~ Pergamon
`
`Leukemia Research Vol. 20, No. 11/12, pp. 891-900, 1996.
`Copyright © 1996 Elsevier Science Ltd. All rights reserved
`Printed in Great Britain
`0145-2126/96 $15.00 + 0.00
`
`PH: SOI45-2126(96)00008-2
`
`MEASUREMENT OF APOPTOSIS, PROLIFERATION AND THREE
`CYTOKINES IN 46 PATIENTS WITH MYELODYSPLASTIC
`SYNDROMES
`
`Vilasini Shetty*, Suneel Mundle*, Sairah Alvi*, Margaret Showel*, LaTanya Broady-Robinson*,
`Saleem Dar*, Raphael Borok*, John Showel *, Stephanie Gregory * , Shelby Rifkint, Sefer Gezer*,
`Agapi Parcharidou *, Parameswaran Venugo~al *, Rohit Shaht, Beatrice Hemandez§,
`Mary Klein~, Devena Alston§, Erwin Robinll, Carlos Dominquez** and Azra Raza*
`*Rush Cancer Institute and the Department of Pathology, Rush-Presbyterian-St. Luke's Medical Center,
`Chicago, Illinois, U.S,A. and tNorthwest Community Hospital, Arlington Heights, Illinois, U.S.A.; tLake
`Forest Hospital, Deerfield, Illinois, U.S.A.; §LaPorte Cancer Treatment Center, LaPorte, Indiana, U.S.A.; ~St.
`Anthony Hospital, Michigan City, Indiana, U.S.A.; IIIngalls Memorial Hospital, Harvey, Illinois, U.S.A.; and
`**The Mount Sinai Comprehensive Care Center, Miami Beach, Florida, U.S.A.
`
`(Received 18 October 1995. Revision accepted 10 January 1996)
`
`Abstract-Extensive apoptosis or programmed cell death (PCD) of both hematopoietic
`(erythroid, myeloid, megakaryocytic) and stromal cells in myelodysplastic syndromes (MDS)
`cancels the high birth-rate resulting in ineffective hematopoiesis and has been demonstrated
`as the probable basis for peripheral cytopenias in MDS by our group. It is proposed that factors
`present in the microenvironment are inducing apoptosis in all the cells whether stromal or
`parenchymal. To investigate this hypothesis further, bone marrow biopsies from 46 MDS
`patients and eight normal individuals were examined for the presence of three cytokines,
`tumor necrosis factor-alpha (TNF-cx), transforming growth factor-beta (TGF-p) and granulocyte
`macrophage-colony stimulating factor (GM-CSF) and one cellular component, macrophages,
`by the use of monoclonal antibodies immunohistochemically. Results showed the presence of
`TNF-cx and TGF-,B in 41/46 and 40/46 cases of MDS respectively, while only 15 cases showed the
`presence of GM-CSF. Further a significant direct relationship was found between the degree of
`TNF-cx and the incidence of PCD (p = 0.0015). Patients who showed high PCD also had an
`elevated TNF-cx level. Thus, the expression of high amounts of TNF-cx and TGF-,B and low
`amounts of the viability factor GM-CSF may be responsible for the high incidence of PCD
`leading to ineffective hematopoiesis in MDS. Future studies will be directed at attempting to
`reverse the lesion in MDS by using anti-TNF-cx drugs such as pentoxifylline. Copyright © 1996
`Elsevier Science Ltd
`
`Key words: Myelodysplastic syndrome, tumor necrosis factor-alpha (TNF-cx), transforming
`growth factor-beta (TGF-,B), granulocyte macrophage-colony stimulating factor (GM-CSF),
`apoptosis and macrophages.
`
`Introduction
`
`Myelodysplastic syndromes (MDS) encompass a group
`of clinical, hematological disorders characterized by
`dysplastic maturation of precursor cells in the bone
`marrow and an approximately 30%
`incidence of
`eventual transformation into acute myeloid leukemia
`(AML) [1-4]. The paradox of this disease is that despite
`a hypercellular or normocellular bone marrow (BM), the
`
`Correspondence to: V. Shetty, Rush Cancer Institute, Rush(cid:173)
`Presbyterian-St. Luke's Medical Center, 2242 West Harrison
`Street, Suite 108, Chicago, IL 60612, U.S.A. (Tel: 312 455
`8474; Fax: 312 455 8479).
`
`patients generally present with peripheral cytopenias [5].
`To address this paradox, we developed unique double
`labeling methods to simultaneously determine the rate of
`proliferation and apoptosis or programmed cell death
`(PCD) [6]. The studies revealed that MDS is an
`extremely proliferative disorder with all three lineages
`of hematopoietic cells synthesizing DNA. At the same,
`time, there was a high rate of intramedullary hemato(cid:173)
`poietic cell death by apoptosis. A large number of
`erythroid, myeloid and megakaryocytic cells were
`undergoing PCD in the BM biopsies. A variety of
`stromal cells such as the fat cells, endothelial cells and
`fibroblasts were also found to undergo PCD simulta(cid:173)
`neously in the microenvironment. We concluded that
`
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`

`892
`
`V. Shetty et at.
`
`high cell death and high cell birth cancel each other
`resulting in ineffective hematopoiesis [7]. Apoptosis
`frequently observed in the stromal cells of the bone
`marrow microenvironment raised questions regarding
`the role of the microenvironment in generating this
`paradox. One hypothesis was that since MDS is a clonal
`disease, perhaps the abnormal clone is producing factors
`which are poisoning the microenvironment so that all
`cells whether stromal or parenchymal are forced to
`undergo PCD. A number of cytokines have been
`identified that inhibit or stimulate the progenitor cells
`either by a direct action on these cells or by modifying
`or modulating the production or actions of other
`molecules. The cytokines measured in this study were
`two inhibitory proteins namely, tumor necrosis factor(cid:173)
`alpha (TNF-a) and transforming growth factor-beta
`(TGF-fj) and one viability factor, granulocyte macro(cid:173)
`phage-colony stimulating factor (GM-CSF), as well as a
`cellular stromal component, the macrophages. It has
`been demonstrated that TNF-a exerts a variety of
`activities on hematopoietic cells which include inhibi(cid:173)
`tion of hematopoietic progenitors in in vitro clonogenic
`assay [8-11], induction of the release of various growth
`factors by stromal cells [12-14] and also has the ability
`to initiate the cascade of cytokines and other factors
`associated with the inflammatory response. GM-CSF on
`the other hand is a pleiotropic cytokine that can
`stimulate the proliferation, maturation and function of
`hematopoietic cells. It is produced by a variety of cell
`types including T-cells, B-cells, macrophages, mast
`cells, endothelial cells and fibroblasts in response to
`cytokine or immune and inflammatory stimuli [15-17].
`The other cytokine of interest
`in
`this study was
`transforming growth factor-beta. TGF-fj are a group of
`growth and differentiation modulation proteins of
`diverse function that induce a variety of biological
`responses on different tissues [18]. While the common
`effects of TGF-fj are both growth stimulatory [19] as
`well as growth inhibitory [20], the latter effect is more
`pronounced upon hematopoietic progenitors. This study
`was undertaken to better understand the biology by
`examining bone marrow biopsies of MDS patients for
`relative amounts of these cytokines using semi-quanti(cid:173)
`tative methods, the hypothesis being that either the
`presence of excessive amounts of TNF-a or TGF-fj and/
`or the relative absence of GM-CSF may account for the
`ineffective hematopoiesis observed in these disorders.
`
`Materials and Methods
`
`The study was carried out on 46 MDS patients, 32
`being males and 14 females. Morphological classifi(cid:173)
`cation as per the French-American-British (FAB) pro(cid:173)
`posal [21] was available on all patients. These details are
`provided in Table 1. Briefly, there were 18 patients in
`
`the refractory anemia (RA) category, six in refractory
`anemia with ringed sideroblasts (RARS), 14 in refrac(cid:173)
`tory anemia with excess of blasts (RAEB), seven in
`refractory anemia with excess of blasts in transformation
`(RAEB-t) and one in chronic myelomonocytic leukemia
`(CMMOL). Similar studies were also carried out on
`eight bone marrow biopsies obtained from patients with
`non-Hodgkin's lymphoma (NHL). These biopsies did
`not show any tumor involvement and were confirmed as
`being normal by two histopathologists. All the above
`patients and the normal biopsies from eight patients with
`NHL received a 1 h infusion of thymidine analogs
`bromodeoxyuridine and/or iododeoxyuridine at 100
`mg/m2 intravenously. Informed consent was taken by all
`patients before the infusion. The infusion protocols were
`reviewed and approved by the Investigative Review
`Board (IRB) of the Rush-Presbyterian-St. Luke's
`Medical Center, National Cancer Institute (NCI) and
`the Food and Drug Administration (FDA). The IUdR
`and BrdU for these studies were supplied by NCI. The
`bone marrow biopsies on these patients have been
`analyzed for studies on cell cycle kinetics and apoptosis.
`
`Detection of cytokines in the microenvironment
`Levels of three cytokines TNF-a, TGF-fj and GM(cid:173)
`CSF were determined semiquantitatively in the bone
`marrow biopsies immunohistochemically. All tissues
`were fixed in Bouin's solution and embedded in glycol
`methacrylate. Two to three micron thick sections were
`obtained and placed on alcian blue coated coverslips.
`The sections were then individually labeled for each
`cytokine using the respective monoclonal antibodies as
`follows : after the tissues were rehydrated in distilled
`water for 10 min, they were incubated with freshly
`diluted 3% H20 2 for 30 min and then with pronase
`1 mg/ml (Calbiochem, La Jolla, CA, U.S.A.) for 45 min.
`Specimens were rinsed carefully with 0.15 M phosphate
`buffered saline (PBS) [0.15 M sodium chloride in 0.1 M
`phosphate buffer, pH 7.5] after each
`incubation.
`Following the last 0.15 M PBS rinse they were placed
`in 0.5 M PBS
`[0.5 M sodium chloride
`in 0.1 M
`phosphate buffer, pH 7.5] for 15 min. The sections were
`treated with 0.5 M PBS containing 1.5% horse serum for
`to block non-specificity. Subsequently
`the
`60 min
`sections were incubated with the respective monoclonal
`anti TGF-fj2/fj3 (1:50) antibody (Oncogene Science
`Inc., Manhasset, NY, U.S.A.) or anti GM-CSF (1:80)
`(Genzyme, Cambridge, MA, U.S.A.) or anti TNF-a
`(1:180) (Prom ega, Madison, WI, U.S.A.) diluted in
`0.5 M PBS containing 1.5% horse serum for 60 min.
`This was followed by incubating the sections with
`biotinylated anti-mouse IgG [diluted (1 :200) in 0.5 M
`PBS with 1.5% horse serum] for 30 min and with the
`avidin-biotin complex or (ABC) reagent. The horse
`serum, biotinylated anti-IgG and ABC complex were
`
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`

`Measurement of apoptosis, proliferation and cytokines in MDS
`
`893
`
`reagents in the Vectastain Elite ABC kit (Vector,
`Burlingham, CA, U.S.A). Mer each of the above
`incubations, specimens were rinsed in 0.5 M PBS. The
`color reaction was then developed using 0.025%, 3,3'(cid:173)
`diamino benzidine
`tetrachloride (DAB) diluted
`in
`100 ml of 0.5 M Tris buffer, pH 7.5, with 0.01 ml 30%
`HzOz for 10 min and rinsed with distilled water. Mter
`processing, the coverslips were mounted on glass slides
`and examined by light microscopy.
`
`Detection of macro phages
`The presence of the macrophages was detected
`immunohistochemically by the method described above.
`The monoclonal antibody used was EBM-11 (Dakopatts,
`Denmark).
`
`Detection of S-phase cells
`The in situ detection of the two thymidine analogs
`IUdR and BrdU administered via intravenous infusions
`was carried out using the protocols and immunohisto(cid:173)
`chemical methods described previously [22]. Mer
`processing and mounting the coverslips with fluoro(cid:173)
`mount, at least 2000 positively S-phase labeled myeloid
`cells were counted to determine the labeling index (LI).
`Erythroid and megakaryocytic cells were excluded.
`
`Measurement of apoptosis using in situ end labeling
`(ISEL) of fragmented DNA
`ISEL was carried out on all the bone marrow biopsies
`of the 46 patients as mentioned in our earlier studies [6].
`Briefly,
`the sections following pre-treatment with
`sodium chloride sodium citrate (SSC) solution at 80°C
`in 0.15 M PBS;
`and with 1% Pronase (1 mglml
`Calbiochem, La Jolla, CA, U.S.A) were incubated with
`a mixture of dATP, dCTP, dGTP (0.01 M, Promega,
`Madison, WI, U.S.A), bio-dUTP (0.001 M, Sigma) and
`DNA Polymerase I (20 Ulml, Promega) at 18°C.
`Incorporation of bio-dUTP was finally visualized using
`avidin-biotin-peroxidase conjugate (Vectastain Elite
`ABC Kit, Vector, Burlingham, CA, U.S.A) and diamino
`benzidine tetrachloride. Thus, cells labeled positively
`for ISEL showed brown staining in their nuclei under the
`light microscope.
`
`Simultaneous detection of apoptosis by in situ end
`labeling and TNF-~
`All the cases were further double-labeled to simulta(cid:173)
`neously detect apoptosis and TNF-~. The sections were
`first treated as described above (ISEL) to label the
`apoptotic cells. Mer staining with DAB, they were
`thoroughly rinsed in distilled water followed by rinsing
`with 0.5 M Tris buffered saline (TBS), pH 7.5 [0.5 M
`NaCI in 0.05 M Tris buffer]. Mer rinsing with 0.5 M
`TBS, the specimens were sequentially treated with 4 N
`HCI for 15 min, the anti TNF-~ monoclonal antibody
`
`diluted 1:180 in 0.5 M TBS containing 0.25% Tween 20
`for 60 min at room temperature, rabbit anti-IgG (Dako,
`Carpenteria, CA, U.S.A) diluted 1:20 in 0.5 M TBS, for
`30 min and with mouse alkaline phosphatase-anti-alka(cid:173)
`line phosphatase antibody complex (AP AAP; Dako)
`diluted 1:40 in 0.5 M TBS for 30 min. The sections were
`thoroughly rinsed with 0.5 M TBS after each of the
`above-mentioned incubations. Tissues were then im(cid:173)
`mersed in the solution prepared as follows-naphthol
`AS-MX phosphate (20 mg) was dissolved in 2 ml of
`N,n-dimethyiformamide and this was added to 100 ml
`0.1 M Tris buffer, pH 8.2 at 20°C. Next, 0.1 ml of 1.0 M
`levamisole was added
`to
`the solution
`to
`inhibit
`endogenous alkaline phosphatase, followed by 100 mg
`of Fast Blue BB salt. This mixture was stirred for 2 min
`and filtered before the sections were immersed. Color
`development took 8--10 min. The specimens were then
`washed in distilled water and mounted in fluoromount.
`The TNF-~ stained blue under the light microscope,
`while ISEL positive cells stained brown.
`
`Interpretation of slides
`All the slides were observed on a televised screen by
`three different investigators (AR. being one of them
`every
`time). A subjective quantitative scale was
`formulated to determine the degree of positivity of the
`different cytokines (TGF-p, TNF-~ and GM-CSF), ISEL
`staining and the cellular component (macrophages) as
`follows: negative, low, intermediate and high.
`
`Low:
`Intermediate:
`High:
`
`less than 1/3 of the biopsy positive
`1/3 to 2/3 of the biopsy positive
`greater than 2/3 of the biopsy positive.
`
`Statistical analysis
`The non-parametric Mann-Whitney U-test was used
`for comparison between two parameters.
`
`Results
`
`Table 1 describes in detail the FAB type, white blood
`cell count (WBC), labeling index (LI), apoptosis and the
`presence of cytokines and macrophages. Results will be
`described under the following sub-headings.
`
`Apoptosis and proliferation
`A high degree of proliferation (Fig. 1a) and apoptosis
`(Fig. 1b) were seen in this group of patients as reported
`previously [7]. Briefly, of the 46 patients studied,
`apoptosis was detected in 42 patients, while three cases
`showed little or no apoptosis and one biopsy was
`unevaluable. Twenty-five patients (54.3%) showed a
`high degree of apoptosis where more than 75% of the
`cells demonstrated ISEL positivity, seven (15.2%)
`showed intermediate and 10 (21.7%) showed low
`
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`
`

`

`894
`
`V. Shetty et al.
`
`Table 1. Studies on cell cycle kinetics, programmed cell death and microenvironment on bone marrow biopsies of patients with
`myelodysplastic syndromes
`
`Patient #
`
`FAB
`
`WBCx109 !1
`
`ISEL
`
`LI%
`
`TGF-fJ
`
`TNF-O(
`
`GM-CSF
`
`Macrophages
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`31
`32
`33
`34
`35
`36
`37
`38
`39
`40
`41
`42
`43
`44
`45
`46
`
`RA
`4.50
`8.40
`RA
`2.70
`RA
`3.90
`RA
`RA
`2.90
`3.10
`RA
`2.50
`RA
`NA
`RA
`15.20
`RA
`1.00
`RA
`3.10
`RA
`8.70
`RA
`1.70
`RA
`3.40
`RA
`3.70
`RA
`6.30
`RA
`NA
`RA
`4.00
`RA
`4.50
`RARS
`7.50
`RARS
`RARS
`38.10
`13.00
`RARS
`1.40
`RARS
`3.10
`RARS
`RAEB
`12.80
`0.70
`RAEB
`3.70
`RAEB
`RAEB
`1.60
`14.60
`RAEB
`1.50
`RAEB
`25.70
`RAEB
`1.60
`RAEB
`4.70
`RAEB
`6.70
`RAEB
`0.80
`RAEB
`6.30
`RAEB
`3.10
`RAEB
`2.50
`RAEB
`RAEB-t
`1.70
`RAEB-t
`2.50
`RAEB-t NA
`RAEB-t
`10.10
`RAEB-t
`1.30
`RAEB-t
`4.90
`RAEB-t
`4.00
`CMMOL 11.80
`
`high
`high
`high
`high
`high
`high
`high
`intermediate
`intermediate
`low
`low
`low
`low
`low
`low
`low
`negative
`bad bx
`high
`high
`high
`high
`intermediate
`intermediate
`high
`high
`high
`high
`high
`high
`high
`high
`high
`intermediate
`low
`low
`negative
`negative
`high
`high
`high
`high
`high
`intermediate
`intermediate
`low
`
`39.60
`26.00
`25.10
`15.00
`25.00
`24.00
`31.50
`46.60
`39.00
`33.10
`25.30
`41.00
`16.00
`35.00
`NA
`NA
`22.90
`NA
`37.00
`30.60
`29.30
`13.00
`49.10
`32.30
`21.30
`24.00
`26.00
`18.00
`33.60
`18.00
`24.50
`23.90
`28.70
`29.00
`38.00
`NA
`14.00
`24.00
`47.00
`32.00
`31.00
`18.00
`28.50
`39.30
`31.00
`19.10
`
`negative
`intermediate
`intermediate
`low
`high
`negative
`low
`high
`intermediate
`intermediate
`low
`high
`low
`intermediate
`negative
`low
`low
`negative
`intermediate
`low
`high
`high
`intermediate
`intermediate
`intermediate
`intermediate
`low
`intermediate
`high
`intermediate
`negative
`intermediate
`low
`high
`intermediate
`low
`negative
`intermediate
`low
`intermediate
`high
`intermediate
`low
`low
`intermediate
`low
`
`high
`high
`high
`high
`intermediate
`high
`high
`low
`high
`intermediate
`low
`high
`negative
`intermediate
`high
`low
`negative
`negative
`intermediate
`high
`low
`intermediate
`intermediate
`high
`high
`high
`high
`intermediate
`low
`high
`high
`high
`negative
`intermediate
`low
`high
`low
`low
`intermediate
`high
`high
`intermediate
`high
`high
`intermediate
`negative
`
`negative
`intermediate
`negative
`low
`intermediate
`negative
`low
`negative
`negative
`negative
`negative
`negative
`negative
`high
`negative
`negative
`negative
`negative
`negative
`negative
`intermediate
`high
`high
`low
`negative
`high
`negative
`low
`negative
`negative
`negative
`negative
`negative
`negative
`negative
`negative
`negative
`negative
`low
`negative
`low
`high
`negative
`high
`negative
`negative
`
`negative
`low
`low
`high
`high
`intermediate
`high
`low
`negative
`negative
`high
`low
`low
`intermediate
`negative
`low
`intermediate
`negative
`intermediate
`high
`low
`low
`intermediate
`negative
`high
`high
`negative
`low
`high
`low
`high
`high
`intermediate
`low
`high
`low
`high
`high
`intermediate
`high
`high
`intermediate
`high
`intermediate
`high
`high
`
`FAB, French-American-British classification; ISEL, in situ end labeling; LI%, labeling index; RA, refractory anemia; RARS,
`refractory anemia with ringed sideroblasts; RAEB, refractory anemia with excess blasts; RAEB-t, refractory anemia with excess
`blasts in transformation; CMMOL, chronic myelomonocytic leukemia; TGF-p, transforming growth factor-beta; TNF-a, tumor
`necrosis factor-alpha; GM-CSF, granulocyte macrophage-colony stimulating factor; NA, not available.
`
`Table 2. Degree of positivity of the different parameters in MDS (n = 46)
`
`Degree of positivity
`
`High
`Intermediate
`Low
`
`ISEL
`
`25.00
`7.00
`10.00
`
`TGF-fJ
`
`TNF-O(
`
`GM-CSF
`
`Macrophages
`
`8.00
`18.00
`14.00
`
`22.00
`11.00
`8.00
`
`6.00
`3.00
`6.00
`
`18.00
`9.00
`12.00
`
`DR. REDDY’S LABS., INC. EX. 1015 PAGE 4
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`

`

`Measurement of apoptosis, proliferation and cytokines in MDS
`
`895
`
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`tt
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`~
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`'1" •
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`
`0
`
`~
`
`~
`
`~ .o~
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`~
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`eo
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`
`Fig. 1. (a) Bone marrow biopsy from an MDS patient showing high percentage of S-phase cells (brown staining in the nucleus)
`detected by virtue of incorporation of IUdR/BrdU in their DNA using specific monoclonal antibody. Original magnification x 400.
`(b) Extremely high incidence of apoptotic cell death involving all types of stromal and hematopoietic cells in the bone marrow of
`MDS patients, detected using ISEL (brown staining). Original magnification x 400.
`
`pOSItIVIty. The normals showed low (6/8) and inter(cid:173)
`mediate (2/8) ISEL positivity. As for the rate of
`proliferation, labeling indices (LI) from bone marrow
`biopsies were available in 41 patients. The median LI
`was 28.7% with a range of 13.0-49.1%. When the LI
`and apoptosis was correlated with
`the WBC, no
`significant relationship was seen.
`
`Localization and degree of positivity of the different
`cytokines in MDS
`TGF-f1 and TNF-a were expressed in varied amounts
`in the bone marrow biopsies (Table 1). Briefly, TGF-f1
`was expressed in 40 patients of the 46 patients studied,
`TNF-a in 41 patients and GM-CSF in 15 patients. Table
`2 summarizes the extent of positivity seen for each of
`the above cytokines. TGF-f1 was seen in the megakaryo(cid:173)
`cytes and blood vessels, primarily along the endothelial
`lining as well as in the interstitial areas (Fig. 2). TNF-a
`on the other hand was demonstrated in monocytes and
`few immature progenitor cells (Fig. 3). GM-CSF was
`localized in the interstitium mainly around the cells in S(cid:173)
`phase (Fig. 4.).
`
`Detection of macro phages
`The macrophages were detected using a specific
`monoclonal antibody (EBM-ll) (Fig. 5a). The degree of
`positivity is depicted in Table 2. Presence of macro(cid:173)
`phages were also revealed by ISEL staining. The
`cytoplasm of the macrophages were filled with ISEL
`positive apoptotic bodies, while the nuclei of these
`macrophages were still intact (Fig. 5b). The present
`phenomena clearly demonstrates the scavenging activity
`of the macrophages present.
`
`Patterns of different parameters studied in different F AB
`categories
`All parameters studied were further analyzed for
`common trends or predominant patterns with respect to
`FAB classification (Table 3). The majority of patients in
`the group of RA category (55.6%) revealed a lower
`degree of apoptosis as compared to the other three, most
`noteworthy being RAEB-t. The most striking observa(cid:173)
`tion seen was that all seven patients with RAEB-t
`showed intermediate to high expression of both TNF-a
`along with high apoptosis. GM-CSF ratings for RAEB
`patients were more likely to be negative than were those
`for RAEB-t patients (P = 0.0486).
`
`Fig. 2. Bone marrow biopsy from an MDS patient showing
`high expression of TGF-f3 (brown) in the interstitium and
`blood vessels, primarily along the endothelial lining. Original
`magnification x 400.
`
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`896
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`V. Shetty et al.
`
`Fig. 3. (a) Presence of TNF-oc in the bone marrow biopsy. Note the high positivity (brown) in the interstitium, and also in few
`immature progenitor cells and monocytes. Original magnification x 400. (b) Bone marrow biopsy from NHL patient, devoid of tumor
`involvement (normal) showing absence of TNF-oc. Original magnification x 400.
`
`Inter-relationships between cytokine expression, apop(cid:173)
`tosis and proliferation
`A significant positive correlation was observed
`between the degree of apoptosis and proliferation in
`our earlier studies [7, 23]. The median labeling index
`(LI) steadily increased to the degree of ISEL from
`undetectable to low to intermediate, reaching statistical
`significance at the intermediate degree (P = 0.007).
`However, the percentage of cells proliferating decreased
`when the patients had more than 75% of cells under(cid:173)
`going cell death. No direct relationship was found
`between the LI and the cytokines. One important finding
`seen pertinent to the macrophages was that patients with
`high macrophages showed a lower rate of proliferation
`than patients who did not
`show macrophages
`(P = 0.0139). This observation appears to implicate that
`macrophages are the source of negative hematopoietic
`
`Fig. 4. Low levels of GM-CSF noted (brown) in the biopsy
`from an MDS patient, mainly around the cells in S-phase.
`Original magnification x 200.
`
`cytokine. There was a highly significant relationship
`between
`incidence of PCD and
`levels of TNF-Cl
`(P = 0.0015). The majority of cases showing greater
`than 75% cells undergoing PCD, expressed intermediate
`to high levels of TNF-Cl. On the other hand GM-CSF was
`conspicuously absent or low in 20/25 cases where
`substantial
`ISELffNF-Cl positivity was detectable.
`Further double labeling to measure apoptosis and
`TNF-Cl simultaneously revealed a great preponderance
`of TNF-Cl specifically around ISEL positive cells (Fig.
`6). The subjective quantitative scale revealed greater
`than 2/3 of the biopsy positive for both TNF-Cl and ISEL.
`
`Results in normal individuals
`Eight normal bone marrow biopsies from patients
`with non-Hodgkin's lymphoma (NHL) were studied.
`Briefly, MDS marrows revealed high expression of the
`two antiproliferative cytokines, i.e. TFG-p and TNF-Cl,
`in conjunction with the high degree of apoptotic cell
`death in these marrows detected by ISEL. On the other
`hand,
`the biopsies of normal
`individuals showed
`complete absence to low levels of both TNF-a and
`TGF-p with low to intermediate degree of apoptotic cell
`death. The level of the expression of GM-CSF remained
`comparable in MDS and normals. The number of
`macrophages was higher in MDS marrows as compared
`to normals.
`
`Discussion
`
`Greater attention is being focused on the study and
`treatment of myelodysplastic syndromes in recent years
`for several reasons. Firstly, the incidence of MDS
`appears to be increasing largely due to the increasing
`numbers of bone marrow transplants being performed on
`patients with malignancies such as lymphomas and
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`Measurement of apoptosis, proliferation and cytokines in MDS
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`897
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`Fig. 5. (a) Detection of macrophages (brown) in the bone marrow biopsy of an MDS patient using specific monoclonal antibody.
`Original magnification x 400. (b) Presence of macrophages also revealed by ISEL staining. Note the cytoplasm of the macrophage
`filled with ISEL positive (brown) apoptotic bodies, while the nuclei of the macrophages are still intact. Original magnification
`x 1000.
`
`breast cancer [24,25]. Secondly, supportive care con(cid:173)
`tinues to be the mainstay of therapy with bone marrow
`transplantation being an option for very few patients,
`since the disease mainly strikes older individuals [26].
`This severe limitation in having almost no options for
`the treatment of these unfortunate patients has resulted
`in renewed interest amongst investigators to examine the
`biology of myelodysplasia more critically. The hope is
`that by understanding more about the basic pathology,
`we will be in a better position to rationally reverse the
`lesion. Finally, recent spectacular advances in cell and
`molecular biology techniques have opened entire new
`areas of research which are sure to provide much
`meaningful information in otherwise dismal fields. One
`such area is related to improved methods for the in situ
`detection of proliferation and apoptosis simultaneously
`and our group has taken advantage of those to document
`cell birth and cell death rates in MDS patients [6,7].
`The present study was designed to interrogate the
`basic biology of MDS by taking the proliferation and
`apoptosis studies one step further. Having demonstrated
`a high incidence of intramedullary apoptotic cell death
`in MDS patients, we extended the studies to evaluate the
`differences in the constituents of the bone marrow
`microenvironment among individual patients. Three
`specific cytokines and one cellular component of the
`bone marrow were targeted for investigation. Two
`cytokines, namely TNF-Q: and TGF-{3, were chosen for
`their possible implication in the excessive death of
`hematopoietic cells, while the presence of a viability
`factor GM-CSF was also determined since its absence
`would have the same effect as increased levels of the
`other two cytokines, i.e. an increase in the incidence of
`apoptosis. Macrophages were chosen because of their
`role not only as scavenger cells, but also as the possible
`
`source of some of these cytokines. The results being
`reported in this paper shed new light on the biology of
`MDS. Excessive levels of TNF-Q: were found in the
`vicinity of cells undergoing programmed cell death in
`the majority of patients. The relationship between TNF(cid:173)
`Q:
`levels and apoptosis was highly
`significant
`(P = 0.(015).
`Based upon these observations, it is possible to
`postulate that in the majority of MDS patients, negative
`cytokines such as TNF-Q: and TGF-{3 may be mediating
`extensive apoptosis and the high proliferation may be
`the body's defense mechanism trying to compensate for
`the high cell death. Alternately, one or more of these
`cytokines may have a dual role in the pathogenesis of
`MDS such that the susceptibility of target cells within
`
`Fig. 6. ISEL and TNF-IX double labeling of MDS bone marrow
`biopsy showing apoptotic death (brown nuclei) and TNF-IX
`(blue) in the interstitium simultaneously. Original magnifica-
`tion x 1000.
`
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`898
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`v. Shelly et al.
`
`88888
`....
`....
`
`(1")NN~.....4
`
`88888
`....40NOO
`
`the same marrow may be completely different to the
`same cytokine. For example, TNF-1X may be a stimulat(cid:173)
`ing cytokine for the premature CD34+ progenitor cells,
`but induce apoptosis as these very cells undergo
`differentiation. This hypothesis is consistent not only
`with our previously reported observations [27], but
`would also explain the paradox of cytopenias (apoptosis
`in maturing cells) despite hypercellular marrows (sti(cid:173)
`mulation of early progenitors). One could also argue that
`yet another alternate explanation for the correlation
`between TNF-1XffGF-p and apoptosis is that a high
`cell-death rate results in stimulation of monocytes/
`macrophages to secrete these cytokines and the positive
`co-relation does not reflect the cause-effect relationship
`we hypothesized. While this is a legitimate possibility,
`our subsequent data do not support this hypothesis. By
`using anti-TNF drugs such as pentoxifylline or its first
`metabolite lisophylline, we and others have shown a
`dramatic reduction in BM and serum TNF-1X levels and
`apoptosis in BM biopsies of MDS patients [28] as well
`as TGF-p levels in murine BM cells [29]. Finally, the
`presence of macrophages in the setting of a high rate of
`apoptosis and significant levels of negative cytokines in
`the BM microenvironment of MDS patients could also
`be either because these cells are the source of these
`cytokines or are appearing in excessive numbers to
`scavenge the dead cells or both. The interesting question
`in this regard would be to determine the lineage of these
`macrophages and demonstrate whether they descend
`from the MDS clone or represent normal cells.
`The question of high rate of proliferation in MDS is
`also an interesting one. Whether this represents a direct
`manifestation of the dual role of TNF-1X (or TGF-P) as
`both a stimulatory and inhibitory cytokine or whether it
`represents a compensatory mechanism to neutralize the
`excessive
`intramedullary apoptosis
`remains
`to be
`determined. If the rate of proliferation decreases with
`anti-TNF therapy,
`it would still not allow us
`to
`distinguish between the two possibilities listed above.
`The best proof could only come from direct in vitro
`studies where sorted CD34+ progenitor cells could be
`stimulated to proliferate while the CD34 - maturing
`subset could be shown to undergo apoptosis with the
`to
`individual bone
`application of TNF-1X directly
`marrows. Such studies are presently underway in our
`laboratory.
`The ultimate result of the above observations in MDS
`is a functional hypoplasia, since the rapid proliferation is
`cancelled by the rapid cellular death, thus accounting for
`the peripheral cytopenia. If this pathological model is
`accepted, then the therapeutic;; implications are very
`clear. Attempts sholiid be made to down-regulate the
`production of TNF-1X which would then restore the
`normal longevity to the dying cells once again. A
`number of anti-TNF agents could be considered for use
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`Measurement of apoptosis, proliferation and cytokines in MDS
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`899
`
`in this context, including such commonly used drugs as
`pentoxifylline or other less commonly used ones such as
`lisophylline, thalidomide and cyclosporin. Most of these
`drugs have successfully been employed for the reduction
`of TNF-a levels in patients with other pathologies [30-
`32]. A point of caution here is that probably none of
`these drugs would affect the leukemic clone