibition
NOTE: SI = strength index. Mean f SEM of six experiments l
Lymphocytotoxins % t/s1 o/o 95171 2/o
Colonies/ 2 X ifIs Cells* 60 f 4 3fl 6fl
ET AL.
Effect of Preplasmapheresis Serum November 1976 and of Anti-B-Cell Serum on in Vitro Colony Formation by Patient’s Recovery Marrow (November 1977)
AB serum + complement Patient’s serum (November 1976) icomplement Anti-B-cell serum + complement
Effect of Random-Donor Platelet Absorption on Inhibitory Activity
AB serum + complement Patient’s serum + complement Patient’s serum (platelet-absorbed) + complement
ANEMIA-FITCHEN
Marrow Incubated with
We have described a patient with aplastic anemia and serologic evidence of SLE who had a complementdependent IgG inhibitor(s) of colony formation by both granulocytic and erythroid I’:ogenitor cells in vitro. Inhibitory activity was distinct from the patient’s erythrocyte antibodies and froim HLA antibodies, as indicated by the failure of red blood cell and random donor platelet absorption to remove the inhibitory activity against normal myeloid progenitors. The inhibitor was also not directed against the recently described B cell antigens present on CFU-C [14]. Repeated plasma exchange was associated with a progressive fall in the circulating level of inhibitor, repopulation of the patient’s marrow with hematopoietic cells and a return to normal of her hematologic status. The improvement could not be attributed to removal of complement. The dramatic hematologic response and the decrease in inhibitor titer that followed plasmapheresis led us to believe that the aplastic anemia was mediated by autoantibodies directed against antigens on hematopoietic stem cells. However, failure to demonstrate inhibitory activity of preplasmapheresis serum on autologous recovery marrow cast doubt on this interpretation. Such an interpretation is tenable only if one postulates antigenic modulation of the patient’s stem cells coincident with plasmapheresis and hematologic recovery. It seems more likely that the inhibitor represents transfusion-induced alloantibody directed against antigens widely distributed on human CFU-C but distinct
Normal Marrow Incubatedwith
IN APLASTIC
Colony Formation at a Tiler of
COMMENTS
TABLE II
INHIBITORS
Colonies/? X lo5 Marrow Cells 63 62 2
from antigens known to be expressed on these cells (“B cell,” HLAJ. If the patient’s inhibitor was in fact alloantibody, then plasmapheresis was only fortuitously related to hematologic recovery. Alternatively, plasmapheresis may have stimulated hematopoiesis by removal of some factor not detectable in our in vitro system. Duckham et al. [16] found a factor in the lipoprotein fraction of 43 per cent of serums from patients with SLE that inhibited colony formation by murine bone marrow, but the relevance of this xenogenic model to human disease is uncertain. Plasmapheresis has been employed successfully in various immune disorders, In patients with Goodpasture’s syndrome, Lockwood and colleagues [li’] were able to decrease antiglomerular basement membrane antibody levels, control pulmonary hemorrhage and improve renal function by use of plasmapheresis in conjunction with immunosuppression. This regimen was also successfplly applied to the treatment of immune complex crescentic nephritis [18]. Verrier Jones et al. ([19] treated eight patients with SLE with plasmapheresis and found clinical and immunochemical improvement in all four who had high levels of circulating immune complexes. The concurrent administration of immunosuppressive agents makes it difficult to interpret the importance of plasmapheresis in these regimens. However, Fraser et al. [ZO] succeeded in reducing antibody titers and preventing intrauterine or neonatal death in 62 per cent of patients with Rhesus isoimmunization by use of plasmapheresis alone. Despite these encouraging results and the apparently beneficial effects of plasma exchange in our patient with aplastic anemia, widespread use of plasmapheresis is probably not warranted [2l] in bone marrow failure disorders unless appropriate studies suggest that abnormal immunoglobins may play a role in their pathogenesis. ACKNOWLEDGMENT We are indebted to Dr. Frederich M. Rarick for referral of the patient and to Drs. Jacob Zighelboim, Gary Siegel and Winston Ho for their suggestions regarding therapy. Carol Le Fevre and Noelle Bersch provided outstanding technical assistance.
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REFERENCES 1. 2. 3.
4.
5.
6.
Speck B. Cluckman E, Haak HL, et al.: Treatment of aplastic anemia by antilymphocyte globulin with and without allogeneic bone-marrow infusions. Lancet 1:1145, 1977. Thomas ED, Storb R. Giblett ER. et al.: Recovery from aulastic anemia following attempted marrow transplantation. Exp Hematol4: 97, 1976. Speck B, Cornu P. Jeannet M, et al.: Autologous marrow recovery following allogeneic marrow transplantation in a patient with severe aplastic anemia. Exp Hematol4: 131. 1976. Territo MC: Autologous bone marrow repo ulation following high dose cyclophosphamide and al Pogeneic marrow transplantation in aplastic anemia. Br J Haematol36: 305. 1977. Sensenbrenner LL. Steele AA, Santos GW: Recovery of hcmatologic competence without engraftment following attempted bone marrow transplantation for aplastic anemia. Report of a case with diffusion chamber studies. Exp Hemat01 5181. 1977. Krantz SB: Pure red-cell aplasia. N Engl ] Med 291: 345, 1974.
7. 8. 9. 10. 11.
542
Chne Ml. Ooelz G. Saxon A. et al.: Autoimmune oardeukopenia.‘hl Engl J Med 295: 1489,1976. Barnett E. Chia D: Ouantification of immunozlobulin G in serum and immune complexes isolated in polyethylene glycol. Annals Rheum Dis 36 (suppl): 26.1977. Pike BL. Robinson A: Human bone marrow colony growth in agar-gel. ] Cell Physiol76: 77, 1970. Golde DW. Cline MJ: Identification of the colony-stimulating cell in human peripheral blood. J Clin Invest 51: 2981, 1972. Terasaki PI, McClelland JD: Microdroplet assay of human
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cytotoxins. Nature 204: 998.1964. 12. Golde DW, Bersch N, Cline MJ: Potentiation of erythropoiesis in vitro bv dexamethasone. I Clin Invest 57: 67.1976. 13. Mittal KK.“Mickey MR, Singal DP. et al.: Serotyping for homo-transplatation. XVIII. Rcfinemeht of microdroplet cytotoxicity test. Transplantation 6: 913, 1968. 14. Cline Ml. Billinv R: Antigens exoressed bv human B Ivmphocytes and”myeloid item cells. ] Exp- Med 146: li43. 1977. 15. Kessler SW: Ra id isolation of antigchs from cells with a staphylococca P protein A-antibody absorbent. Parameters of the interaction of antibody-antigen complexes with protein A. ] ImmunoJ 115: 1617, 1975. 16. Duckham DJ, Rhync RL Jr, Smith RE, et al.: Retardation of colony growth of in vitro bone marrow culture usin sera from patients with F&y’s syndrome, disseminated Pupus erythematosus (SLE). rheumatoid arthritis and other disease states. Arthritis Rheum 18: 323. 1975. 17. Lockwood CM, Pearson TA. Rees AJ. et al.: Immunosuppression and plasma-exchange in the treatment of Goodpasture’s syndrome Lancet 1: 711. 1976. 18. Lockwood CM, et al.: Plasma-exchange and immunosdppression in the treatment of fulminating immune-complex crescentic nephritis. Lancet 1: 63, 1977. 19. Verrier Jones J. Bucknall RC, Cumming RH, et al.: Plasmanheresis in the management of acute mstemic IUDUS erythematosus? Lancet 7: 709.1976. . 20. Fraser ID, Bennett MO, Bothamlcv JE, et al.: Intensive antenatal plasmaphercsis in severekhesus isoimmunizatlon. Lancet 1: 6. 1976. 21. Editorial: Plasmapheresis and immunosuppression. Lancct 1: 113, 1976.
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