Leukemia Research Vol. 14, No. 3, pp. 279-286, 1990. Printed in Great Britain,
0145-2126/90 $3.00 + .00 Pergamon Press plc
PURE RED CELL APLASIA ASSOCIATED WITH B CELL LYMPHOMA: DEMONSTRATION OF BONE MARROW COLONY INHIBITION BY SERUM IMMUNOGLOBULIN ROXANNE ALTER,* SHANTARAM S. JOSHI,t JOSEPH D. VERDIRAME~ and DENNIS D. WEISENBURGER* *Departments of Pathology and Microbiology, tAnatomy, and $Internal Medicine, University of Nebraska Medical Center, Nebraska, U.S.A.
(Received 18 September 1989. Accepted 30 September 1989) Abstract--A 58-year old male with follicular small cleaved B cell lymphoma developed pure red cell aplasia (PRCA) during chemotherapy. To understand the etiology of the PRCA, we studied the effects of patient sera on the progenitor cell colony formation of normal human bone marrow cells in vitro. We demonstrated a marked inhibition of normal bone marrow progenitor cell colony formation by patient sera, but not pooled normal human sera. Immunoglobulin was then precipitated from patient sera for similar studies. The majority of the precipitated immunoglobulin was of the IgG type. The immunoglobulin fraction markedly inhibited normal bone marrow progenitor cell colony formation, whereas the non-immunoglobulin fraction was not inhibitory. The presence of inhibitory serum immunoglobulin correlated with the hematologic status of the patient. We conclude that the development of PRCA in patients with B cell lymphoma may be due to a serum IgG inhibitor of bone marrow progenitor cell growth.
Key words: Pure red cell aplasia, erythropoiesis, B cell lymphoma, bone marrow progenitor cells, immunoglobulin, serum inhibitor, colony forming units.
INTRODUCTION
some cases, the P R C A is due to antibodies directed against erythroid progenitor cells or growth factors [1]. More recently, the role of the cellular immune system in promoting and suppressing erythropoiesis has been an area of intense interest [9]. In this study, we isolated and partially characterized a serum inhibitor of bone m a r r o w progenitor cell growth from a patient with B cell nonHodgkin's lymphoma. The in-vitro activity of the serum inhibitor correlated with the hematologic status of the patient.
ACQUIRED pure red cell aplasia ( P R C A ) is a rare hematologic disorder that is characterized by severe erythroid hypoplasia of the b o n e marrow and the absence of a reticulocytosis [1]. Approximately 40% of the reported cases of P R C A have been associated with a t h y m o m a . O t h e r cases have been associated with immunologic or a u t o i m m u n e disorders such as myasthenia gravis, thyroiditis, systemic lupus erythematosis and r h e u m a t o i d arthritis [1]. Only a few cases of P R C A have been associated with lymphoproliferative disorders such as B cell nonHodgkin's l y m p h o m a [2], H o d g k i n ' s disease [3], and T or B cell chronic lymphocytic leukemia [4-8]. In
CASE REPORT AND METHODS
Case report In September of 1985, a 58-year old white male was admitted to the hospital with generalized lymphadenopathy and the recent onset of night sweats. A left cervical lymph node biopsy specimen showed follicular non-Hodgkin's lymphoma of the small cleaved lymphocytic type. Immunohistochemical stains of frozen tumor tissue revealed the presence of monoclonal B cells bearing surface IgM of the lambda light chain type. The hemoglobin was 16.3 g/dl and the white cell count was 6.4 x 103/cmm, with 48% segmented neutrophils, 10% bands, 28% lymphocytes, 9% monocytes, 4% eosinophils and 1% b asophils. The platelet count was 164 x 103/cmm. A bone marrow specimen was positive for lymphoma. In the bone marrow smears, large
Abbreviations: BFU-E, burst forming units---erythrocytes; COP, cyclosphosphamide, vincristine, prednisone; COP-BLAM, cyclophosphamide, vincristine, prednisone, bleomycin, Adriamycin, procarbazine; CFU-E, colony forming units---erythrocytes; CFU-GEMM, colony forming units--granulocyte/erythrocyte/monocyte/megakaryocytes; CFU-GM, colony forming units--granulocyte/ monocyte; PRCA, pure red cell aplasia; SAS, saturated ammonium sulfate; SDS, sodium dodecyl sulfate. Correspondence to: Dr Dennis D. Weisenburger, Department of Pathology & Microbiology, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105, U.S.A. 279
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numbers of atypical lymphoid cells were present. The myeloid and erythroid series were relatively decreased in number, but maturation was normal. Iron stores were also normal. Figure 1 shows the peripheral blood counts during the patients' clinical course. After the initial diagnosis, written informed consent was obtained and the patient was treated with cyclophosphamide, vincristine, and prednisone (COP). After seven courses of chemotherapy, the white cell count was 4.8 x 103/cmm, and the hemoglobin was 10g/dl with a reticulocyte count of less than 0.1%. At the same time, the lymphoma had responded dramatically to chemotherapy. However, a bone marrow aspirate now showed aplasia of the erythroid series, whereas myeloid precursors and megakaryocytes were present in normal numbers. Atypical lymphoid cells still comprised 20% of the nucleated cells in the marrow aspirate smears. At this time, the patient received six cycles of cyclophosphamide, vincristine, prednisone, bleomycin, adriamycin and procarbazine (COPBLAM). In April of 1986, serum and bone marrow samples were obtained for cell culture in vitro and inhibition studies. The bone marrow aspirate at that time revealed normal cellularity, with early recovery of the erythroid series. The marrow aspirate smears also showed persistent involvement by lymphoma. The white cell count was 4.3 x 103/ cmm, and the hemoglobin was 10.6 g/dl with a reticulocyte count of 1.5%. As the hemoglobin level and reticulocyte counts continued to improve, a second serum sample for in-vitro culture studies was obtained in May. The white cell count was 2.6 x 103/cram, and the hemoglobin level was 11.4 g/dl with a reticulocyte count of 3.4%. A third serum sample for in-vitro culture studies was obtained in November of 1986. At that time, the patient was well with a hemoglobin of 13.3 g/dl and a reticulocyte count of2.5%. Although he had persistent lymphoma in the bone marrow, there was no other evidence of disease.
Bone marrow culture assays Peripheral blood and bone marrow mononuclear cells were separated from freshly-drawn heparinized blood and bone marrow, respectively, by density sedimentation using lymphocyte separation medium [10[. The colony forming units--granulocyte/fnonocyte (CFU-GM) assay was performed according to the agar method of Robinson and Pike [11]. In this assay, 1 x 105 mononuclear cells in 1 ml of 0.3% in McCoy's medium was layered onto an underlayer of normal or patient peripheral blood leukocytes (1 x 106 normal or patient peripheral blood leukocytes in 1 ml of 0.5% agar in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and antibiotics). The cultures were incubated at 37°C with 5% CO~ and 95% air in a humidified atmosphere for 14 days. At the end of this period, groups of 50 or more cells were scored as colonies. The colony forming units--granulocyte/ erythrocyte/monocyte/megakaryocyte (CFU-GEMM) assay was performed using a modification of the method of Fauser and Messner [12]. In this assay, 5 x 104 cells were plated in 1 ml of 1.2% methylcellulose with 2.0 I.U. of human urinary erythropoietin (Cancer Research Laboratories, Vancouver, British Columbia). Groups of greater than 50 cells were scored as colonies on day 14. In the same assay, the colony forming u n i t s I e r y t h r o c y t e (CFU-E) and burst forming u n i t s I e r y t h r o c y t e (BFU-E) were scored on days 7 and 16, respectively. For the serum inhibition assays, we added either 0.1 ml of serum from
the patient or 0.1 ml of pooled normal sera to the above culture dishes.
Precipitation of immunoglobulin Immunoglobulin was precipitated from patient sera and normal pooled sera using saturated ammonium sulfate (SAS). The sera was mixed slowly with SAS ( p H 7.0) at a ratio of 6 : 4, and the mixture was incubated for 15 minutes at room temperature to allow complete precipitation. The mixture was then centrifuged at 1500 r.p.m, and the supernatant was collected. The precipitate was then reconstituted to the original volume with phosphate buffered saline. The reconstituted precipitate was then reprecipitated with SAS. Both the final reconstituted precipitate (immunoglobulin-rich fraction) and the first supernatant (non-immunoglobulin fraction) were dialyzed against phosphate buffered saline, and the purified fractions were characterized and used in the in-vitro colony inhibition assays. Electrophoretic analysis The immunoglobulin and non-immunoglobulin fractions were analyzed on sodium dodecyl sulfate (SDS) polyacrylamide gel (BioRadLab, Richmond, CA) according to the method of Laemmli [13]. Equal volumes of immunoglobulin and non-immunoglobulin fractions were loaded on 12% SDS polyacrylamide gel and electrophoresed for 4-6 hours, After electrophoresis, the gel was stained with Coomassie Blue (Sigma Chemical Co., St Louis, MO), and then destained with a solution consisting of 25% methanol, 10% acetic acid and 65% water. Ouchterloney immunodiffusion The type of immunoglobulin present in the SAS precipitate was determined by Ouchterloney gel immunodiffusion [14]. Fifty microliters of the immunoglobulin or non-immunoglobulin fractions were placed in the center wells of 1% BactoAgar (Difco, Detroit, MI) in a Petri dish. Antisera to human immunogloblins G (IgG) or M (IgM) were placed in the outer wells of the Petri dishes. The Petri dishes were then incubated at room temperature in a humidified atmosphere for 48 hr, and then examined for the development of specific immunoprecipitation lines using a light box. Precipitation lines were photographed directly without staining. RESULTS
Bone marrow cultures using patient sera T h e effects of p a t i e n t s e r a on t h e g r o w t h o f n o r m a l b o n e m a r r o w C F U - G M is s h o w n in T a b l e 1. T h e p a t i e n t s e r u m s a m p l e s u s e d in this assay w e r e o b t a i n e d at t h r e e d i f f e r e n t t i m e s d u r i n g the clinical course (Fig. 1). P o o l e d n o r m a l s e r a was u s e d as t h e c o n t r o l sera. T h e r e was d r a m a t i c i n h i b i t i o n o f n o r m a l b o n e m a r r o w C F U - G M f o r m a t i o n by p a t i e n t s e r u m o b t a i n e d at the t i m e o f m a r k e d e r y t h r o i d h y p o p l a s i a of the b o n e m a r r o w . A s t h e h y p o p l a s i a r e s o l v e d , C F U - G M g r o w t h r e t u r n e d to n o r m a l . W h e n p a t i e n t b l o o d m o n o n u c l e a r cells w e r e u s e d as f e e d e r s to p r o v i d e colony s t i m u l a t i n g factors, C F U - G M form a t i o n was mildly d e c r e a s e d , b u t r e t u r n e d to n o r m a l with the a d d i t i o n of n o r m a l sera. In t h e a b s e n c e o f
281
P u r e r e d cell a p l a s i a a n d B cell l y m p h o m a T A B L E 1. EFFECTS OF PATIENT SERUM AND P E R I P H E R A L BLOOD MONONUCLEAR CELL FEEDERS ON NORMAL BONE M A R R O W CFU-GM FORMATION
Mean number of CFU-GM colonies* NBM + NS NBM + PS1 NBM + PS2
NBM Feeder Feeder Feeder Patient
No. I t No. 2t No. 3t feeder:~
180 209 123 64
+- 8 -+ 13 +- 9 - 16
143 - 22 179 +- 14 158 - 11 162 -+ 9
0 0 0 9 -+ 1
NBM + PS3
89 -+ 10 13 +- 1 76 +- 17 ND
ND 192 -+-2 146 - 3 ND
CFU-GM, colony forming units--granulocyte/monocyte; * mean --+-standard error; t normal peripheral blood feeder layers; ~: patient peripheral blood mononuclear cells from 4/86 used in feeder layers; NBM, normal bone marrow; NS, normal pooled sera; PS1, patient serum from 4/86; PS2, patient serum from 5/86; PS3, patient serum from 11/86; ND, not done.
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moolobin (gm/dl)
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White o e h (XlO+/cmm) -0
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4
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MONTHS
FIG. 1. Peripheral blood counts during the clinical course of a patient with pure red aplasia and B-cell lymphoma; • represents the transfusion of packed red blood cells; * indicates the times when serum samples were obtained for inhibition studies; COP = cyclophosphamide, vincristine, and prednisone; COP-BLAM = cyclophosphamide, vincristine, prednisone, bleomycin, adriamycin and procarbazine.
patient serum, patient bone m a r r o w formed normal numbers of C F U - G M colonies (data not shown). The effects of patient sera on the formation of erythroid and non-erythroid colonies by normal bone marrow in the C F U - G E M M assay are shown in Table 2. Patient serum obtained at the time of erythroid hypoplasia showed the greatest colony inhibition as c o m p a r e d to the two later sera. As the patient's hematologic status improved, decreasing inhibition of colony formation was seen. B F U - E colony formation was the most markedly inhibited by the first patient serum sample, but returned to normal with subsequent serum samples.
Characterization of the serum inhibitor Serum immunoglobulin was precipitated using
saturated a m m o n i u m sulfate, and the immunoglobulin and non-immunoglobulin fractions were analyzed by electrophoresis. Figure 2 shows the electrophoretic analysis of the immunoglobulin (lane A) and non-immunoglobulin (lane B) fractions. The non-immunoglobulin fraction consisted of albumin and other serum proteins, without the 160,000 mol. wt band seen in the immunoglobulin fraction. Thus, the electrophoretic analysis d e m o n s t r a t e d that the non-immunoglobulin fraction did not contain significant amounts of immunoglobulin, whereas the immunoglobulin fraction consisted predominantly of immunoglobulin. Figure 3 shows the Ouchterloney immunodiffusion pattern of the immunoglobulin fraction. The majority of immunoglobulin in the immunoglobulin fraction was of the I g G type. The
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R. ALTER et al.
TABLE 2. EFFECTS OF PATIENT SERA ON THE FORMATION OF ERYTHROID AND NON-ERYTHROID COLONIES BY NORMAL BONE MARROW
NBM CFU-E CFU-GEMM BFU-E
Red 54-+ 1 73 -+ 4 106-+4
Other 11+-3 48 + 6 54-+7
NBM + NS Red 71-+11 80 -+ 1 128 + 2
Mean number of colonies* NBM + PS1 NBM + PS2
Other 65-+8 56 -+ 2 67-+3
Red 31-+2 32 -+ 2 28-+2
Other 40 + 3 32 + 1 37 + 1
Red 57+2 51 -+ 1 112+3
Other 48-+6 36 -+ 3 61-+-3
NBM + PS3 Red ND 82 + 1 107-+2
Other ND 46 -+ 3 54-+3
* Mean -+ standard error; CFU-E, colony forming units--erythroid, scored on day 7; CFU-GEMM, colony forming units--granulocyte/erythrocyte/monocyte/megakaryocyte, scored on day 14; BFU-E, burst forming units--erythroid, scored on day 16; NBM, normal bone marrow; NS, normal pooled wera; PS1, patient serum from 4/86; PS2, patient serum from 5/86; PS3, patient serum from 11/86; Red, colonies with hemoglobinization; Other, colonies without hemoglobinization; ND, not done. non-immunoglobulin fraction did not contain detectable immunoglobulin (not shown). Bone marrow cultures using the serum immunoglobulin fraction The immunoglobulin and non-immunoglobulin fractions were then tested for their inhibitory activity in the bone marrow colony assays. Table 3 shows that the serum inhibitor was present in the immunoglobulin fraction, but not in the non-immunoglobulin fraction. The first serum immunoglobulin fraction resulted in prominent inhibition of normal colony growth in all three assays. As the hematologic status of the patient improved, decreasing inhibition of colony formation by the immunoglobulin fraction was seen. The non-immunoglobulin fraction did not inhibit normal bone marrow colony formation significantly.
DISCUSSION Pure red cell aplasia (PRCA) is a well-defined clinical disorder that may arise by at least three different immunopathogenic mechanisms. These include antibodies to erythroblasts [15-19] or erythropoietin-responsive cells [20], antibodies to erythropoietin [21, 22], and T-cell suppression of erythropoiesis [6]. In adults with PRCA, all three mechanisms have been demonstrated. In transient erythroblastopenia of childhood, however, it appears that most cases are due to an IgG inhibitor directed against erythroid progenitor cells [23]. In this report we describe a patient who developed P R C A after receiving chemotherapy for follicular B cell non-Hodgkin's lymphoma. To our knowledge, this is the only reported study of P R C A associated with follicular B-cell lymphoma using bone marrow culture techniques. We have clearly demonstrated the presence of a serum inhibitor to bone marrow colony formation. Additional studies revealed that
the inhibitor was a serum immunoglobulin of the IgG type. The presence of the inhibitor correlated well with the hematologic status of the patient. Colony inhibition was demonstrated in all of the assays used, and was most pronounced using the first immunoglobulin fraction (PSI-Ig) in the CFU-E assay (Table 3). Interesingtly, inhibition of myeloid colony formation in the C F U - G M and C F U - G E M M assays (non-hemoglobinized colonies) was also seen. This latter finding indicates the presence of cross-reacting inhibition in vitro, which was not evident in vivo. Similar bone marrow culture studies of a patient with B-cell chronic lymphocytic leukemia and P R C A have been reported by Mangan & D'Alessandro [5]. In their studies, an inhibitor directed against the erythroid colony forming unit (CFU-E) was demonstrated. Further studies, however, suggested that the suppression of CFU-E formation resulted from a Tcell derived inhibitor. A patient with T-cell chronic leukemia and P R C A has also been described by Hansen et al. [8]. Erythroid burst forming units (BFU-E) were shown to be inhibited by suppressor T-lymphocytes. The mechanisms by which these Tcell inhibitor factors induced bone marrow colony inhibition were not studied further. To our knowledge, no other case of P R C A due to a serum immunoglobulin inhibitor in a patient with a lymphoproliferative disorder has been reported. In conclusion, we have identified and characterized a serum immunoglobulin inhibitor of bone marrow colony formation in vitro, the presence of which correlated well with the hematologic status of our patient. Although we do not know the exact origin of the inhibitor, we presume that it was not produced by the tumor cells, since the lymphoma was of the lgM type and persisted throughout the clinical course. We speculate that the inhibitor was produced by non-neoplastic B cells as a result of altered regulation of the immune system, perhaps secondary to chemotherapy. When the patient was treated with
A
B
C
FIG. 2. Electrophoretic pattern of the immunoglobulin and non-immunoglobulin fractions precipitated from patient serum; lane A, immunoglobulin fraction (arrow indicates the immunoglobulin band); lane B, non-immunoglobulin fraction; lane C, marker proteins.
283
FIG. 3. Ouchterloney immunodiffusion of the immunoglobulin fraction of patient serum with antibodies to human IgM and IgG; well A, anti-human IgM; well B, patient immunoglobulin fraction; well C, anti-human IgG.
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Pure red cell aplasia and B cell lymphoma
285
more intensive chemotherapy, the P R C A resolved in the presence of persistent lymphoma. These findings suggest that i m m u n e dysregulation may play a role in the etiology of unexplained or persistent cytopenias associated with chemotherapy. Additional studies of similar patients are clearly needed to further our understanding of this p h e n o m e n o n .
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0145-2126/90 $3.00 + .00 Pergamon Press plc
PURE RED CELL APLASIA ASSOCIATED WITH B CELL LYMPHOMA: DEMONSTRATION OF BONE MARROW COLONY INHIBITION BY SERUM IMMUNOGLOBULIN ROXANNE ALTER,* SHANTARAM S. JOSHI,t JOSEPH D. VERDIRAME~ and DENNIS D. WEISENBURGER* *Departments of Pathology and Microbiology, tAnatomy, and $Internal Medicine, University of Nebraska Medical Center, Nebraska, U.S.A.
(Received 18 September 1989. Accepted 30 September 1989) Abstract--A 58-year old male with follicular small cleaved B cell lymphoma developed pure red cell aplasia (PRCA) during chemotherapy. To understand the etiology of the PRCA, we studied the effects of patient sera on the progenitor cell colony formation of normal human bone marrow cells in vitro. We demonstrated a marked inhibition of normal bone marrow progenitor cell colony formation by patient sera, but not pooled normal human sera. Immunoglobulin was then precipitated from patient sera for similar studies. The majority of the precipitated immunoglobulin was of the IgG type. The immunoglobulin fraction markedly inhibited normal bone marrow progenitor cell colony formation, whereas the non-immunoglobulin fraction was not inhibitory. The presence of inhibitory serum immunoglobulin correlated with the hematologic status of the patient. We conclude that the development of PRCA in patients with B cell lymphoma may be due to a serum IgG inhibitor of bone marrow progenitor cell growth.
Key words: Pure red cell aplasia, erythropoiesis, B cell lymphoma, bone marrow progenitor cells, immunoglobulin, serum inhibitor, colony forming units.
INTRODUCTION
some cases, the P R C A is due to antibodies directed against erythroid progenitor cells or growth factors [1]. More recently, the role of the cellular immune system in promoting and suppressing erythropoiesis has been an area of intense interest [9]. In this study, we isolated and partially characterized a serum inhibitor of bone m a r r o w progenitor cell growth from a patient with B cell nonHodgkin's lymphoma. The in-vitro activity of the serum inhibitor correlated with the hematologic status of the patient.
ACQUIRED pure red cell aplasia ( P R C A ) is a rare hematologic disorder that is characterized by severe erythroid hypoplasia of the b o n e marrow and the absence of a reticulocytosis [1]. Approximately 40% of the reported cases of P R C A have been associated with a t h y m o m a . O t h e r cases have been associated with immunologic or a u t o i m m u n e disorders such as myasthenia gravis, thyroiditis, systemic lupus erythematosis and r h e u m a t o i d arthritis [1]. Only a few cases of P R C A have been associated with lymphoproliferative disorders such as B cell nonHodgkin's l y m p h o m a [2], H o d g k i n ' s disease [3], and T or B cell chronic lymphocytic leukemia [4-8]. In
CASE REPORT AND METHODS
Case report In September of 1985, a 58-year old white male was admitted to the hospital with generalized lymphadenopathy and the recent onset of night sweats. A left cervical lymph node biopsy specimen showed follicular non-Hodgkin's lymphoma of the small cleaved lymphocytic type. Immunohistochemical stains of frozen tumor tissue revealed the presence of monoclonal B cells bearing surface IgM of the lambda light chain type. The hemoglobin was 16.3 g/dl and the white cell count was 6.4 x 103/cmm, with 48% segmented neutrophils, 10% bands, 28% lymphocytes, 9% monocytes, 4% eosinophils and 1% b asophils. The platelet count was 164 x 103/cmm. A bone marrow specimen was positive for lymphoma. In the bone marrow smears, large
Abbreviations: BFU-E, burst forming units---erythrocytes; COP, cyclosphosphamide, vincristine, prednisone; COP-BLAM, cyclophosphamide, vincristine, prednisone, bleomycin, Adriamycin, procarbazine; CFU-E, colony forming units---erythrocytes; CFU-GEMM, colony forming units--granulocyte/erythrocyte/monocyte/megakaryocytes; CFU-GM, colony forming units--granulocyte/ monocyte; PRCA, pure red cell aplasia; SAS, saturated ammonium sulfate; SDS, sodium dodecyl sulfate. Correspondence to: Dr Dennis D. Weisenburger, Department of Pathology & Microbiology, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105, U.S.A. 279
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R. ALTERet al.
numbers of atypical lymphoid cells were present. The myeloid and erythroid series were relatively decreased in number, but maturation was normal. Iron stores were also normal. Figure 1 shows the peripheral blood counts during the patients' clinical course. After the initial diagnosis, written informed consent was obtained and the patient was treated with cyclophosphamide, vincristine, and prednisone (COP). After seven courses of chemotherapy, the white cell count was 4.8 x 103/cmm, and the hemoglobin was 10g/dl with a reticulocyte count of less than 0.1%. At the same time, the lymphoma had responded dramatically to chemotherapy. However, a bone marrow aspirate now showed aplasia of the erythroid series, whereas myeloid precursors and megakaryocytes were present in normal numbers. Atypical lymphoid cells still comprised 20% of the nucleated cells in the marrow aspirate smears. At this time, the patient received six cycles of cyclophosphamide, vincristine, prednisone, bleomycin, adriamycin and procarbazine (COPBLAM). In April of 1986, serum and bone marrow samples were obtained for cell culture in vitro and inhibition studies. The bone marrow aspirate at that time revealed normal cellularity, with early recovery of the erythroid series. The marrow aspirate smears also showed persistent involvement by lymphoma. The white cell count was 4.3 x 103/ cmm, and the hemoglobin was 10.6 g/dl with a reticulocyte count of 1.5%. As the hemoglobin level and reticulocyte counts continued to improve, a second serum sample for in-vitro culture studies was obtained in May. The white cell count was 2.6 x 103/cram, and the hemoglobin level was 11.4 g/dl with a reticulocyte count of 3.4%. A third serum sample for in-vitro culture studies was obtained in November of 1986. At that time, the patient was well with a hemoglobin of 13.3 g/dl and a reticulocyte count of2.5%. Although he had persistent lymphoma in the bone marrow, there was no other evidence of disease.
Bone marrow culture assays Peripheral blood and bone marrow mononuclear cells were separated from freshly-drawn heparinized blood and bone marrow, respectively, by density sedimentation using lymphocyte separation medium [10[. The colony forming units--granulocyte/fnonocyte (CFU-GM) assay was performed according to the agar method of Robinson and Pike [11]. In this assay, 1 x 105 mononuclear cells in 1 ml of 0.3% in McCoy's medium was layered onto an underlayer of normal or patient peripheral blood leukocytes (1 x 106 normal or patient peripheral blood leukocytes in 1 ml of 0.5% agar in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and antibiotics). The cultures were incubated at 37°C with 5% CO~ and 95% air in a humidified atmosphere for 14 days. At the end of this period, groups of 50 or more cells were scored as colonies. The colony forming units--granulocyte/ erythrocyte/monocyte/megakaryocyte (CFU-GEMM) assay was performed using a modification of the method of Fauser and Messner [12]. In this assay, 5 x 104 cells were plated in 1 ml of 1.2% methylcellulose with 2.0 I.U. of human urinary erythropoietin (Cancer Research Laboratories, Vancouver, British Columbia). Groups of greater than 50 cells were scored as colonies on day 14. In the same assay, the colony forming u n i t s I e r y t h r o c y t e (CFU-E) and burst forming u n i t s I e r y t h r o c y t e (BFU-E) were scored on days 7 and 16, respectively. For the serum inhibition assays, we added either 0.1 ml of serum from
the patient or 0.1 ml of pooled normal sera to the above culture dishes.
Precipitation of immunoglobulin Immunoglobulin was precipitated from patient sera and normal pooled sera using saturated ammonium sulfate (SAS). The sera was mixed slowly with SAS ( p H 7.0) at a ratio of 6 : 4, and the mixture was incubated for 15 minutes at room temperature to allow complete precipitation. The mixture was then centrifuged at 1500 r.p.m, and the supernatant was collected. The precipitate was then reconstituted to the original volume with phosphate buffered saline. The reconstituted precipitate was then reprecipitated with SAS. Both the final reconstituted precipitate (immunoglobulin-rich fraction) and the first supernatant (non-immunoglobulin fraction) were dialyzed against phosphate buffered saline, and the purified fractions were characterized and used in the in-vitro colony inhibition assays. Electrophoretic analysis The immunoglobulin and non-immunoglobulin fractions were analyzed on sodium dodecyl sulfate (SDS) polyacrylamide gel (BioRadLab, Richmond, CA) according to the method of Laemmli [13]. Equal volumes of immunoglobulin and non-immunoglobulin fractions were loaded on 12% SDS polyacrylamide gel and electrophoresed for 4-6 hours, After electrophoresis, the gel was stained with Coomassie Blue (Sigma Chemical Co., St Louis, MO), and then destained with a solution consisting of 25% methanol, 10% acetic acid and 65% water. Ouchterloney immunodiffusion The type of immunoglobulin present in the SAS precipitate was determined by Ouchterloney gel immunodiffusion [14]. Fifty microliters of the immunoglobulin or non-immunoglobulin fractions were placed in the center wells of 1% BactoAgar (Difco, Detroit, MI) in a Petri dish. Antisera to human immunogloblins G (IgG) or M (IgM) were placed in the outer wells of the Petri dishes. The Petri dishes were then incubated at room temperature in a humidified atmosphere for 48 hr, and then examined for the development of specific immunoprecipitation lines using a light box. Precipitation lines were photographed directly without staining. RESULTS
Bone marrow cultures using patient sera T h e effects of p a t i e n t s e r a on t h e g r o w t h o f n o r m a l b o n e m a r r o w C F U - G M is s h o w n in T a b l e 1. T h e p a t i e n t s e r u m s a m p l e s u s e d in this assay w e r e o b t a i n e d at t h r e e d i f f e r e n t t i m e s d u r i n g the clinical course (Fig. 1). P o o l e d n o r m a l s e r a was u s e d as t h e c o n t r o l sera. T h e r e was d r a m a t i c i n h i b i t i o n o f n o r m a l b o n e m a r r o w C F U - G M f o r m a t i o n by p a t i e n t s e r u m o b t a i n e d at the t i m e o f m a r k e d e r y t h r o i d h y p o p l a s i a of the b o n e m a r r o w . A s t h e h y p o p l a s i a r e s o l v e d , C F U - G M g r o w t h r e t u r n e d to n o r m a l . W h e n p a t i e n t b l o o d m o n o n u c l e a r cells w e r e u s e d as f e e d e r s to p r o v i d e colony s t i m u l a t i n g factors, C F U - G M form a t i o n was mildly d e c r e a s e d , b u t r e t u r n e d to n o r m a l with the a d d i t i o n of n o r m a l sera. In t h e a b s e n c e o f
281
P u r e r e d cell a p l a s i a a n d B cell l y m p h o m a T A B L E 1. EFFECTS OF PATIENT SERUM AND P E R I P H E R A L BLOOD MONONUCLEAR CELL FEEDERS ON NORMAL BONE M A R R O W CFU-GM FORMATION
Mean number of CFU-GM colonies* NBM + NS NBM + PS1 NBM + PS2
NBM Feeder Feeder Feeder Patient
No. I t No. 2t No. 3t feeder:~
180 209 123 64
+- 8 -+ 13 +- 9 - 16
143 - 22 179 +- 14 158 - 11 162 -+ 9
0 0 0 9 -+ 1
NBM + PS3
89 -+ 10 13 +- 1 76 +- 17 ND
ND 192 -+-2 146 - 3 ND
CFU-GM, colony forming units--granulocyte/monocyte; * mean --+-standard error; t normal peripheral blood feeder layers; ~: patient peripheral blood mononuclear cells from 4/86 used in feeder layers; NBM, normal bone marrow; NS, normal pooled sera; PS1, patient serum from 4/86; PS2, patient serum from 5/86; PS3, patient serum from 11/86; ND, not done.
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FIG. 1. Peripheral blood counts during the clinical course of a patient with pure red aplasia and B-cell lymphoma; • represents the transfusion of packed red blood cells; * indicates the times when serum samples were obtained for inhibition studies; COP = cyclophosphamide, vincristine, and prednisone; COP-BLAM = cyclophosphamide, vincristine, prednisone, bleomycin, adriamycin and procarbazine.
patient serum, patient bone m a r r o w formed normal numbers of C F U - G M colonies (data not shown). The effects of patient sera on the formation of erythroid and non-erythroid colonies by normal bone marrow in the C F U - G E M M assay are shown in Table 2. Patient serum obtained at the time of erythroid hypoplasia showed the greatest colony inhibition as c o m p a r e d to the two later sera. As the patient's hematologic status improved, decreasing inhibition of colony formation was seen. B F U - E colony formation was the most markedly inhibited by the first patient serum sample, but returned to normal with subsequent serum samples.
Characterization of the serum inhibitor Serum immunoglobulin was precipitated using
saturated a m m o n i u m sulfate, and the immunoglobulin and non-immunoglobulin fractions were analyzed by electrophoresis. Figure 2 shows the electrophoretic analysis of the immunoglobulin (lane A) and non-immunoglobulin (lane B) fractions. The non-immunoglobulin fraction consisted of albumin and other serum proteins, without the 160,000 mol. wt band seen in the immunoglobulin fraction. Thus, the electrophoretic analysis d e m o n s t r a t e d that the non-immunoglobulin fraction did not contain significant amounts of immunoglobulin, whereas the immunoglobulin fraction consisted predominantly of immunoglobulin. Figure 3 shows the Ouchterloney immunodiffusion pattern of the immunoglobulin fraction. The majority of immunoglobulin in the immunoglobulin fraction was of the I g G type. The
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R. ALTER et al.
TABLE 2. EFFECTS OF PATIENT SERA ON THE FORMATION OF ERYTHROID AND NON-ERYTHROID COLONIES BY NORMAL BONE MARROW
NBM CFU-E CFU-GEMM BFU-E
Red 54-+ 1 73 -+ 4 106-+4
Other 11+-3 48 + 6 54-+7
NBM + NS Red 71-+11 80 -+ 1 128 + 2
Mean number of colonies* NBM + PS1 NBM + PS2
Other 65-+8 56 -+ 2 67-+3
Red 31-+2 32 -+ 2 28-+2
Other 40 + 3 32 + 1 37 + 1
Red 57+2 51 -+ 1 112+3
Other 48-+6 36 -+ 3 61-+-3
NBM + PS3 Red ND 82 + 1 107-+2
Other ND 46 -+ 3 54-+3
* Mean -+ standard error; CFU-E, colony forming units--erythroid, scored on day 7; CFU-GEMM, colony forming units--granulocyte/erythrocyte/monocyte/megakaryocyte, scored on day 14; BFU-E, burst forming units--erythroid, scored on day 16; NBM, normal bone marrow; NS, normal pooled wera; PS1, patient serum from 4/86; PS2, patient serum from 5/86; PS3, patient serum from 11/86; Red, colonies with hemoglobinization; Other, colonies without hemoglobinization; ND, not done. non-immunoglobulin fraction did not contain detectable immunoglobulin (not shown). Bone marrow cultures using the serum immunoglobulin fraction The immunoglobulin and non-immunoglobulin fractions were then tested for their inhibitory activity in the bone marrow colony assays. Table 3 shows that the serum inhibitor was present in the immunoglobulin fraction, but not in the non-immunoglobulin fraction. The first serum immunoglobulin fraction resulted in prominent inhibition of normal colony growth in all three assays. As the hematologic status of the patient improved, decreasing inhibition of colony formation by the immunoglobulin fraction was seen. The non-immunoglobulin fraction did not inhibit normal bone marrow colony formation significantly.
DISCUSSION Pure red cell aplasia (PRCA) is a well-defined clinical disorder that may arise by at least three different immunopathogenic mechanisms. These include antibodies to erythroblasts [15-19] or erythropoietin-responsive cells [20], antibodies to erythropoietin [21, 22], and T-cell suppression of erythropoiesis [6]. In adults with PRCA, all three mechanisms have been demonstrated. In transient erythroblastopenia of childhood, however, it appears that most cases are due to an IgG inhibitor directed against erythroid progenitor cells [23]. In this report we describe a patient who developed P R C A after receiving chemotherapy for follicular B cell non-Hodgkin's lymphoma. To our knowledge, this is the only reported study of P R C A associated with follicular B-cell lymphoma using bone marrow culture techniques. We have clearly demonstrated the presence of a serum inhibitor to bone marrow colony formation. Additional studies revealed that
the inhibitor was a serum immunoglobulin of the IgG type. The presence of the inhibitor correlated well with the hematologic status of the patient. Colony inhibition was demonstrated in all of the assays used, and was most pronounced using the first immunoglobulin fraction (PSI-Ig) in the CFU-E assay (Table 3). Interesingtly, inhibition of myeloid colony formation in the C F U - G M and C F U - G E M M assays (non-hemoglobinized colonies) was also seen. This latter finding indicates the presence of cross-reacting inhibition in vitro, which was not evident in vivo. Similar bone marrow culture studies of a patient with B-cell chronic lymphocytic leukemia and P R C A have been reported by Mangan & D'Alessandro [5]. In their studies, an inhibitor directed against the erythroid colony forming unit (CFU-E) was demonstrated. Further studies, however, suggested that the suppression of CFU-E formation resulted from a Tcell derived inhibitor. A patient with T-cell chronic leukemia and P R C A has also been described by Hansen et al. [8]. Erythroid burst forming units (BFU-E) were shown to be inhibited by suppressor T-lymphocytes. The mechanisms by which these Tcell inhibitor factors induced bone marrow colony inhibition were not studied further. To our knowledge, no other case of P R C A due to a serum immunoglobulin inhibitor in a patient with a lymphoproliferative disorder has been reported. In conclusion, we have identified and characterized a serum immunoglobulin inhibitor of bone marrow colony formation in vitro, the presence of which correlated well with the hematologic status of our patient. Although we do not know the exact origin of the inhibitor, we presume that it was not produced by the tumor cells, since the lymphoma was of the lgM type and persisted throughout the clinical course. We speculate that the inhibitor was produced by non-neoplastic B cells as a result of altered regulation of the immune system, perhaps secondary to chemotherapy. When the patient was treated with
A
B
C
FIG. 2. Electrophoretic pattern of the immunoglobulin and non-immunoglobulin fractions precipitated from patient serum; lane A, immunoglobulin fraction (arrow indicates the immunoglobulin band); lane B, non-immunoglobulin fraction; lane C, marker proteins.
283
FIG. 3. Ouchterloney immunodiffusion of the immunoglobulin fraction of patient serum with antibodies to human IgM and IgG; well A, anti-human IgM; well B, patient immunoglobulin fraction; well C, anti-human IgG.
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Pure red cell aplasia and B cell lymphoma
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more intensive chemotherapy, the P R C A resolved in the presence of persistent lymphoma. These findings suggest that i m m u n e dysregulation may play a role in the etiology of unexplained or persistent cytopenias associated with chemotherapy. Additional studies of similar patients are clearly needed to further our understanding of this p h e n o m e n o n .
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