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NEOPLASIA
From the Haematological Malignancy Diagnostic Service,
Department of Haematology, Leeds General Infirmary, Leeds, United
Kingdom.
Interleukin-6 (IL-6) is reported to be central to the pathogenesis
of myeloma, inducing proliferation and inhibiting apoptosis in
neoplastic plasma cells. Therefore, abrogating IL-6 signaling is of
therapeutic interest, particularly with the development of humanized
anti-IL-6 receptor (IL-6R) antibodies. The use of such antibodies
clinically requires an understanding of IL-6R expression on neoplastic
cells, particularly in the cycling fraction. IL-6R expression levels
were determined on plasma cells from patients with myeloma (n = 93)
and with monoclonal gammopathy of undetermined significance (MGUS) or
plasmacytoma (n = 66) and compared with the levels found on normal
plasma cells (n = 11). In addition, 4-color flow cytometry was used
to assess the differential expression by stage of differentiation and
cell cycle status of the neoplastic plasma cells. IL-6R alpha chain
(CD126) was not detectable in normal plasma cells, but was expressed in
approximately 90% of patients with myeloma. In all groups, the
expression levels showed a normal distribution. In patients with MGUS
or plasmacytoma, neoplastic plasma cells expressed significantly higher
levels of CD126 compared with phenotypically normal plasma cells from the same marrow. VLA-5 Interleukin-6 (IL-6) was initially identified as a
factor that promoted differentiation of B-lymphocytes into
class-switched immunoglobulin-secreting plasma cells, without inducing
proliferation.1 For normal circulating plasma cell
precursors, the molecule is a critical survival factor.2,3
In contrast to its effects on normal B-cells, IL-6 induces
proliferation of myeloma cell lines and freshly isolated myeloma plasma
cells, but does not induce immunoglobulin secretion.4,5
Kawano et al6 have demonstrated that myeloma cells at an
early stage of differentiation are more proliferative in response to
IL-6. This "immature" fraction can be identified because it lacks
VLA-5 (CD49e) expression, has plasmablastic morphology, and produces
low levels of immunoglobulin.6 In addition, IL-6 can
inhibit the apoptotic effects of anti-fas antibodies or dexamethasone
on myeloma cell lines, but not of agents that induce DNA damage such as
doxorubicin or etoposide.7,8 IL-6 can function in both an
autocrine and paracrine fashion,9 but most evidence
indicates that its primary mode of action in vivo is paracrine in
nature.10,11 Transgenic mice that overexpress IL-6 have an
excess of plasma cells and polyclonal immunoglobulins, but do not
develop overt myeloma.12,13
The central role of IL-6 in the pathogenesis of myeloma has led to a
number of therapeutic strategies to abrogate IL-6
signaling.14 Clinical studies with murine anti-IL-6
antibodies showed evidence of disease regression in some patients, but
these were not maintained, probably because of the development of a
human anti-mouse reaction.15 Phase 1 studies of chimeric
anti-IL-6 in patients refractory to second-line therapy showed
effective blocking of IL-6 signals, but again no clinical response by
standard criteria.16 A possible reason for the lack of
clinical response is the inability to neutralize serum IL-6 completely,
as therapy appears most effective in patients with low serum IL-6
levels.17 Serum IL-6 receptor (IL-6R) levels are increased
to a much lesser extent than IL-6 levels in patients with
myeloma,18 and immunotherapeutic strategies are currently focused on the receptor for IL-6 because it is more effective to target
the receptor than the IL-6 molecule.19
The IL-6 receptor is a complex of 2 molecules, CD126 and gp130
(CD130).20 The alpha-chain CD126 is a glycoprotein that
contains the ligand-binding site.21 Although this molecule
is sufficient for low-affinity binding, high-affinity binding and
signal transduction require the presence of the CD130
molecule.20 CD130 is also the common signal transduction
unit for IL-11, oncostatin M, ciliary neurotropic factor, and leukemia
inhibitory factor.22 The soluble form of CD126 that is
generated by both normal and myeloma cells23 can bind IL-6
and mediate signal transduction through membrane-bound CD130.20 Thus, successful anti-CD126 immunotherapy must
inhibit the binding of IL-6 to both soluble and membrane CD126. A
number of studies have assessed the levels of serum CD126 in myeloma and monoclonal gammopathy of undetermined significance
(MGUS).24,25 However, the use of therapeutic strategies in
the clinical environment also demands a full understanding of the
pattern of expression of the membrane IL-6R.
Although receptor transcripts can be detected in the majority of
myeloma plasma cells,26 early flow cytometric studies were unable to detect surface expression.27 However, recent
studies using more sensitive phycoerythrin (PE)-conjugated
antibodies, which are unaffected by endogenous IL-6, have been able to
demonstrate CD126 and CD130 expression by plasma cells from most
patients with myeloma and MGUS.28
The aim of this study was to determine whether it is possible to
identify differences in IL-6R expression between normal and neoplastic
plasma cells that might explain their differential responses to IL-6. A
central part of this approach was to compare expression between normal
and neoplastic plasma cells from individual patients with MGUS, as well
as to assess the differential expression in precursor and cycling
plasma cell fractions.
Patients
Sample preparation and fluorescence-activated cell sorter
(FACS) analysis
Plasma cells were identified using a sequential gating strategy, shown
in Figure 1. An initial region (R1) was
set around cells expressing a high level of CD38 and CD138, and then a
second region (R2) was set on the light scatter of gated
CD38+CD138+ cells. A third region (R3)
was set around the cells satisfying both R1 and R2 for CD38 and CD45
expression. The final plasma cell gate used was a combination of R2 and
R3 (CD38, CD45, and scatter characteristics). CD138 was not used for
the final plasma cell gate because the level of expression is not
sufficient to separate plasma cells from other leukocytes, particularly
in the peripheral blood.29
The expression of CD138 (positive control) and CD3 (negative
control) was assessed to ensure that the gated cells were plasma cells
with minimal contamination. The expression of CD19-PE and CD56-PE was then used to distinguish between normal and
neoplastic plasma cells. The former are consistently
CD19+CD56
For analysis of cytoplasmic light-chain expression, leukocytes
were incubated with antibodies to surface antigens as described above.
The cells were then washed twice with FACSFlow containing 0.3% BSA,
incubated with 100 µL of FACSLyse (BD Biosciences) for 10 minutes,
and washed twice with FACSFlow containing 5% BSA. For
permeabilization, the cells were incubated with 100 µL of FACSFlow
containing 5% BSA and 0.05% NP-40 (Sigma-Aldrich) for 5 minutes and
washed twice with FACSFlow containing 5% BSA. The cells were then
incubated with 10 µL antibodies to intracellular antigens for 20 minutes, washed twice with FACSFlow containing 0.3% BSA, and acquired
and analyzed as above. Representative plots are shown in Figure
3.
For 4-color analysis of VLA-5 and Ki-67 expression, the same gating strategy and antibodies were used, but CD38-APC and CD45-PE/Cy5 were used to identify plasma cells. Assessment of VLA-5 expression was performed using fluorescein isothiocyanate (FITC)-conjugated anti-VLA-5, with no other changes to the methodology. For assessment of nuclear Ki-67 expression, we labeled the cells with PE, PE/Cy5, and APC conjugates as above and then incubated them overnight in 1% paraformaldehyde/FACSFlow (Sigma-Aldrich). The cells were then washed twice and incubated with 0.5% Triton X-100/FACSFlow (Sigma-Aldrich/BD Biosciences) for 10 minutes, washed once, and incubated with Ki-67-FITC, then washed twice and analyzed as described above. Because there was significant overlap between negative control and test histograms for anti-CD126 fluorescence, standard methods of analysis (ie, percentage positive and mean fluorescence intensity) were considered unsuitable. Therefore, Kolmogorov-Smirnov (K-S) analysis was used to assess the level of expression. CD126 and CD130 expression on plasma cells (gated as described above) was compared with control expression by K-S analysis with CELLQuest software. Briefly, the cumulative test and control distributions were compared, and the largest difference (D) between the 2 was calculated. This method has a number of advantages over other analyses (see Discussion) and is the standard method of comparing the difference between 2 overlapping histograms. To determine the reproducibility of the assay, we analyzed cell lines K620, U266b, JJN3, and JIM-1 in triplicate and produced 9 D values for each antigen on each cell line. The coefficient of variation for these values was consistently less than 5%. Cell culture Leukocytes were prepared from freshly aspirated (within 24 hours) bone marrow cells by density gradient centrifugation (Lymphoprep; Nycomed Amersham, Bucks, United Kingdom). Plasma cells were then magnetically purified with CD138 microbeads (Miltenyi Biotec, Surrey, United Kingdom). A total of 5 × 104 cells were incubated for 24 hours in 200 µL RPMI 1640 containing 5% fetal calf serum; 10 µg/mL dexamethasone; 0.1 µg/mL IL-6 (Sigma-Aldrich); and humanized anti-IL-6R (MRA, derived fron anti-CD126 clone PM-1, kindly provided by Chugai Pharma Europe, London, United Kingdom) at a concentration of 100, 10, 1, or 0.1 µg/mL. A control well, lacking both MRA and IL-6, was also prepared. All tests were performed in triplicate. After incubation, the cells were washed and incubated with 10 µL each CD38-PE/Cy5, 7-AAD (Sigma-Aldrich 1:20), and CD45-FITC. The cells were washed again, and 10 µL of 1:100 FITC CaliBRITE beads (BD Biosciences) was added to each well. The cells were acquired as described above. Plasma cells were identified by CD38 versus CD45 expression, and the proportion of viable (7-AAD ) plasma cells to beads was calculated for each
well. Test values (mean ± SE) were then reported as a percentage
of the mean of the dexamethasone-only control.
Antibodies CD3 (OKT3)-PE, CD19 (FMC7)-PE, CD38 (OKT8)-PE/Cy5/APC, and CD45 (4B2)-FITC/PE/Cy5 were prepared from hybridoma supernatant and conjugated and titered in-house. MRA (humanized anti-CD126) was conjugated and titered in-house. Other antibodies, which were used according to the manufacturers' recommendations, were VLA-5-FITC (SAM-1; Serotec), CD138-PE (B-B4; Serotec), CD56-PE (MY-31; Becton Dickinson), Ki-67-FITC (MIB-1; Coulter), CD126-PE (M91; Immunotech), and CD130-PE (AM64; Pharmingen).
Myeloma plasma cells have significantly higher levels of CD126 expression than normal plasma cells, which express negligible levels of CD126 Bone marrow plasma cells from patients with myeloma at presentation (n = 93) and from normal controls (n = 11) were assessed for expression of CD126. For normal plasma cells, CD126 fluorescence intensity was virtually identical to the control (median D = 0; range, 0-0.2). CD126 expression was significantly increased in myeloma patients (median D = 0.45; range, 0-0.99; Mann-Whitney U test, P < .0001) (Figure 4).
In both myeloma patients and normal individuals, the level of CD126 expression in the group as a whole showed a normal distribution (Anderson-Darling, P < .01). To determine a reference for the level of CD126 expression, we assessed the K-S D value for the U266b cell line. This cell line has been shown to express approximately 27 000 binding sites.20 Of the myeloma patients analyzed, 10% showed expression above this level, 13% expressed CD126 at control levels, and the remainder had an intermediate level of expression. To determine whether the different levels of surface CD126 result in
functional differences, we compared the ability of exogenous IL-6 to
rescue CD126+ and CD126
VLA-5 fraction and the
cycling fraction, identified by Ki-67 expression. This analysis was
performed in 10 patients with myeloma at presentation. The majority of
plasma cells lacked VLA-5 expression (median 88%; range, 44% to
98%). Coexpression studies indicated that the Ki-67+ cells
were restricted to the VLA-5 fraction, with more than
95% of the Ki-67+ plasma cells lacking VLA-5 expression in
all cases. The Ki-67+ fraction represented a median of 4%
(range, 0%-39%) of plasma cells, which is consistent with previous
studies.32
The VLA-5
There is no correlation between disease stage or serum  2m level. Patients with stage I
or II disease showed CD126 expression (median D = 0.62; range,
0.0-0.90) similar to that of patients with stage III disease (median
D = 0.46; range, 0.0-0.95; Mann-Whitney U test,
P > .05). Furthermore, patients with high levels (more
than 4.2 mg/L) of serum 2m did not show a higher level of CD126
expression (median D = 0.48; range, 0.03-0.90) compared with patients
with low 2m levels (median D = 0.40; range, 0.0-0.69; Mann-Whitney
U test, P > .05). Thus, CD126 expression does
not correlate with any major prognostic factor.
In MGUS and solitary plasmacytoma, CD126 is expressed on neoplastic, but not normal, plasma cells We and others have previously shown that patients with MGUS and solitary plasmacytoma have 2 populations of bone marrow plasma cells, one with a normal phenotype and another with a neoplastic phenotype.29,35 We therefore used this flow cytometric approach to compare the level of expression of CD126 on normal and malignant plasma cells derived from the same patient. Plasma cells were discriminated by their CD45 expression; they were considered normal if more than 80% of the cells were CD19+CD56,
and neoplastic if more than 80% of the cells were CD19
or CD19+CD56+. Only cases with more than 100 events in each population were included. The gating strategy is
demonstrated in Figures 1 and 2. Figure 3 shows representative plots of
cytoplasmic light-chain restriction and immunophenotype in patients
with myeloma and MGUS.
In 19 of 39 cases, it was possible to identify these 2 populations and
to directly compare the level of CD126 expression on normal and
neoplastic cells from the same individual. Plasma cells with a
neoplastic phenotype expressed significantly higher levels of CD126
(median D = 0.25; range, 0-0.69) than their normal counterparts (median D = 0.02; range, 0-0.28; Wilcoxon matched-pairs signed ranks,
P = .0035) (Figure 7).
Furthermore, expression of CD126 by the normal-phenotype plasma cells
in patients with MGUS or plasmacytoma was not significantly different
from that of plasma cells from normal donors (Mann-Whitney U
test, P = .4031). However, expression of CD126 by the
neoplastic plasma cells from MGUS was significantly lower than that of
plasma cells from myeloma (Mann-Whitney U test,
P = .0048).
Normal plasma cells express CD130, but neoplastic plasma cells express significantly higher levels Bone marrow plasma cells from patients with myeloma at presentation (n = 46), with MGUS or solitary plasmacytoma (n = 26), and from normal controls (n = 8) were also assessed for expression of the CD130 molecule. The results are shown in Figure 4. In contrast to the CD126 molecule, there was clear expression of CD130 by normal plasma cells. K-S analysis indicated that the normal level of expression was a median D value of 0.68 (range, 0.58-0.79; 95% confidence upper limit = 0.71). Plasma cells from MGUS or SP patients showed significantly higher levels (median D = 0.78; range, 0.51-0.99; Mann-Whitney U test, P = .023; 77% of patients above the normal range). Plasma cells from patients with myeloma also showed a higher level of CD130 expression than both normal and MGUS plasma cells (median D = 0.92; range, 0.41-0.99; Mann-Whitney U test, P = .0013 for normal and P = .0286 for MGUS; 83% of patients above the normal range). Although there was no correlation between CD126 and CD130 expression, CD130 was always expressed by cells with detectable levels of CD126 expression.
IL-6 is essential for differentiation of normal B cells into plasma cells1 and has also been demonstrated to be an essential survival factor for circulating plasma cell precursors.2 CD126 is detectable on the surface of circulating precursors from patients with reactive plasmacytoses.3 However, despite having a highly sensitive assay for detection of the receptor, we can find little or no evidence of expression on normal plasma cells. This strongly suggests that the CD126 chain is down-regulated during terminal differentiation of normal B-cells. In contrast, plasma cells from more than 80% of patients with myeloma, MGUS, or plasmacytoma show IL-6R expression above control levels. These data clearly demonstrate that the normal down-regulation of CD126 does not occur in neoplastic plasma cells. The proportion of myeloma patients expressing CD126 is consistent with previous studies that have detected CD126 mRNA in approximately 70% of patients with myeloma26 and surface receptor expression in 60% of patients.28 We have analyzed CD126 expression using the D value generated by K-S analysis, which has previously been shown to correlate directly with the number of receptor molecules determined by a radioligand-binding assay in a similar setting.36 K-S analysis is highly sensitive to small shifts in expression, and this may explain why the results of this study are more compatible with reverse transcriptase-polymerase chain reaction analysis than previous flow cytometry studies. Patients with MGUS or plasmacytoma, who have a mixture of normal and neoplastic plasma cells detectable by flow cytometry, provided an internal control population for this study. The difference in CD126 expression seen between normal and neoplastic plasma cells in MGUS patients is similar to the difference in expression seen in plasma cells from normal individuals and those from patients with myeloma. The overexpression of CD126 in neoplastic cells therefore provides a
possible mechanism for the differential responses of neoplastic and
normal plasma cells to IL-6. However, it is less clear whether the
major effect in vivo is proliferative or antiapoptotic. Kawano et
al6,37 have demonstrated that the VLA-5 Ki-67+ cells have significantly lower CD126 expression than
their noncycling counterparts, and in one patient with 30%
Ki-67+ plasma cells, there was no detectable CD126
expression. The proliferative capacity of the myeloma cells clearly
does not correlate with CD126 expression. This is not consistent with
studies that showed increased proliferation of neoplastic cells in
response to IL-6.5,37,38 It is possible that CD126
expression is transient and occurs only before entry into the cell
cycle. However, studies suggesting a proliferative role for IL-6 are
mostly based on tritiated-thymidine incorporation, in which decreased
apoptosis could produce similar results to increased proliferation. The
effects of IL-6 have been analyzed in vivo, and myeloma patients having
infusion of IL-6 showed no significant alteration in plasma cell
labeling index.39 We did not find increased CD126
expression in patients with more aggressive stage III disease, and
CD126 expression did not correlate with high serum A key finding of this study is the differential expression of CD126 in phenotypically normal and neoplastic plasma cells within the same individuals with MGUS and solitary plasmacytoma. This demonstrates that the level of CD126 expression is not a function of the marrow environment, but of the neoplastic plasma cells. Several studies have suggested that the marrow microenvironment plays a key role in the pathogenesis of myeloma. Lokhorst et al41 have shown that stromal layers depleted of plasma cells from myeloma patients produce higher levels of IL-6 than those from normal donors. Furthermore, the level of IL-6 produced correlates with disease stage in myeloma patients.41 In addition, the recent but controversial findings of Rettig et al suggested that infection of stromal cells by HHV-8,42 which produces a functional homologue of IL-6,43 may be central to the pathogenesis of myeloma. The finding that up-regulation of CD126 expression is present on neoplastic but not normal plasma cells from the same marrow aspirate indicates that aberrant expression is inherent to the neoplastic clone. Thus, stromal overproduction of IL-6 may inhibit apoptosis or promote growth of the neoplastic clone, but the primary abnormality is within the plasma cells, as their normal counterparts retain CD126 expression identical to that from normal individuals. In summary, the results suggest that a key feature of myeloma plasma cells compared with normal plasma cells is a failure to down-regulate CD126. This overexpression is unlikely to be the result of an arrest in maturation at a stage in which this receptor would normally be expressed because even the most "mature" myeloma plasma cells overexpress CD126. The overexpression is also not induced by stromal cells because neoplastic plasma cells from patients with MGUS express higher levels of CD126 than normal plasma cells from the same marrow. Increased CD126 expression is therefore inherent to the neoplastic cells, and, in combination with other studies, our results suggest that the major role of IL-6 in vivo is antiapoptotic. The significance of these results in the context of CD126 immunotherapy is 2-fold. First, the difference in expression between normal and neoplastic plasma cells supports the use of anti-CD126 antibodies for treatment of myeloma. However, this will require measurement of surface CD126 expression levels in each patient because those who lack CD126 expression are unlikely to benefit. Further studies are required to determine the relation between membrane and soluble CD126. Second, the therapeutic effects of CD126 antagonism may be either to induce apoptosis in malignant cells or to sensitize the cells to the apoptotic effects of other chemotherapeutic agents.
Submitted December 23, 1999; accepted July 20, 2000.
Supported in part by research funding from Chugai Pharma Europe, Yorkshire Cancer Research, and the Leukaemia Research Fund.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Gareth Morgan, Department of Haematology, University of Leeds, Leeds General Infirmary, Great George St, Leeds, West Yorkshire, lS1 3EX, United Kingdom; e-mail: garethm{at}pathology.leeds.ac.uk.
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Abstract
Introduction
