(CANCER RESEARCH 48, 4812-4816, September 1, 1988]
Abnormal Blood Monocytes in Chronic Lymphocytic Leukemia1
Dimitri Flieger2 and H. W. LömsZiegler-Heitbrock3
Institute for Immunology, University of Munich, Goethestr. 31, ÄMünchen2, Federal Republic of Germany
ABSTRACT
Blood monocytes were analyzed in 28 patients with chronic lymphocytic leukemia without previous cytotoxic therapy and without recent
infection. Using monoclonal antibodies and flow cytometry, monocytes
identified by LeuM3 or My4 were low in percentage (2.3%), but absolute
numbers were increased in many patients with values exceeding the
normal range (120 to 510/nl) in seven of 28 patients. Monocytosis was
more prominent in patients with high leukemic counts, but there was no
correlation to clinical stages. Monocytopenia was evident with less than
50 IA-iiM.Vcells/ill in three patients.
Two-color fluorescence was used for the analysis of cell surface expres
sion of major histocompatibility complex (MHC) Class II molecules,
complement receptors, and Fc receptors on the LeuM3* monocytes.
Compared to cells from control donors, there was an increase for MHC
class II antigens, complement receptors, and Fc receptors on the mono
cytes in chronic lymphocytic leukemia, in terms of both the percentage
of positive cells among the LeuM3* monocytes and of fluorescence
intensity. This increase was not restricted to patients with monocytosis
nor were the molecules always upregulated concomitantly. The increase
of antigen expression on LeuM3* monocytes was more than 50% (1.5fold) in seven of 22 patients for MHC Class II antigens, in seven of 16
patients for complement receptor and in six of 12 patients for Fc receptor.
A similar decrease of antigen expression was observed only in one patient
for MHC Class II and in one patient for complement receptor expression.
Monocytosis and increased expression of monocyte cell surface anti
gens described for a large portion of patients might be causally involved
in the immunodeficiency in chronic lymphocytic leukemia.
INTRODUCTION
Multiple abnormalities of the immune system have been
detected in CLL." Among these are antibody deficiency (1-4),
alterations in T-cell subsets (5-9), defective primary responses
of both T- and B-cells (10, 11), and defective NK cell activity
(12, 13) going along with the absence of CD16+ lymphocytes
(14). These abnormalities substantially contribute to the clinical
problems of CLL patient, including frequent infections (15-17)
and secondary malignancies (18-20). Whether these abnormal
ities are governed by several independent events or by a single
element which influences T-cells, B-cells, and NK cells has not
been determined. Monocytes with their variety of regulatory
functions (for review, see Ref. 21) could form such an element.
In CLL, however, little is known about monocytes, probably
because of the difficulty of identifying and of purifying these
cells in the presence of overwhelming leukemic cell counts.
Hence we determined monocyte numbers and relevant cell
surface molecules in a group of previously untreated patients
by using monoclonal antibodies and flow cytometry, thus allow
ing for analysis of more than 200,000 cells per sample. Using
this approach we could demonstrate numeric and phenotypic
alterations in many CLL patients.
MATERIALS
AND METHODS
Patient Populations. The study included 28 patients, 18 men and 10
women, with a mean age of 63.4 yr (range, 48 to 82 yr), with B-CLL
as assessed by clinical, morphological, and immunological criteria (Ba1, Bl, Leu-1, Hd6, surface immunoglobulin). Twenty patients never
received any chemotherapy or cortisone, and the remaining 8 patients
had not been treated for at least 12 wk prior to this study. No patient
had an infection at the time of study. Staging (Table 1) was performed
according to Binet et al. (22). Briefly, Stages A through C are all
characterized by lymphocytosis in both blood and bone marrow; in
addition, Stage A includes lymphadenopathy of up to 2 lymph node
areas; Stage B, lymphadenopathy of 3 or more lymph node areas; and
Stage C patients exhibit anemia and/or thrombocytopenia. Control
donors were healthy volunteers between the ages of 52 and 76 (mean,
62.4 yr).
Cell Separation and Storage. Peripheral blood mononuclear cells
were separated from heparinized blood by Ficoll-Hypaque (Pharmacia,
Uppsala, Sweden) density gradient sedimentation, according to routine
procedures (23). Aliquots of cells were stored in liquid nitrogen after
controlled freezing in the presence of 10% dimethyl sulfoxide and 10%
heat-inactivated fetal calf serum. Previous experiments had shown that
these procedures do not result in change of cellular properties. Directly
before use, mononuclear cells were rapidly thawed in a 37°Cwater bath
and washed twice.
Monoclonal Antibodies. MAB LeuM3-FITC (Ref. 24; purchased
from Becton-Dickinson), My4 (Ref. 25; purchased from Coulter Im
munology), and M42 (kindly provided by Professor E. P. Rieber)
identify the majority of monocytes. MAB L243 recognizes HLA-DR
Class II MHC antigens (26), MAB M522 recognizes the C3bi receptor
(CR3) on monocytes (27, 28), and for identification of the Fc receptor,
HIGG was used. Immunoglobulin on monocytes was determined with
a biotinylated mouse anti-human immunoglobulin MAB (Tago, Burlingame, ÇA).As isotype controls, MABs of the same isotype purchased
from the same company were used. For the preparation of HIGG, 100
mg of human IgG (Miles Laboratories, Uppsala, Sweden) were treated
at 62"C for 30 min and afterwards applied to a Sepharose 4B column
(Pharmacia, Freiburg, Federal Republic of Germany). Fractions con
taining oligomers of IgG were pooled, vacuum dialyzed, and titrated
for their ability to inhibit Fc receptor-mediated nonspecific binding.
MAB L243, M522, HIGG, and their isotype controls were biotinylated
with D-biotin-W-hydroxysuccinimide ester (Boehringer Mannheim,
Federal Republic of Germany).
Immunofluorescence. In order to inhibit nonspecific binding of the
MAB to the monocyte surface, 1 x 10' PBM were preincubated with
25 M' of HIGG for 30 min. For two-color immunofluorescence, an
appropriate dilution of LeuM3-FITC (25 n\) and of a biotinylated MAB
(25 /il) was added, followed by incubation for 30 min on ice. After twice
washing with PBS/2.5% fetal calf serum/0.02% NaN3, the cells were
incubated with 50 /¿I
of avidin phycoerythrin (1:3; Becton Dickinson)
for development of the biotinylated MAB. MAB My4 used for singlecolor fluorescence was developed in the second incubation with goat
anti-mouse FITC (Tago, Burlingame, CA). The cells were fixed with
1% paraformaldehyde dissolved in PBS and analyzed with an EPICS
V flow cytometer (Coulter Electronics) with 488-nm excitation wave
Received 3/10/88; revised 5/17/88; accepted 5/26/88.
The costs of publication of this article were defrayed in part by the payment
length and photomultiplier tubes at 900 to 1250 V as described (29).
of page charges. This article must therefore be hereby marked advertisement in
At least 200,000 cells were analyzed for both specific MAB and isotype
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
control. Data analysis was performed with the MDADS software of the
1Supported by W. Sander Stiftung and Deutsche Krebshilfe.
2This work is part of the doctoral thesis of D. F.
EPICS V. For estimation of changes in cell size, we used forward-angle
3To whom requests for reprints should be addressed.
light scatter signals. Standard beads of 7.5 and 9.99 /im in diameter
4 The abbreviations used are: CLL, chronic lymphocytic leukemia; CR3, com
(Flow Cytometry Standard Corporation and Coulter Electronics, re
plement receptor type 3; H1GG, heat-aggregated immunoglobulin; MAB, mono
spectively), i.e., with roughly a 2-fold difference in cell surface area,
clonal antibody; MHC, major histocompatibility complex; PBM, peripheral blood
gave a 6 channel difference on the 64 channel linear scale. For light
mononuclear cells; NK, natural killer; FITC, fluorescein isothiocyanate; PBS,
phosphate-buffered saline; IL-1, interleukin 1; TNF, tumor necrosis factor.
scatter analysis of monocytes, the control and CLL samples were
4812
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ABNORMAL BLOOD MONOCYTES IN CLL
patients with values of up to 1200 LeuM3+ monocytes//*!.
1200
Numbers of monocytes exceeding the control range were found
in 7 of 28 CLL samples (Fig. 1). Monocytosis was more evident
in patients with high leukemic cell counts. Patients with more
than 50,000 WBC//il had in average 672 LeuM3+ cells/Ml (P <
1000••
0.01). Further monocytosis was also more prominent in later
stages (cf. Table 1). Eighteen patients had monocyte counts
within the control range, 4 patients had moderately decreased
monocyte numbers, and 3 patients (Patients D. E., D. M., and
N. J.) had clear-cut monocytopenia with less than 50 LeuM3+
500400
300
200
too
Fig. 1. Absolute monocyte numbers in CLL patients. Given is the number of
LeuM3-positive cells///! of peripheral blood. The open box indicates the control
range (±1SD). Results were obtained by fluorescence-activated cell sorter analysis
of at least 200,000 cells per sample with high leukocyte counts.
studied side by side with the identical excitation conditions and the
identical gain for the scatter detector. The absolute number of monocytes is expressed as LeuM3+ cells recovered after isolation and storage
by using the formula: % of LeuM3+ cells x mononuclear cells recovered
from 1 ^1 of blood/100.
Statistics. For statistical evaluation Student's t test was used.
RESULTS
In peripheral blood of patients with CLL, percentages of
LeuM3+ monocytes are low (Table 1). Absolute numbers ex
pressed in cells per ¿¿1
of blood are, however, increased in many
cells/^l.
When using two other monoclonal antibodies for definition
of monocytes (My4 and M42), very similar monocyte numbers
were determined (Table 1).
In order to study functionally relevant cell surface structures
on monocytes in CLL, we used two-color immunofluorescence
analysis. Monocytes were defined with the fluorescein-conjugated LeuM3 MAB, and MHC Class II antigens, C3bi receptors
(CR3), and Fc receptors were identified with biotinylated re
agents that were visualized with avidin phycoerythrin.
An example of the flow cytometry analysis for MHC Class
II antigens on 400,000 cells of a CLL sample is shown in Fig.
2C compared with a control sample (Fig. 2A). The population
of green fluorescent LeuM3+ cells on the jc-axis is strongly
positive for yellow fluorescent MHC Class II antigens on the
j>-axis (Fig. 2C). In Fig. 2, B and D, the low background staining
of LeuM3+ cells with a y2a isotype control is depicted. The
Table 1 Immunofluorescence data of CLL patients compared to controls
òlfMonocyte'340108145<10304432201901301091627808463316708231220107067965184831193343851
positive (%)
PatientA.N.B.C.B.
Stage*
PBM*BAACACABCAAACABABACBCABAAddBBBddnddnndnn/dBndnnBBan/dn8.163.42103.421.76.17.4484.893988.16.872915.48.3156219108
levels'
R.D.E.D.
G.D.M.F.
A.G.J.G.
K.H.B.H.H.H.
12.1+5.1-0.6+9.7+3.1+3+
P.H.
R.K.
H.K.J.L.J.L.
12.7+2.7-0.4+6.9+4.7+0.4+
T.N.J.P.
1.7+0.3+2.2-2-5.1+4.2-0.5+2.8+0.6-1.7_—M
J.P.O.R.
A.S.
H.S.
R.W.
A.W.E.W.
L.W.
T.Z.
A.Age59526758507074626972524862535275727373756072495948738264SexMFMFFFFMMFMMFMMMFMMMFFMMMMMMTherapy"(n)nn(n)n(n)nBBBn(n)(n)n(n)naBBBnnn(n)nBn
Mean:
63.4
50.9
352.5
2.3
2.1
1.5
Control donors: n = 12; 8 M; 4 F
Mean (range): 62.4 (52-76)
1.2 ±0.5 233 ±120 19.7 ±7.3
19.6 ±5.7 21.4 ±7.6
" Therapy: n, none; (n), none for at least 12 wk prior to investigation.
* Staging according to J. L. Binet, 1981.
' Immunoglobulin levels: n, normal; d, decreased.
rfPBM x lO'/rnl separated from 1 ml of blood.
' Monocytes (LeuM3* cclls)/fil of blood.
'il. intensity of staining on L.cuM.V cells compared to staining obtained in the respective control donor, 6.5 channels of difference on log scale reflect 2-fold
difference in antigen density; 3 channels reflect change by 50%.
* —,not done.
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ABNORMAL BLOOD MONOCYTES IN CLL
for the CLL patient was 9.7 channels higher in this log scale
display. Compared to control, this reflects a 3-fold-higher an
tigen expression on the CLL monocytes. Fig. 3C shows the
specific staining for C3bi receptor (CR3) on LeuM3+ mono
Leu M 3
Fig. 2. Expression of Class II MHC antigens on monocytes in CLL. Twocolor ¡mmunofluorescence for LeuM3/L243 (A, C) compared to LeuM3/UPC
(B, D) as isotype control. A and B, control donor; C and D, CLL Patient H. R.
Results for the CLL sample were derived from fluorescence-activated cell sorter
analysis of 400,000 cells.
projections of yellow fluorescence gated on the LeuM3+ cells
are used for further evaluation.
Fig. 3A compares specific staining for MHC Class II antigens
on LeuM3+ monocytes in the CLL patient and the control
donor depicted in Fig. 2. Percentages of positive cells were
higher in this CLL patient compared to control (cf. legend to
Fig. 3). More importantly, mean specific fluorescence intensity
cytes in another CLL patient and a control. The monocytes of
this patient express 2-fold more C3bi receptor compared to
control. Histograms of the isotype controls for MABs L243
and M522 (Fig. 3, B and O, respectively) show an identical
staining pattern in CLL and control donors.
Additional data, including expression of Fc receptors, are
given in Table 1. Fluorescence intensity was 1.5-fold (50%)
higher than in controls in 7 of 22 CLL patients for MHC Class
II antigen expression (P < 0.01), in 7 of 16 CLL patients for
C3bi receptor expression (P < 0.01), and in 6 of 12 CLL
patients for Fc receptors (P < 0.01). The higher signal for Fc
receptors on CLL monocytes was not due to a decreased en
dogenous loading of Fc receptors by serum antibody, since
expression of opsonized immunoglobulin on monocytes was
also increased in many CLL patients (data not shown).
The expression of cell surface molecules on CLL monocytes
was not always upregulated together. Patient H. H., for in
stance, expressed only increased MHC Class II antigen, and
Patient S. R., only increased C3bi receptor. An increased
expression of at least one of the cell surface molecules studied
was found in 14 of 22 patients.
Increased expression of cell surface molecules was found in
patients with normal and with increased monocyte numbers
(e.g., Patient A. N. and Patient H. R.). Further monocytosis
was not always correlated with upregulated cell surface mole
cules (Patient W. L.), indicating that the enhanced expression
of Class II molecules, complement, and Fc receptors is not
directly related to monocyte number.
An estimation of size of LeuM3+ monocytes with forwardangle light scatter signals (30, 31) revealed no difference be
tween monocytes of CLL patients and controls with a mean
channel of 32.4 ±4.6 for CLL monocytes and 32.2 ±2.6 for
control monocytes (n = 10). Since forward-angle light scatter
correlates with cell size, this indicates that the increased antigen
expression per cell might be due to an increased antigen density
on the cell surface.
'-"i
A
Fig. 3. Expression of Class II MHC and
CR3 antigens on monocytes in CLL. Yellow
fluorescence for L243 (A) and M522 (C) gated
on LeuM3 cells. In A, the CLL Patient H. R.
(right curve) has an increased L243 (MHC
Class II) expression compared to a control
donor (left curve). The percentage of Class IIpositive cells among the LeuM3* monocytes
was 84.5% for the CLL patient and 53.6% for
the control donor. The isotype control (UPC)
in B shows the same fluorescence intensity for
both CLL and control. Another CLL Patient
S. R. in C shows increased M522 (C3bi recep
tor) expression compared to control, while the
percentage of M522-positive cells was almost
identical (97.4% and 99.0%). Their isotype
controls (MOPC) are shown in D. Left, antiClass II; right, anti-CR3.
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ABNORMAL BLOOD MONOCYTES IN CLL
tion of TNF in many CLL samples.6 Such functional studies
DISCUSSION
Analysis of monocytes in CLL is difficult, since in the pres
ence of high numbers of leukemic B-cells, these cells form only
a few percentages of the mononuclear cells. In one previous
study, Zeya et al. (32, 33) found a decreased cellular enzyme
content of monocytes in CLL after density gradient separation
using Percoli. The study, however, is difficult to interpret,
because of the problem of clearly identifying and purifying the
monocytes. Contaminating leukemic lymphocytes, which also
have decreased enzyme activities in CLL, could account for the
observed deficiency.
For our analysis we used MABs like LeuM3 and M42, which
to our knowledge exclusively react with monocytes. The MAB
My4, however, does react with CLL B-cells (Ref. 34; Footnote
5). The specific fluorescence intensity, however, is approxi
mately 30-fold lower on CLL B-cells compared to the mono
cytes (data not shown). Hence, clear identification of the in
tensely stained monocytes was possible in every instance.
In order to reliably analyze the small percentage of monocytes
within the leukemic sample, we used immunofluorescence and
flow cytometry and could analyze more than 200,000 cells per
sample (the same number of cells was also analyzed in the
isotype control samples). Since no purification procedures in
volving 37°Cincubations are required in our procedure, artifi
cial alterations of cell surface molecules can be excluded.
Using this approach in our studies, monocytosis was evident
in 7 of 28 patients. This increase of monocytes in peripheral
blood could either be a reaction in response to increased cellular
decay that requires removal of debris, or it could be the result
of frequent infections. In a retrospective analysis, however, we
could not detect an increased report of infectious disease in
patients with and without monocytosis. Whatever the reason
for the monocytosis might be, these cells could have regulatory
effects on other cells of the immune system in CLL. Hence, to
learn more about the functional status of these monocytes, we
performed two-color immunofluorescence analysis in flow cy
tometry.
Irrespective of the presence of monocytosis we found an
increased expression of MHC Class II antigens, of C3bi recep
tors, and of Fc receptors on monocytes in CLL. The 3 patients
with the most elevated Class II antigen expression on mono
cytes (A. N., H. B., H. R.; cf. Table 1) all exhibited hypogammaglobulinemia. Furthermore, there was a positive correlation
between C3bi receptor expression and stage of disease (P <
0.01). This points to a possible clinical relevance of these
findings. The increase of Fc and C3bi receptor expression might
hint towards a higher phagocytic capacity for opsonized infec
tious agents. In light of the prevalent antibody deficiency in
CLL (1-4), such a higher receptor expression might at least
partially compensate for the reduced protective capacity due to
the low antibody level.
Expression of MHC Class II antigens together with IL-1
secretion is a critical element in antigen presentation to T-cells.
Cell-mediated immune response, when looking at skin reactivity
to neoantigens, like dinitrofluorobenzene, is, however, strongly
reduced in CLL (10). This might indicate that other compo
nents of this reaction are defective. To elucidate the mecha
nisms involved in this process in CLL, additional studies on
monocyte-derived mediators like prostaglandin \-'.:. IL-1, and
TNF are required.
In the parallel study on TNF (which in many instances is
concomitantly regulated with IL-1) we found defective produc
together with the phenotypic analysis reported herein might
help in answering the question of whether monocytes form a
central regulatory element in the immunodeficiency in CLL.
ACKNOWLEDGMENTS
The authors acknowledge the provision of patient samples by Dr.
Eggert (Krankenhaus Schwabing, Munich). Professor Emmerich (Kran
kenhaus rechts der Isar, TU-University, Munich), Professor Thiel
(Medizinische Klinik, LMU-University, Munich), and Professor Killer
(Krankenhaus Großhadern,Munich); the provision of monoclonal an
tibodies by Professor E. P. Rieber (Institute for Immunology, Munich);
and the assistance in flow cytometry by T. Schlunk.
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1987.
4816
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1988 American Association for Cancer Research.
Abnormal Blood Monocytes in Chronic Lymphocytic Leukemia
Dimitri Flieger and H. W. Löms Ziegler-Heitbrock
Cancer Res 1988;48:4812-4816.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1988 American Association for Cancer Research.