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Advances in Brief |
Laboratory of Oncology, G. Gaslini Institute, 16147 Genova [I. A., R. G., A. C., P. F., V. P.]; Department of Experimental Medicine, Section of Human Anatomy, University of Genova, Genova 16132 [F. G.]; and Service of Pathology, S. Martino Hospital, Genova 16132 [M. T.], Italy
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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and IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-10, IL-12 p35 and p40, IL-13, IL-15; IFN-
, G-CSF, GM-CSF,
M-CSF; TNF, LT-, TGF-ß1, and LIF. Most of these cytokines have a
well-characterized functional activity on human B cells
(2)
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Normal B-cell subsets representing the normal counterparts of FL, MCL, and MZL were isolated from tonsils. Twenty-five tonsils were obtained from patients undergoing tonsillectomy for inflammatory disorders, following informed consent.
MNCs were isolated by a Ficoll-Hypaque density gradient and were T-cell depleted by E rosetting. Macrophages and natural killer cells were subsequently removed by incubation with CD68 and CD56 mAbs, respectively, followed by immune rosetting. Neoplastic B lymphocytes were further purified by positive selection of malignant B cells according to the expression of monotypic immunoglobulin light chains (4) . Naive B lymphocytes were isolated as IgD+ cells from tonsil B lymphocyte suspensions by immune rosetting. The IgD- B-cell fractions were further separated into CD38+ (GC) cells and CD38- (memory) cells (5) . In some experiments, GC B cells were isolated by a Percoll density gradient, followed by removal of IgD+, CD39+ B cells (4) . All of the steps for tonsil B-cell subset isolation were performed at 4°C to prevent spontaneous apoptosis of GC B cells.
CD3, CD19, CD38, CD56, and CD68 mAbs were from Becton Dickinson (San Jose, CA). CD39 mAb was from PharMingen (San Diego, CA), whereas the anti-IgD mAb was from Dako (Glostrup, Denmark). Flow cytometric analysis was carried out as reported (4) .
Normal or malignant B lymphocytes were cultured for 4–24 h in RPMI
1640 (Seromed-BiochromKG, Berlin, Germany) supplemented with 10% FCS
(Seromed), in the presence or absence of CD40 mAb (1 µg/ml;
Immunotech, Marseille, France) in combination with recombinant IL-4 (10
ng/ml; Genzyme, Cambridge, MA). Normal B-cell fractions were also
incubated as above with: (a) anti-
(1
µg/106 cells; Southern Biotechnology
Associates, Birmingham, AL) and anti-
(1
µg/106 cells) immunoglobulin light chain mAbs
(Southern Biotechnology Associates); or (b) PMA (10 ng/ml;
Sigma Chemical Co., St. Louis, MO) in combination with calcium
ionophore (250 ng/ml; Sigma).
Supernatants from B cells cultured in the presence or absence of CD40
mAb and IL-4 were tested in triplicate for IL-6, IL-8, IL-10, TNF, and
IFN- production by ELISA (Biosource, Camarillo, CA) or for G-CSF by
the same technique (R&D Systems, Minneapolis, MN). The presence of
IL-2, IL-7, and IL-15, which share the common chain of the IL-2
receptor for signal transduction (6)
, was investigated
using the CTLL-2 bioassay (7)
. In this assay, recombinant
IL-2, used as positive control, induces cell proliferation in a
dose-dependent manner (7)
. The identification of
biologically active cytokine(s) in supernatants, which were tested in
this study at the final concentration of 10% v/v, rests upon
neutralization with anti-cytokine antibodies.
RNA was extracted from freshly isolated or cultured cells using
Ultraspec (Biotecx Laboratory, Inc., Houston, TX) and subjected to
RT-PCR as reported (8
, 9)
. Primer sequences and profiles
of amplification were described in previous studies (8
, 9)
, with the exception of the following: IL-1,
5'-TGACTCAGAGGAAGAAATCATC and 3'-TGGGCAACTGATGTGAAATAG; IL-1ß,
5'-CTACGAATCTCCGACCACCAC and 3'-CGCTTTTCCATCTTCTTCTTTG; IL-7,
5'-ATGTTCCATGTTTCTTTTAGG and 3'-TCAGTGTTCTTTAGTGCCCATC; IL-13,
5'-GACCACGGTCATTGCTCTCAC and 3'-GTGTCTCGGACATGCAAGCTG; G-CSF,
5'-TGTGCCACCTACAAGCTGTGC and 3'-CCCAGTTCTTCCATCTGCTGC; LIF,
5'-TCTTGGCGGCAGGAGTTGTG and 3'-GTCGGCGGTGGCGTTGAGC; IL-15,
5'-CTGTTTCAGTGCAGGGCTTCC and 3'-GAATCAATTGCAATCAAGAAGTG; M-CSF,
5'-CTGCGTCCGAACTTTCTATGAG and 3'-CAGCAAGACCAGGATGACACT; LT-,
5'-TGACACCACCTGAACGTCTC and 3'-GAGAAACCATCCTGGAGGAAG; CD3,
5'-GGTTCGGTACTTCTGACT and 3'-TGGTTTTGACTTGTTCTG; CD19,
5'-ACCTCCTCGCCTCCTCTTC and 3'-TCCCCTTCCTCTTCTTCTG; and CD68,
5'-CATCCAACAAGCAATAGCA and 3'-CTGAGCCGAGAATGTCCACT. The amplification
profile was 94°C for 1 min, annealing 60°C (IL-1 and IL-1ß),
65°C (LT- and M-CSF), 48°C (CD3), 57°C (CD19), and 54°C
(CD68) for 1 min, 55°C (IL-7) or 70°C (IL-13 and G-CSF and LIF) for
2 min, and extension at 72°C for 1 min. Each cycle of amplification
was repeated 32 times. Ten µl of each sample were electrophoresed
through a 1.5% agarose gel containing ethidium bromide. The
specificity of amplification products was verified by confirming the
known bp sequence length and by Southern blot.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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The expression of the CD3, CD68, and
CD19 genes in normal or neoplastic B-cell fractions
was next investigated by RT-PCR. CD3 is selectively expressed in T
cells, CD68 is a specific macrophage marker, and CD19 expression is
restricted to the B-cell lineage. CD3 and CD68 mRNA were never found
in normal B-cell suspensions or in B-lymphoma cells, whereas they were
detected in tonsil MNCs (Fig. 1)
. In contrast, CD19 transcripts were found in purified normal and
neoplastic B-cell suspensions, as well as in tonsil MNCs (Fig. 1)
. The
results shown in Fig. 1
refer to one representative case of MCL, FL,
and MZL each and to a normal B-lymphocyte suspension. Normal and
malignant B-cell fractions that contained even minute amounts of
contaminant cell types, as assessed by flow cytometry and RT-PCR, were
excluded from the study.
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To check the correspondence between cytokine mRNA accumulation and
protein secretion, normal B-cell suspensions were cultured for 24 h with or without CD40 mAb and IL-4. Supernatants were tested for IL-6,
IL-8, IL-10, G-CSF, TNF, and IFN- by ELISA and for IL-2, IL-7, and
IL-15 by the CTLL-2 bioassay. Although activated B-cell supernatants
contained recombinant IL-4, RPMI 1640 supplemented with the same IL-4
concentration present in the CTLL assay (1 ng/ml) failed to induce cell
proliferation, which was instead clearly detectable in the presence of
recombinant IL-2 (data not shown). These experiments were not carried
out with lymphoma cells because of the paucity of purified B
lymphocytes obtained.
Cytokine Gene Expression in Neoplastic B Cells from MCL and in
Naive Tonsil B Lymphocytes.
Fig. 2
shows the patterns of cytokine gene expression in neoplastic B cells
from two representative MCL cases and in two naive
(IgD+) tonsil B-cell fractions, tested
immediately after isolation (see also Table 1
). Expression of IL-1, IL-1ß, IL-6, IL-7, IL-8, IL-10, IL-12 p35,
IL-15, LIF, TNF, TGF-ß1, IFN-, LT-, GM-CSF, and M-CSF was
observed consistently in both malignant and normal B cells (Fig. 2)
.
IL-3, IL-4, IL-5, and IL-13 transcripts were never detected, either in
MCL or naive B cells. IL-12 p40 and G-CSF mRNA were not expressed in
any MCL sample but were always found in naive B cells (Fig. 2)
. These
results were confirmed in 3 additional MCL cases and in 13 naive B-cell
suspensions. The IL-2 transcript was found consistently in naive B
cells but only in 2 of 5 MCL samples (Fig. 2
and data not shown).
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IL-6 and IL-8 were detected in the supernatants from both unstimulated
and CD40 mAb plus IL-4-activated naive B cells at the following
concentrations: IL-6, unstimulated cells, 533–1000 pg/ml,
stimulated cells, 1000–2100 pg/ml, ranges from three different
experiments; IL-8, unstimulated cells, 430–750 pg/ml, stimulated cells
980-1420 pg/ml, ranges from three different experiments. In contrast,
IL-2, IL-7, IL-10, IL-15, G-CSF, TNF, and IFN- were below the
threshold of detection.
Cytokine Gene Expression in Neoplastic B Cells from FL and in GC
Tonsil B Lymphocytes.
Fig. 3
shows the patterns of cytokine gene expression in neoplastic B cells
from two representative FL cases and in two GC
(CD38+, IgD-) tonsil
B-cell fractions, tested after isolation (see also Table 1
). Expression
of IL-1, IL-1ß, IL-6, IL-7, IL-8, IL-10, IL-12 p35, IL12 p40,
IL-15, LIF, TNF, TGF-ß1, IFN-, LT-, and GM-CSF was found
consistently in both malignant and normal B cells (Fig. 3)
. IL-3, IL-4,
IL-5, IL-13, and M-CSF transcripts were never observed, either in FL or
GC B cells. G-CSF mRNA was present in GC B cells but absent from FL B
cells. These results were confirmed in 8 FL cases and in 13 GC B-cell
suspensions. The IL-2 transcript was detected always in GC B cells
but only in 2 of 10 FL samples (Fig. 3
and data not shown).
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Next, FL or GC B cells were stimulated in vitro with CD40 mAb and IL-4 before being tested for cytokine gene expression. In GC B cells, the only change observed was the induction de novo of IL-13 mRNA expression, whereas in FL cells IL-5, IL-13, and G-CSF transcripts were detected (data not shown). Again, incubation of GC B cells with cross-linking anti-immunoglobulin mAbs or with PMA and calcium ionophore was ineffective at inducing de novo expression of any cytokine gene (data not shown).
IL-6, IL-8, and G-CSF were found in supernatants from unstimulated and
CD40 mAb plus IL-4-activated GC B cells. The ranges detected in three
different experiments are the following: unstimulated cells, 238–533
pg/ml IL-6, 750-1090 pg/ml IL-8, and 100–170 pg/ml G-CSF; stimulated
cells, 207–309 pg/ml IL-6, 1200–1340 pg/ml IL-8, and 200–300 pg/ml
G-CSF. IL-2, IL-7, IL-10, IL-15, TNF, and IFN- were below the
threshold of detection.
Cytokine Gene Expression in Neoplastic B Cells from MZL and in
Memory Tonsil B Lymphocytes.
Fig. 4
shows the patterns of cytokine gene expression in neoplastic B cells
from two representative MZL cases and in two memory
(CD38-, IgD-) tonsil
B-cell fractions, tested after isolation (see also Table 1
). Expression
of IL-1, IL-1ß, IL-6, IL-7, IL-8, IL-10, IL-12 p35, IL12 p40,
IL-15, LIF, TNF, TGF-ß1, IFN-, and LT- was observed
consistently in both malignant and normal B cells (Fig. 4)
. IL-3, IL-4,
IL-5, and IL-13 transcripts were never detected, either in MZL or
memory B cells. IL-2, G-CSF, M-CSF, and GM-CSF mRNA were always present
in memory B cells but absent from MZL B cells. These results were
confirmed in 3 additional MZL cases and in 13 memory B-cell
suspensions. A summary of cytokine gene expression studies in freshly
isolated B-cell subsets and in their malignant counterparts is shown in
Table 1
. In vitro stimulation of memory B cells or MZL B
cells with CD40 mAb and IL-4 induced de novo IL-13 mRNA
expression, whereas no effect was observed after culture with
anti-immunoglobulin mAbs or PMA and calcium ionophore (data not shown).
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were below the threshold of detection. |
Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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The results obtained from the analysis of freshly isolated normal
B-cell subsets indicate that naive, GC, and memory B lymphocytes
display similar profiles of cytokine gene expression. Thus, IL-1,
IL-1ß, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12 p35, IL-12 p40, IL-15,
LIF, TNF, TGF-ß1, IFN-, LT-, G-CSF, and GM-CSF were expressed
in all samples, whereas IL-3, IL-4, IL-5, and IL-13 transcripts were
consistently absent from the three major B-cell subsets. M-CSF mRNA was
detected in naive and memory B cells but not in GC B cells.
IL-13 mRNA induction was observed in all B-cell subsets upon culture with CD40 mAb and IL-4 but not with anti-immunoglobulin mAbs, suggesting that, in our experimental conditions, costimulatory interactions between CD40 on B cells and CD40 ligand on T cells, rather than B-cell receptor triggering, are of primary importance in the induction of cytokine gene transcription. This conclusion is reinforced by the finding that, in naive B cells, CD40 and IL-4 stimulation, but not surface immunoglobulin cross-linking, also induced the transcripts of IL-3 and IL-5.
Malignant B cells from MCL, FL, and MZL displayed overall profiles of cytokine gene expression similar to that observed in their normal counterparts. However, some holes in the repertoire of neoplastic B cells were detected and found to be reproducibly associated with a given lymphoma category.
In comparison with naive B cells, freshly purified MCL cells consistently lacked G-CSF and IL-12 p40 mRNA. The latter finding indicates that MCL cells cannot assemble the IL-12 heterodimer (10) .
In freshly isolated FL cells, at variance with GC B cells, G-CSF mRNA was absent, although it became detectable after CD40 and IL-4 stimulation. Notably, G-CSF was identified previously as an autocrine and/or paracrine antiapoptotic factor for GC B cells (11) .
Freshly isolated MZL lacked IL-2, G-CSF, GM-CSF, and M-CSF mRNA in comparison with memory B cells. The functional relevance of these findings is thus far unknown and deserves further investigation. Minor differences between malignant B cells and their normal counterparts were observed as for de novo cytokine mRNA induction after CD40 and IL-4 stimulation. Activated B lymphocytes can present antigen to T cells (12) . Murine studies support the hypothesis that this B cell-T cell interaction drives Th2 cell differentiation (13) .
In this study, human CD40- and IL-4-stimulated B cells of all subsets
were found to express the mRNA of cytokines that are instrumental for
the induction of Th1 differentiation, i.e., IFN-, IL-12
p35, and IL-12 p40 (10)
, whereas they lacked the
transcript of IL-4, an essential regulator of Th2 commitment
(14)
. IFN- was not detected in culture supernatants
from unstimulated or activated B cells, but it is conceivable that
additional signals, such as those delivered by IL-12, are required for
induction of IFN- synthesis and release (10)
. Taken
together, these findings would support the hypothesis that, in
secondary lymphoid organs where most antigen-specific immune responses
occur, activated B cells can promote Th1 differentiation. However,
current models postulate that antigen-presenting B cells are committed
to maintain the Th1 or Th2 phenotype of already differentiated
CD4+ cells through IL-12 or IL-6 release,
respectively, rather than to promote such differentiation
(15)
.
Production of IL-6 and IL-8 from the three major B-cell subsets and of G-CSF from GC B cells was detected both in the absence of stimuli and after CD40 mAb and IL-4 stimulation, whereas IL-2, IL-7, IL-10, IL-15, or TNF were not found in any supernatant. These results may be related to cytokine mRNA instability or to the requirement of additional signals for the induction of synthesis and release of the corresponding proteins.
Cytokines, such as IL-6, TNF, and IFN-, have been shown to be
hyper-produced by malignant B lymphocytes from lymphoproliferative
disorders other than those investigated here and to prolong survival or
promote proliferation of tumor cells (2
, 16, 17, 18)
. The
issue of cytokine production by lymphoma cells could not be here
addressed because of limitations in the number of cells available.
In conclusion, this study demonstrates that the holes in the repertoire of cytokines expressed by malignant B cells versus their normal counterparts differ consistently in MCL, FL, and MZL and appear to represent molecular fingerprints of each type of lymphoma. Functional studies addressing the potential relevance of these findings for tumor growth are warranted.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from
Associazione Italiana per la Ricerca sul Cancro and Ministero della
Sanità, Progetti Finalizzati (to V. P.). 
2 To whom requests for reprints should be
addressed, at Laboratory of Oncology, G. Gaslini Institute, Largo G.
Gaslini, 5, 16147 Genova, Italy. Phone: 39-010-5636342; Fax:
39-010-3779820; E-mail: vitopistoia{at}ospedale-gaslini.ge.it
3 The abbreviations used are: REAL, Revised
European American Lymphoma; GC, germinal center; MCL, mantle cell
lymphoma; FL, follicular lymphoma; MZL, marginal zone lymphoma; RT-PCR,
reverse-transcription PCR; IL, interleukin; G-CSF, granulocyte-colony
stimulating factor; M-CSF, macrophage-CSF; GM-CSF,
granulocyte/macrophage-CSF; TNF, tumor necrosis factor; LT,
lymphotoxin; TGF, transforming growth factor; LIF, leukemia inhibitory
factor; MNC, mononuclear cell; PMA, phorbol myristate acetate; Th, T
helper; mAb, monoclonal antibody; CD, cluster designation; G3PDH,
glyceraldehyde-3-phosphate dehydrogenase; mAb, monoclonal antibody.
Received 10/16/00. Accepted 12/28/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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T-cell lymphoma arising in a kidney transplant recipient.. Haematologica, 65: 256-262, 2000.
, and IL-10: role of B cells in the maintenance of T cell responses.. J. Exp. Med., 189: 1-11, 1999. inhibits apoptotic cell death in B cell chronic lymphocytic leukemia.. J. Exp. Med., 177: 213-218, 1993.This article has been cited by other articles:
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  D. de Jong Molecular Pathogenesis of Follicular Lymphoma: A Cross Talk of Genetic and Immunologic Factors J. Clin. Oncol., September 10, 2005; 23(26): 6358 - 6363. [Abstract] [Full Text] [PDF]
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 M. Hoogendoorn, J. Olde Wolbers, W. M. Smit, M. R. Schaafsma, I. Jedema, R. M.Y. Barge, R. Willemze, and J.H. F. Falkenburg Primary Allogeneic T-Cell Responses against Mantle Cell Lymphoma Antigen-Presenting Cells for Adoptive Immunotherapy after Stem Cell Transplantation Clin. Cancer Res., July 15, 2005; 11(14): 5310 - 5318. [Abstract] [Full Text] [PDF]
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