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Increased proliferation of eosinophil clusters in myelodysplastic syndromes
Pergamon 0145-2126(95)00083-6
INCREASED
PROLIFERATION OF EOSINOPHIL CLUSTERS MYELODYSPLASTIC SYNDROMES
IN
Michiaki Koike, Taijiro Ishiyama, Akihiro Yokoyama, Keiichirou Kawakami, Takeshi Nakamaki, Shigeru Tomoyasu and Nobuyoshi Tsuruoka Department of Hematology, Showa University School of Medicine, Tokyo, Japan (Received 25 January 1995. Accepted 25 May 1995) Abstract-A patient with myelodysplastic syndromes (MDS) developed eosinophilia during treatment with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). To study the mechanism of this eosinophilia, we investigated the proliferation of eosinophil colony-forming units (CFU-Eo) in nine patients and four healthy controls. Eosinophil clusters increased significantly in the patients (P
units, granulocyte-macrophage
colony-stimulating
various hematological disorders [4-71, including aplastic anemia (AA) and myelodysplastic syndromes (MDS). In these conditions, administration of rhGMCSF results in an increase in circulating neutrophils, eosinophils and monocytes [S, 91. In addition, when patients with primary MDS are treated with GM-CSF, eosinophilia is commonly observed [lo, 111. We encountered a patient with refractory anemia (RA) who developed severe eosinophilia as a result of rhGM-CSF therapy. To examine the mechanism of this eosinophilia, we investigated the proliferation of eosinophil colony-forming units (CFU-Eo) in the present study.
Introduction Eosinophil production and function are regulated by cytokines such as granulocyte-macrophage colonystimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 [l-3]. The cytokine GM-CSF is a stimulator of pluripotent as well as granulocytic/monocytic precursor cells. The gene for GM-CSF has been cloned, and recombinant human (rh) GM-CSF is now available in amounts sufficient for clinical use in the treatment of Abbreviations: AA, aplastic anemia;BFU-E, erythroid burstforming units; CFU-Eo, eosinophil colony-forming units; CFU-GM, granulocyte/macrophage colony forming units; GM-CSF, granulocyte-macrophage colony-stimulating factor; FCS, fetal calf serum; G-CSF, granulocyte colony-stimulating factor; Hb, hemoglobin; IL, interleukin; MD& myelodysplastic syndromes;MN, mononuclear; NCC, nuclear cell count; PHALCM, phytohemagglutinin-lymphocyte conditioned medium; RA, refractory anemia; RAEB, RA with excess of blasts; RAEB-T, RA with excess of blasts in transformation; RARS,
Case Report A 70-year-old woman developed fatigue in June 1991 and was admitted to our hospital with anemia in October (Table 1, patient No. 5). On admission, she had purpura on the extremities, but physical examination was otherwise normal. The hemoglobin (Hb) was 10.1 g/dl, the white blood cell count (WBC) was 2OOO/pl (Seg. 28%, Lymph. 64, Mono. 3, Eos. 1, and Baso. 4), and the platelet count was 1.7 x 104/p1. The bone marrow
RA with ring sideroblasts; rh, recombinant human; WBC, white blood cell count. Correspondence to: Michiaki Koike, M.D., Department of Hematology, Showa University School of Medicine, l-5-8 Hatanodai, Shinagawa, Tokyo, Japan (Tel: 03 3784 8244; Fax: 03 3784 8250). 915
916
M. Koike et al.
the peripheral blood (Fig. 1). However, the Hb and platelet count did not improve. When granulocyte colony-stimulating factor (G-CSF) was administered at 125 and 250 pg/day, the WBC increased without eosinophilia. Although the red blood cell count failed to respond, the platelet count increased gradually (Fig. 2) and she was discharged. Materials and Methods
Fig. 1. Hypogranular eosinophils in an MDS patient treated with GM-CSF (Patient No. 5) (x 1000).
nuclear
cell count (NCC)
was 11.2 x 104/p1 (myelo-
blasts 1.3% and megakaryocytes 32/ul). Severe dysplasia of the erythroblastic, and megakaryocytic
granulocytic
series was observed. She also had a
number of abnormal karyotypes (- 8, - 12, - 17, - 18, lq-, 22q, +4mar). The patient was diagnosed with IL4 on the basis of these findings. When GM-CSF was administered at a dose of 125 pi/day to improve pancytopenia (informed consent was obtained),
an increased WBC
and eosino-
philia resulted in numerous hypogranular eosinophils in
WBC/“I ‘0°06000-
I By
_I GY-CSF(125rg/d.sy)
EM
uEu
Patients Nine patients aged 66.4kl4.5 years (mean +S.D.) with MDS: five with RA, one RA with ring sideroblasts (RARS), one RA with excess of blasts (RAEB), one RAEB featuring eosinophilia and one RA with excess of blasts in transformation (RAEB-T) were studied (Table 1). Four MDS patients had an abnormal karyotype, and three patients died during follow-up. Four normal controls were studied for comparison. Reagents The rhG-CSF was obtained from Chugai Pharmaceutical Company (Tokyo, Japan) and was stored at -20°C until use. The rhGM-CSF was obtained from Schering-Plough (Osaka, Japan). Assays for GM-CSF, IL-3 and IL-5 The GM-CSF concentrations in serum from normal controls and MDS patients were measured by ELISA (Otsuka Assay Kit; Otsuka Corp., Otsuka, Japan, according to the manufacture’s directions). Interleukin-3 and IL-5 concentrations in serum from the MDS patient with eosinophilia (patient No. 9) were measured by Dr H. Enokihara (Third Department of Internal Medicine, Dokkyo University School of Medicine) using ELISA. The detection limits of the assays were 20 pgiml for GM-CSF, 50 pg/ml for IL-3 and 78 pgiml for IL-5. Preparation of bone marrow mononuclear cells (MN cells) Approximately 3 ml of bone marrow was collected in a heparinized syringe. Samples were diluted 2:l with RPM1 1640, and one part of the diluted bone marrow was gently overlaid on to three parts of Lymphoprep (sodium metrizoate-Ficoll solution, Nygaard, Oslo, Norway). Samples were centrifuged at 450xg for 30 min, and the MN cells at the interface were collected and washed twice with RPM1 1640.
OCT.
NOV.
DEC. 1991
JAN.
1992
Fig. 2. Clinical course in the MDS patient with severe eosinophilia with GM-CSF therapy (Patient No. 5). Erythoid series (E), myeloid series (M), stab (St), segment (Seg), eosinophil (Eos), platelet (PLT), concentrated (Cone), red blood cell (RBC).
Preparation of phytohemagglutinin-lymphocyte conditioned medium (PHA-LCM) Approximately 10 ml of peripheral blood was collected in a heparinized syringe and diluted 1:l with RPM1 1640. Samples were centrifuged at 450xg for 30 min, MN cells at the interface were collected, and washed twice with RPM1 1640. Then 1 x lo6 cells were resuspended in 1.0 ml of RPM1 1640 containing 30 g of PHA (Difco Laboratories, Stanmor, Australia) and cultured for 7 days in a humidified 5% CO* incubator at 37°C. The culture suspensions were centrifuged at 1OOOxg for 5 min, and the supernatant was collected as PHA-LCM. Eosinophil colony formation Eosinophil colony formation was assessed according to the methods of Metcalf and Horie [12, 131. Peripheral blood MN
917
CFU-Eo in MDS Table 1. Clinical profile of the patients with MDS Patient Age/sex No.
PB*% eosino
BMP% eosino
BM% blast
13.2 2.4 10.6 34.2 1.7
3.0 3.0 0.0 4.0 1.0
2.6 0.8 0.1 1.9 -
1.0 0.3 0.0 1.5 1.3
9.2
4.1
4.0
1.6
12.4 8.4 7.4
0.9 4.0 10.1
1.0 1.0 16.0
0.5 49.8
Diagnosis
WBC/pl
73/M 59/F 85/M 38/F 70/F
RA RA RA RA RA
3000 3800 3100 4500 2000
4.6 6.3 8.0 6.3 10.1
6
76/F
RARS
3400
7 8 9
48/F 69/F 80/M
3200 1600 2800
RAEB RAEB-T RAEB (eosinonhilial
Hb g/d1 Plt x 104/pl
Abnormal chromosome
7q+?8 normal -5,-8,-12 1q - ,22q - ,+mar -8,-12,-17,-18 0.9 5q - ,7q - ,+mar -12,-13,-20 5.3 +8,lp+ 21.4 normal 5.8 i(lW
Duration of survival (month) 23+overt 12+ 36 dead 6+ 10 dead 8 dead
lO+ 6+overt 11+
*PB = peripheral blood. TBM = bone marrow.
cells (1 x 106) were plated in 1.0 ml of RPM1 1640 containing 7.5% fetal calf serum (FCS), 7.5% PHA-LCM, and 0.3% agar gel (Bactoagar, Difco). The cells were cultured in Petri dishes for 14 days in a 5% CO2 incubator at 37°C. The dose response to G-CSF and GM-CSF was determined under varying concentrations (data not shown) in continuous exposure.
apple-green cytoplasm. Discrete aggregates of 40 or more positive cells were scored as colonies, and aggregates of less than 40 positive cells were scored as clusters (Fig. 3).
Statistical analysis Statistical analysis was performed using the Student’s t-test with P
Identification of eosinophil colonies The agar culture was tipped gently on to microscope slides, air-dried, and stained for 1 h with Luxol-Fast-Blue. The stain consisted of 0.1 g of stain powder (Gurr MBS, Searle Diagnostic, High Wycombe, U.K.) dissolved in 70% ethanol saturated with urea. The slides were washed with tap water for 15 min, followed by staining with hematoxylin for 2 min [14, 151. Under the microscope, stained cells were readily identified by their bright
Results Effects of G-CSF and GM-CSF on eosinophil colony formation in normal controls As shown in Fig. 4, there were 3.90 + 2.4 eosinophil colonies and 27.5 f 23.8 clusters per dish in the control
Fig. 3. Left: eosinophil cluster (x600); colony (x 600).
Right: eosinophil
M. Koike et al.
m
eosinophil
colonies
0
eosinophil
clusters
T
ti
Control
T
G-CSF
T
T
i
-
%I Fig. 4. Effects of G-CSF and GM-CSF on eosinophil colony formation in normal controls. The data are shown as mean f SD. for eosinophil colonies and clusters/lo6 peripheral cells. group. Culture with GM-CSF increased the number of eosinophil colonies (PC 0.01) but not clusters. G-CSF had no effect on either.
ony forming units (CFU-GM) and erythroid burstforming units (BFU-E) has been reported in MDS [16191. The present study found that eosinophil cluster formation was increased in MDS. We also found that GM-CSF induced proliferation of eosinophil clusters in MDS patients more than in normal controls. Our results may be explained by an increase in abnormal eosinophil progenitor cells that show hyper-responsiveness to GMCSF. Recent studies have clearly documented that eosinophil production is regulated by GM-CSF, IL-5 and IL-3. However, we found that serum cytokine concentrations in MDS patients were within the normal range. The GM-CSF-stimulated neutrophils in MDS generally remain dysplastic in appearance during and following treatment [20]. Similarly, we found many hypogranular eosinophils in MDS patient No. 5 during treatment with GM-CSF. Duhamel [21] has reported eosinophilia in 37% of MDS patients, and Rios et al. [22] found increased eosinophils in 26% of biopsies from MDS patients. In these studies, eosinophilia has shown a certain relationship to survival by univariate analysis. Estey et al. reported that MDS patients who failed to respond to high dose GM-CSF, and who
x=MEB kJahwMe)
Effects of G-CSF and GM-CSF on eosinophil colony formation in MDS patients As shown in Fig. 5, MN cells from MDS patients yielded 5.0 f 6.8 eosinophil colonies and 100.0 + 75.9 clusters per dish. Eosinophil clusters in MDS were significantly increased (P < O.Ol), but eosinophil colonies were not significantly increased than normal controls. The MN cells from the MDS patient with eosinophilia (patient No. 9) gave rise to 26.0 eosinophil colonies and 232.0 clusters. The GM-CSF increased the number of eosinophil colonies (0.01
232
m
eo&ophil
colonies
0
eesinophil clusters
a
x240
180
n=8
O
4 G.
Fig. 5. Effects of G-CSF and GM-CSF on eosinophil colony formation in MDS patients including RAEB with eosinophilia (patient No. 9). The data are shown as meanfS.D. for eosinophil colonies and clusters/lo6 peripheral cells.
CFU-Eo in MDS &ml
Serum
GM-CSF
loo-
500 $"gd
-'
0
00 0 F
0
MDS
Normal n=4
n=6
Figure 6. GM-CSF, IL-5 and IL-3 concentrations in serum from controls and MDS patients showed a rise in eosinophils rather than neutrophils, may constitute a distinct subset of the myelodysplastic population with unique biological and prognostic features [23]. Thus it is possible that defective myeloid maturation potential in MDS may result in increased abortive eosinophil colony formation. We identified one RAEB patient with eosinophilia and increased CFU-Eo, but serum IL-3, IL-5 and GMCSF concentrations were not increased. Thus, eosinophilia was likely to be due to abnormal eosinophil progenitor cells rather than elevated cytokine concentrations. Future investigations may determine if increased eosinophil cluster and colony formation is related to the prognosis of MDS. Acknowledgements-We wish to thank Dr H. Enokihara (Third Department of Internal Medicine, Dokkyo University School of Medicine) for measuring the IL-5 and IL-3 concentrations. We also acknowledge with thanks the technical assistance of Mrs Y. Murata (Juntendo University School of Medicine). We thank Mr Albert Simpson for checking the English manuscript. References 1. Fermand J. P., Mitjavila M. T., Couedic J. P., Tsapis A., Berger R., Modigliani R., Seligmann M., Brouet J. C. & Vainchenker W. (1993) Role of granulocyte-macrophage colony-stimulating-factor, interleukin-3 and interleukin-5 in the eosinophilia associated with T cell lymphoma. Br. J. Haematol. 83, 359. 2. Lopez A. F., Williamson J. & Gamble J. R. (1986) Recombinant human granulocyte-macrophage colonystimulating factor stimulates in vitro mature human neutrophil and eosinophil function surface receptor expression and survival. J. Clin. Invest. 78, 1220. 3. Clutterbuck E. J. & Sanderson C. J. (1990) Regulation of
919
human eosinophilia precursor production by cytokine: a comparison of recombinant human interleukin-1 (rhIL-1), rhIL-3, rhIL-5, rhIL-6 and rh granulocyte-macrophage colony-stimulating factor. Blood 75, 1774. 4. Golde D. W. & Gasson J. C. (1988) Cytokines: myeloid growth factors. In Inflammation. Basic Principles and Clinical Correlates (Gallin J. I., Goldstein I. M. & Synderman R., Eds). New York, Raven Press. 5. Clark S. C. & Kamen R. (1987) The human hemopoietic colony stimulating factors. Science 236, 1229. 6. Metcalf D. (1986) The molecular biology and functions of the granulocyte-macrophage colony-stimulating factors. Blood 67, 257. 7. Gasson J. C., Weisbart R. H., Gerber S. E., Clark S. C. & Golde D. W. (1984) Purified human granulocyte-macrophage colony-stimulating factor direct action on neutrophils. Science 226, 1339. 8. Champlin S. D., Nimer S. D., Ireland P., Oette D. H. & Golde D. W. (1989) Treatment of refractory aplastic anemia with recombinant human granulocyte-macrophage colony-stimulating factor. Blood 73, 694. 9. Antin J. H., Smith B. R., Holmes W. & Rosenthal D. S. (1988) Phase I/II study of recombinant human granulocyte-macrophage colony-stimulating factor in aplastic anemia and myelodysplastic syndrome. Blood 72, 705. 10. Willemze R., Lelv N. van der, Zwierzina H., Suciu G., Gerhartz H., Laba; B., Visani GI, Peetermans M. E., Jacobs A., Stryckmans P., Fenaux P., Haak H. L., Ribeiro M. M., Baumelou E., Baccarani M., Mandeelli F., Jaksic B., Louwagie A., Thyss A., Hayat M., Cataldo F. de, Stern A. C. & Zittoun R. on behalf of the EORTC Leukemia Cooperative Group (1992) A randomized phase I/II multicenter study of recombinant human granulocytemacrophage colony-stimulating factor (GM-CSF) therapy for patients with myelodysplastic syndromes and a relatively low risk of acute leukemia. Ann. Hematol. 64, 173. 11 Thompson J. A., Lee D. J., Kidd P., Rubin E., Kaufmann J., Bonnen E. M. & Fefer A. (1989) Subcutaneous granulocyte macrophage colony stimulating factor in patients with myelodysplastic syndrome: toxicity, pharmacokinetics and hematological effects. J. Clin. Oncol. 7, 629. 12. Johnson G. R. & Metcalf D. (1979) Detection of a new type of mouse eosinophil colony by Luxol-Fast-Blue staining. Expl Hematol. 8, 549. 13. Horie S., Sugimoto M. & Wakabayasi Y. (1989) In vitro growth of eosinophil colonies from normal human peripheral blood. Acta. Haematol. 52, 608. 14. Shoham D., David B. & Rozenszajn A. (1974) Cytochemical and morphologic identification of macrophages and eosinophils in tissue cultures of normal human bone marrow cells. Blood 49, 835. 15. Dresch C., Johnson G. R. & Rozenszajn A. (1974) Cytochemical and morphologic identification of macrophages and eosinophils in tissue cultures of normal human bone marrow. Blood 49, 221. 16. Juvonen E., Partanen S., Knuutil A. & Ruutu T. (1989) Colony formation by megakaryocyte progenitors in myelodysplastic syndromes. Eur. J. Haematol. 42, 389. 17. Koeffler H. P. (1986) Myelodysplastic syndromes (preleukemia). Semin. Hematol. 23, 284. 18. Tennant G. B., Bowen D. R. & Jacobs A. (1991) Colonycluster ration and cluster number in culture of circulating myeloid progenitors as indicators of high-risk myelodysplasia. Br. J. Haematol. 23, 284.
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19. Yoshida Y. (1987) Biology of myelodysplastic syndromes. Intl J. Cell Cloning 23, 284. 20. Gradisher W. J., Le Beau M. M., O’Laughlin R., Vardiman J. W. & Larson R. A. (1992) Clinical and cytogenetic responses to granulocyte-macrophage colony-stimulating factor in therapy-related myelodysplasia. Blood 80, 2463. 21. Duhamel C. (1974) Histopathologie deLa Moelle. Osseuse Masson, Paris. 22. Rios A., Carizo M. A., Vallespi T., Sanz G., Torrabadella M., Comis F., Ruiz C. & Sanmiguel J. F. (1990) Bone
marrow biopsy in myelodysplastic syndromes: morphological characteristics and contribution to the study of prognostic factors. Br. J. Haematol. 75, 26. 23. Estey E. H., Kurzrock R., Talpaz M., Mccredie K. B., O’Brein S., Kantarjian H. M., Keating J., Deisseroth A. B. & Gutterman J. U. (1991) Effects of low doses of recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) in patients with myelodysplastic syndromes. Br. J. Haematol. 77, 291.
INCREASED
PROLIFERATION OF EOSINOPHIL CLUSTERS MYELODYSPLASTIC SYNDROMES
IN
Michiaki Koike, Taijiro Ishiyama, Akihiro Yokoyama, Keiichirou Kawakami, Takeshi Nakamaki, Shigeru Tomoyasu and Nobuyoshi Tsuruoka Department of Hematology, Showa University School of Medicine, Tokyo, Japan (Received 25 January 1995. Accepted 25 May 1995) Abstract-A patient with myelodysplastic syndromes (MDS) developed eosinophilia during treatment with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). To study the mechanism of this eosinophilia, we investigated the proliferation of eosinophil colony-forming units (CFU-Eo) in nine patients and four healthy controls. Eosinophil clusters increased significantly in the patients (P
units, granulocyte-macrophage
colony-stimulating
various hematological disorders [4-71, including aplastic anemia (AA) and myelodysplastic syndromes (MDS). In these conditions, administration of rhGMCSF results in an increase in circulating neutrophils, eosinophils and monocytes [S, 91. In addition, when patients with primary MDS are treated with GM-CSF, eosinophilia is commonly observed [lo, 111. We encountered a patient with refractory anemia (RA) who developed severe eosinophilia as a result of rhGM-CSF therapy. To examine the mechanism of this eosinophilia, we investigated the proliferation of eosinophil colony-forming units (CFU-Eo) in the present study.
Introduction Eosinophil production and function are regulated by cytokines such as granulocyte-macrophage colonystimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 [l-3]. The cytokine GM-CSF is a stimulator of pluripotent as well as granulocytic/monocytic precursor cells. The gene for GM-CSF has been cloned, and recombinant human (rh) GM-CSF is now available in amounts sufficient for clinical use in the treatment of Abbreviations: AA, aplastic anemia;BFU-E, erythroid burstforming units; CFU-Eo, eosinophil colony-forming units; CFU-GM, granulocyte/macrophage colony forming units; GM-CSF, granulocyte-macrophage colony-stimulating factor; FCS, fetal calf serum; G-CSF, granulocyte colony-stimulating factor; Hb, hemoglobin; IL, interleukin; MD& myelodysplastic syndromes;MN, mononuclear; NCC, nuclear cell count; PHALCM, phytohemagglutinin-lymphocyte conditioned medium; RA, refractory anemia; RAEB, RA with excess of blasts; RAEB-T, RA with excess of blasts in transformation; RARS,
Case Report A 70-year-old woman developed fatigue in June 1991 and was admitted to our hospital with anemia in October (Table 1, patient No. 5). On admission, she had purpura on the extremities, but physical examination was otherwise normal. The hemoglobin (Hb) was 10.1 g/dl, the white blood cell count (WBC) was 2OOO/pl (Seg. 28%, Lymph. 64, Mono. 3, Eos. 1, and Baso. 4), and the platelet count was 1.7 x 104/p1. The bone marrow
RA with ring sideroblasts; rh, recombinant human; WBC, white blood cell count. Correspondence to: Michiaki Koike, M.D., Department of Hematology, Showa University School of Medicine, l-5-8 Hatanodai, Shinagawa, Tokyo, Japan (Tel: 03 3784 8244; Fax: 03 3784 8250). 915
916
M. Koike et al.
the peripheral blood (Fig. 1). However, the Hb and platelet count did not improve. When granulocyte colony-stimulating factor (G-CSF) was administered at 125 and 250 pg/day, the WBC increased without eosinophilia. Although the red blood cell count failed to respond, the platelet count increased gradually (Fig. 2) and she was discharged. Materials and Methods
Fig. 1. Hypogranular eosinophils in an MDS patient treated with GM-CSF (Patient No. 5) (x 1000).
nuclear
cell count (NCC)
was 11.2 x 104/p1 (myelo-
blasts 1.3% and megakaryocytes 32/ul). Severe dysplasia of the erythroblastic, and megakaryocytic
granulocytic
series was observed. She also had a
number of abnormal karyotypes (- 8, - 12, - 17, - 18, lq-, 22q, +4mar). The patient was diagnosed with IL4 on the basis of these findings. When GM-CSF was administered at a dose of 125 pi/day to improve pancytopenia (informed consent was obtained),
an increased WBC
and eosino-
philia resulted in numerous hypogranular eosinophils in
WBC/“I ‘0°06000-
I By
_I GY-CSF(125rg/d.sy)
EM
uEu
Patients Nine patients aged 66.4kl4.5 years (mean +S.D.) with MDS: five with RA, one RA with ring sideroblasts (RARS), one RA with excess of blasts (RAEB), one RAEB featuring eosinophilia and one RA with excess of blasts in transformation (RAEB-T) were studied (Table 1). Four MDS patients had an abnormal karyotype, and three patients died during follow-up. Four normal controls were studied for comparison. Reagents The rhG-CSF was obtained from Chugai Pharmaceutical Company (Tokyo, Japan) and was stored at -20°C until use. The rhGM-CSF was obtained from Schering-Plough (Osaka, Japan). Assays for GM-CSF, IL-3 and IL-5 The GM-CSF concentrations in serum from normal controls and MDS patients were measured by ELISA (Otsuka Assay Kit; Otsuka Corp., Otsuka, Japan, according to the manufacture’s directions). Interleukin-3 and IL-5 concentrations in serum from the MDS patient with eosinophilia (patient No. 9) were measured by Dr H. Enokihara (Third Department of Internal Medicine, Dokkyo University School of Medicine) using ELISA. The detection limits of the assays were 20 pgiml for GM-CSF, 50 pg/ml for IL-3 and 78 pgiml for IL-5. Preparation of bone marrow mononuclear cells (MN cells) Approximately 3 ml of bone marrow was collected in a heparinized syringe. Samples were diluted 2:l with RPM1 1640, and one part of the diluted bone marrow was gently overlaid on to three parts of Lymphoprep (sodium metrizoate-Ficoll solution, Nygaard, Oslo, Norway). Samples were centrifuged at 450xg for 30 min, and the MN cells at the interface were collected and washed twice with RPM1 1640.
OCT.
NOV.
DEC. 1991
JAN.
1992
Fig. 2. Clinical course in the MDS patient with severe eosinophilia with GM-CSF therapy (Patient No. 5). Erythoid series (E), myeloid series (M), stab (St), segment (Seg), eosinophil (Eos), platelet (PLT), concentrated (Cone), red blood cell (RBC).
Preparation of phytohemagglutinin-lymphocyte conditioned medium (PHA-LCM) Approximately 10 ml of peripheral blood was collected in a heparinized syringe and diluted 1:l with RPM1 1640. Samples were centrifuged at 450xg for 30 min, MN cells at the interface were collected, and washed twice with RPM1 1640. Then 1 x lo6 cells were resuspended in 1.0 ml of RPM1 1640 containing 30 g of PHA (Difco Laboratories, Stanmor, Australia) and cultured for 7 days in a humidified 5% CO* incubator at 37°C. The culture suspensions were centrifuged at 1OOOxg for 5 min, and the supernatant was collected as PHA-LCM. Eosinophil colony formation Eosinophil colony formation was assessed according to the methods of Metcalf and Horie [12, 131. Peripheral blood MN
917
CFU-Eo in MDS Table 1. Clinical profile of the patients with MDS Patient Age/sex No.
PB*% eosino
BMP% eosino
BM% blast
13.2 2.4 10.6 34.2 1.7
3.0 3.0 0.0 4.0 1.0
2.6 0.8 0.1 1.9 -
1.0 0.3 0.0 1.5 1.3
9.2
4.1
4.0
1.6
12.4 8.4 7.4
0.9 4.0 10.1
1.0 1.0 16.0
0.5 49.8
Diagnosis
WBC/pl
73/M 59/F 85/M 38/F 70/F
RA RA RA RA RA
3000 3800 3100 4500 2000
4.6 6.3 8.0 6.3 10.1
6
76/F
RARS
3400
7 8 9
48/F 69/F 80/M
3200 1600 2800
RAEB RAEB-T RAEB (eosinonhilial
Hb g/d1 Plt x 104/pl
Abnormal chromosome
7q+?8 normal -5,-8,-12 1q - ,22q - ,+mar -8,-12,-17,-18 0.9 5q - ,7q - ,+mar -12,-13,-20 5.3 +8,lp+ 21.4 normal 5.8 i(lW
Duration of survival (month) 23+overt 12+ 36 dead 6+ 10 dead 8 dead
lO+ 6+overt 11+
*PB = peripheral blood. TBM = bone marrow.
cells (1 x 106) were plated in 1.0 ml of RPM1 1640 containing 7.5% fetal calf serum (FCS), 7.5% PHA-LCM, and 0.3% agar gel (Bactoagar, Difco). The cells were cultured in Petri dishes for 14 days in a 5% CO2 incubator at 37°C. The dose response to G-CSF and GM-CSF was determined under varying concentrations (data not shown) in continuous exposure.
apple-green cytoplasm. Discrete aggregates of 40 or more positive cells were scored as colonies, and aggregates of less than 40 positive cells were scored as clusters (Fig. 3).
Statistical analysis Statistical analysis was performed using the Student’s t-test with P
Identification of eosinophil colonies The agar culture was tipped gently on to microscope slides, air-dried, and stained for 1 h with Luxol-Fast-Blue. The stain consisted of 0.1 g of stain powder (Gurr MBS, Searle Diagnostic, High Wycombe, U.K.) dissolved in 70% ethanol saturated with urea. The slides were washed with tap water for 15 min, followed by staining with hematoxylin for 2 min [14, 151. Under the microscope, stained cells were readily identified by their bright
Results Effects of G-CSF and GM-CSF on eosinophil colony formation in normal controls As shown in Fig. 4, there were 3.90 + 2.4 eosinophil colonies and 27.5 f 23.8 clusters per dish in the control
Fig. 3. Left: eosinophil cluster (x600); colony (x 600).
Right: eosinophil
M. Koike et al.
m
eosinophil
colonies
0
eosinophil
clusters
T
ti
Control
T
G-CSF
T
T
i
-
%I Fig. 4. Effects of G-CSF and GM-CSF on eosinophil colony formation in normal controls. The data are shown as mean f SD. for eosinophil colonies and clusters/lo6 peripheral cells. group. Culture with GM-CSF increased the number of eosinophil colonies (PC 0.01) but not clusters. G-CSF had no effect on either.
ony forming units (CFU-GM) and erythroid burstforming units (BFU-E) has been reported in MDS [16191. The present study found that eosinophil cluster formation was increased in MDS. We also found that GM-CSF induced proliferation of eosinophil clusters in MDS patients more than in normal controls. Our results may be explained by an increase in abnormal eosinophil progenitor cells that show hyper-responsiveness to GMCSF. Recent studies have clearly documented that eosinophil production is regulated by GM-CSF, IL-5 and IL-3. However, we found that serum cytokine concentrations in MDS patients were within the normal range. The GM-CSF-stimulated neutrophils in MDS generally remain dysplastic in appearance during and following treatment [20]. Similarly, we found many hypogranular eosinophils in MDS patient No. 5 during treatment with GM-CSF. Duhamel [21] has reported eosinophilia in 37% of MDS patients, and Rios et al. [22] found increased eosinophils in 26% of biopsies from MDS patients. In these studies, eosinophilia has shown a certain relationship to survival by univariate analysis. Estey et al. reported that MDS patients who failed to respond to high dose GM-CSF, and who
x=MEB kJahwMe)
Effects of G-CSF and GM-CSF on eosinophil colony formation in MDS patients As shown in Fig. 5, MN cells from MDS patients yielded 5.0 f 6.8 eosinophil colonies and 100.0 + 75.9 clusters per dish. Eosinophil clusters in MDS were significantly increased (P < O.Ol), but eosinophil colonies were not significantly increased than normal controls. The MN cells from the MDS patient with eosinophilia (patient No. 9) gave rise to 26.0 eosinophil colonies and 232.0 clusters. The GM-CSF increased the number of eosinophil colonies (0.01
232
m
eo&ophil
colonies
0
eesinophil clusters
a
x240
180
n=8
O
4 G.
Fig. 5. Effects of G-CSF and GM-CSF on eosinophil colony formation in MDS patients including RAEB with eosinophilia (patient No. 9). The data are shown as meanfS.D. for eosinophil colonies and clusters/lo6 peripheral cells.
CFU-Eo in MDS &ml
Serum
GM-CSF
loo-
500 $"gd
-'
0
00 0 F
0
MDS
Normal n=4
n=6
Figure 6. GM-CSF, IL-5 and IL-3 concentrations in serum from controls and MDS patients showed a rise in eosinophils rather than neutrophils, may constitute a distinct subset of the myelodysplastic population with unique biological and prognostic features [23]. Thus it is possible that defective myeloid maturation potential in MDS may result in increased abortive eosinophil colony formation. We identified one RAEB patient with eosinophilia and increased CFU-Eo, but serum IL-3, IL-5 and GMCSF concentrations were not increased. Thus, eosinophilia was likely to be due to abnormal eosinophil progenitor cells rather than elevated cytokine concentrations. Future investigations may determine if increased eosinophil cluster and colony formation is related to the prognosis of MDS. Acknowledgements-We wish to thank Dr H. Enokihara (Third Department of Internal Medicine, Dokkyo University School of Medicine) for measuring the IL-5 and IL-3 concentrations. We also acknowledge with thanks the technical assistance of Mrs Y. Murata (Juntendo University School of Medicine). We thank Mr Albert Simpson for checking the English manuscript. References 1. Fermand J. P., Mitjavila M. T., Couedic J. P., Tsapis A., Berger R., Modigliani R., Seligmann M., Brouet J. C. & Vainchenker W. (1993) Role of granulocyte-macrophage colony-stimulating-factor, interleukin-3 and interleukin-5 in the eosinophilia associated with T cell lymphoma. Br. J. Haematol. 83, 359. 2. Lopez A. F., Williamson J. & Gamble J. R. (1986) Recombinant human granulocyte-macrophage colonystimulating factor stimulates in vitro mature human neutrophil and eosinophil function surface receptor expression and survival. J. Clin. Invest. 78, 1220. 3. Clutterbuck E. J. & Sanderson C. J. (1990) Regulation of
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