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Keywords:

  • CLL;
  • interleukin-6;
  • TNF;
  • B cell;
  • leukaemia

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

The production of the cytokines interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) in B-CLL cells from 24 patients at different stages of chronic lymphocytic B-cell leukaemia (B-CLL) was investigated in vitro. In the majority of these cases, low spontaneous IL-6 production was measured. Mitogenic stimulation with phorbol 12-myristate 13-acetate (PMA) or PMA plus interleukin-2 (IL-2) resulted in a tremendous increase in TNF-α and IL-6 production in cells representing early stage (Binet A) disease. In contrast, very little, if any, production took place in cells from patients with advanced stage (Binet C) B-CLL. The results from stage B patients were intermediate. The most remarkable difference was recorded in PMA-stimulated (1 ng/ml) IL-6 production. In stimulated 72 h cultures, IL-6 concentrations were 1280 ± 1080 pg/ml for Binet A (n = 11), 757 ± 597 pg/ml for Binet B (n = 8) and 46.0 ± 84.0 pg/ml for Binet C (n = 5). The differences in IL-6 production between stage C v B and stage C v A were both statistically significant (P = 0.025). Similar effects, but to a lesser extent, were observed in TNF-α production. These results suggest that the varying capacity to produce IL-6 and TNF-α may play a role in B-CLL progression and in clinical manifestations of the disease.

Chronic lymphocytic leukaemia of the B-cell type (B-CLL) is a disease characterized by expansion of mature-appearing B-lymphocytes in the peripheral blood and bone marrow ( Dighiero et al, 1991 ). The malignant monoclonal B cells display several phenotypic and functional differences compared with their normal cellular counterparts (reviewed by Zaknoen & Kay, 1990). B-CLL cells typically express low-density surface membrane immunoglobin, mostly IgM or IgD or both and a single κ or λ light-chain. Accepted requirements for diagnosis also include a specific immunophenotype, absolute lymphocytosis and infiltration of the bone marrow by lymphocytes ( Bennet et al, 1989 ; Matutes et al, 1994 ). Although the aetiology and pathogenesis of the disease is still in many respects unknown, recent data have provided important information about factors related to disease progression and cellular mechanisms involved in the leukaemic process. The lack of spontaneous in vitro proliferation of malignant B cells supports the theory that expansion of the monoclonal B-cell population is largely dependent on exogenous growth stimulation provided by environmental cells and autocrine growth factors ( Collins et al, 1989 ). Cytokines such as TNF-α, interleukin-1β (IL-1β), IL-2, IL-4, IL-6, IL-8, IL-10 and interferon-α have been proposed to play a role in the regulation of growth and death of leukaemic B cells ( Aderka et al, 1993 ; Buschle et al, 1993 ; Dancescu et al, 1992 ; Di Celle et al, 1994 ; Fluckiger et al, 1994 ; Larsson et al, 1993 ; Panayiotides et al, 1994 ; Van Kooten et al, 1992 ). In some studies, B cells have also been shown to express functional receptors for some of these cytokines, such as TNF-α, IL-6 and IL-2, suggesting the existence of possible autocrine cytokine loops ( Cordingley et al, 1988 ; Fluckiger et al, 1993 ; Lavabre-Bertrand et al, 1995 ). Exogenously administered TNF may also show a co-stimulatory effect on mitogen-activated normal human B cells as well as providing a proliferative signal to leukaemic B cells in B-CLL ( Digel et al, 1989 ). Recent reports emphasize the role of TNF-α as an autocrine and paracrine growth factor for B-CLL cells and the role of IL-6 as an inhibitory factor for this TNF-α-induced proliferation ( Reittie et al, 1996 ). Some reports suggest that there is constitutive spontaneous expression of IL-6 and TNF-α in CLL cells of B-CLL patients, the mechanism and meaning of which are still unknown ( Biondi et al, 1989 ; Foa et al, 1990 ). The present study was designed to investigate interleukin-6 and TNF-α production in B-CLL cells from patients at different stages of disease, in order to determine possible connections between these cytokines and disease aggressiveness, and prognostic parameters.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Patients

Clinical specimens were obtained after informed consent from 24 consecutive patients referred to the CLL out-patient clinic of Tampere University Hospital. Clinical and haematological data are presented in Table I. The diagnosis and staging of CLL were based on standard clinical, morphological and immunophenotypical criteria ( Bennet et al, 1989 ; Binet et al, 1981 ; Matutes et al, 1994 ; Rai et al, 1975 ). Case 9 had previously been treated with leukeran (chlorambucil) and cases 8, 10 and 24 with leukeran and prednisolone. Cases 2, 12 and 21 had also been treated with more intensive protocols comprising cyclophosphamide, adriamycin, vincristine and prednisolone (CHOP).

Samples and cell separation

Peripheral blood mononuclear cells were isolated from heparinized (Noparin, Nova Nordisk, Dagsvaerd, Denmark) blood samples by centrifugation over a Lymphoprep layer (Nycomed, Oslo, Norway) with a density of 1.077 g/ml (400 g, 40 min). The cells were washed twice with phosphate-buffered saline (PBS, pH 7.4) and once with complete culture medium consisting of RPMI 1640, 20 m m Hepes (ICN Biomedical, Costa Mesa, Calif.), 10% heat-inactivated fetal calf serum (Gibco BRL, Eggenstein, Germany), 2 m ml-glutamine, and antibiotics (Gibco; 50 units penicillin/ml and 50 μg streptomycin/ml). Cell counting was performed by using Technicon H*1 or H*2 analysers (Bayer Diagnostica). The proportion of monocytes was always less than 2%. Cell viability was determined haemocytometrically by trypan blue dye exclusion.

Immunophenotyping

Immunophenotyping was performed by flow cytometry (EPICS C, Coulter Electronics, Hialeah, Calif.) using commercial mouse monoclonal antibodies and respective immunoglobulin isotype controls as recommended by the manufacturers. The antibodies were as follows: FITC/PE-labelled Simultest Isotype control IgG1/IgG2a (clones X40/X39), LeucoGATE CD45/14 (anti-HLe-1/Leu-M3), anti-κ/anti-λ (clones TB28-2/1-155-2), anti-CD4/anti-CD8 (Leu-3a/Leu-2a); FITC-labelled anti-CD2 (Leu-5b), anti-CD5 (Leu-1), anti-CD22 (Leu-14), anti-CD25 (IL-2R); PE-labelled anti-CD19 (Leu-12), anti-CD20 (Leu-16), anti-CD23 (Leu-20); all from Becton Dickinson (Mountain View, Calif.). FITC-labelled anti-FMC7 was purchased from Dako (Glostrup, Denmark) and PE-labelled anti-CD2 from Immunotech (Marseille, France). The B-cell nature of all CLL cases was confirmed as described previously ( Matutes et al, 1994 ). The number of clonal B cells in the Lymphoprep isolates was always >90%, and the proportion of CD2-positive cells varied between 0 and 6%.

Culture conditions, mitogens and cytokines

Cells were cultured in RPMI culture medium (above) at a concentration of 2 × 106/ml in 24-well flat-bottomed plates (Nunclon Delta S1, Nunc, Roskilde, Denmark). Phorbol 12-myristate 13-acetate was used as a mitogen (PMA; Sigma, St Louis, Mo.) at a concentration of 1 ng/ml, with and without interleukin-2 (10 U/ml, recombinant human IL-2, Boehringer Mannheim, Mannheim, Germany). Leukaemic B cells were tested for their capacity to release TNF-α and IL-6, both spontaneously and after various stimuli. After 72 h in culture, cell supernatants were collected, centrifuged and stored at −20°C until cytokine determination.

ELISAs

Interleukin-6 and TNF-α concentrations were determined by using commercially available enzyme-linked immunosorbent assays (Pelikine Compact human IL-6 ELISA kit and Pelikine compact human TNF-α ELISA kit; CLB, Amsterdam), following the manufacturer's instructions. The optical density of individual wells was determined with a ‘Multiscan Biochromatic 348’ (Titertek) spectrophotometer. The detection limit of the IL-6 assay was 0.6 pg/ml and for the TNF-α assay it was 1.4 pg/ml.

Intracellular IL-6 detection

Expression of intracellular IL-6 was studied by flow cytometry (Becton Dickinson Immunocytometry Systems, Palo Alto, Calif.) in leukaemic cells. Cells (2 × 106/ml) were stimulated with PMA at a concentration of 1 ng/ml. After 6, 24 and 72 h in culture, the cells were stained using a commercially available cytostain kit (Cytofix/Cytoperm kit, Pharmingen, San Diego, Calif.), following the manufacturer's protocol. The protein transport inhibitor Brefeldin-A (BFA; Sigma, 10 μg/ml) had been added to the cell cultures 6 h prior to staining. To demonstrate specificity of staining, an IL-6 ligand block control was prepared by pre-incubating FITC-conjugated rat anti-human IL-6 mAb (clone MQ2-6A3, Pharmingen) with a molar excess of IL-6 protein (recombinant human IL-6, CLB). Quadrant markers for bivariate dot blots were set, based on non-specific PE-labelled surface isotype control (IgG1, Becton Dickinson) and ligand-blocked FITC-labelled anti-IL-6 mAb.

Statistical analysis

Mean values were compared by using Student's t-test.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

IL-6 production

In almost all cases (21/24) low (mean 57.8, SD 91.4 pg/ml, range 4–436 pg/ml) spontaneous IL-6 production was observed in unstimulated cell cultures. After stimulation with PMA (1 ng/ml) the cells of Binet class C patients showed significantly (P < 0.05) lower IL-6 production compared with cells of Binet class A or B patients (Fig 1). Co-stimulatory experiments with PMA and IL-2 produced similar results. With PMA stimulation, IL-6 production was 28-fold in Binet group A compared with group C (P = 0.025) and 16-fold in Binet group B compared with group C (P = 0.025). Only a minor, 2-fold difference in IL-6 production was found between the cells of Binet classes A and B (P = 0.232). The reduction in IL-6 production was also demonstrated when Rai's classification ( Rai et al, 1975 ) was used (data not shown). The results were also related to the cell immunophenotype score ( Table I) and the treatment previously given, but no clear correlations between these parameters and IL-6 production were found. Neither were there correlations between the percentage of CD2- or CD14-positive cells and measured IL-6 protein levels.

image

Figure 1. ), B (n = 8) and C (n = 5). Ordinate: IL-6 concentration in picograms per millilitre; abscissa: stimulus.

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Intracellular IL-6 detection

In order to determine the cellular source of IL-6 protein, cells from one index patient were selected for intracytoplasmic cytokine detection studies (see Materials and Methods). After stimulus with PMA (1 ng/ml) the most distinct IL-6 response was seen at the 6 h time point in cells representing CD19-positive leukaemic cells (Fig 3). Percentages of IL-6-producing CD19 cells were 37.2% in 6 h culture, 17.5% in 24 h culture and 15.6% in 72 h culture. Percentages of IL-6-positive CD2 cells were < 1.8% at all time points.

TNF-α production

With one exception (1/24), TNF-α concentrations were below the assay limit (<1.4 pg/ml) in unstimulated cell cultures. After mitogenic activation with PMA, low TNF-α concentrations were observed in samples from Binet class A and Binet class B patients. None of the Binet class C patients' cells responded to PMA stimulus alone. In co-stimulatory experiments with PMA + IL-2, a clear increasing trend in TNF-α concentrations was observed in all groups (Fig 2). When TNF-α concentrations were compared within the Binet stages, TNF-α production was 20-fold in Binet group A compared with group C (P = 0.303), 9-fold in Binet group B compared with group C (P = 0.064) and 2-fold (P = 0.413) between Binet groups A and B, respectively. As was the case with IL-6, the results showed the same trend when Rai's staging system ( Rai et al, 1975 ) was applied. No correlation between cell immunophenotype score ( Table I), treatment or percentage of CD2- or CD14-positive cells, and TNF-α production was found. Neither was there correlation between TNF-α and IL-6 production (data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

We have shown that B-CLL cells constitutively release low levels of IL-6 and that activated release of this protein is significantly lower in more advanced forms of the disease. The effect was pronounced after mitogenic exposure to PMA or to PMA with IL-2. There are reports that IL-6 inhibits TNF-α-induced proliferation of B-CLL cells ( Aderka et al, 1993 ; Van Kooten et al, 1993 ) and that IL-6 production is lowered in B-CLL cells of patients with progressive disease ( Aguilar-Santelises et al, 1991 ). Interleukin-6 is known to be a B-cell growth and differentiation factor and it is generally found to be exceptionally highly expressed in many autoimmune diseases (reviewed by Kishimoto, 1992). On the other hand, it has been shown that IL-6 is essential for normal B-cell development ( Rieckmann et al, 1991 ) and for normal megakaryocyte maturation ( Han et al, 1991 ).

The concentration of IL-6 has been found to be decreased in the serum of CLL patients compared with that in cases of other B-cell neoplasms ( Denizot et al, 1995 ). It is also known that growth of B-CLL cells is induced by TNF-α and TNF-βin vitro and inhibited by IL-6, whereas anti-IL-6 and anti-IL-6R antibodies potentiate B-cell proliferation ( Aderka et al, 1993 ). This is in contrast to the growth stimulation effects of IL-6 in other B-cell neoplasms ( Barut et al, 1990 ; Burger et al, 1994 ).

It has been suggested that CLL might result from a step-wise process in which, under the influence of mutational agents, the CD5-positive B cell would eventually undergo malignant transformation and be driven to monoclonal proliferation ( Rozman & Montserrat, 1995). However, neoplastic CD5-positive B lymphocytes appear to accumulate because of the inhibition of programmed cell death (apoptosis) rather than via straightforward proliferation ( Gale et al, 1994 ). It is known that the absence of growth factors induces apoptosis of CLL B cells ( Collins et al, 1989 ) and cytokines partly regulate cell death by acting as survival signals ( Buschle et al, 1993 ; Dancescu et al, 1992 ; Panayiotidis et al, 1994 ). On the other hand, prolonged cell survival could also result from a lack of specific apoptosis-inducing factors in the cell milieu ( Fluckiger et al, 1994 ). Autocrine secretion or aberrant levels of certain cytokines could be responsible for delayed apoptosis and extended survival of malignant cells. On this basis, one may speculate that the low constitutive IL-6 release observed in B-CLL cells is an autoinhibitory (physiological) reaction to abundant B-cell expansion. If so, impaired ability to produce IL-6 could lead to disease progression and accumulation of malignant cells in the blood.

As the definitive role of IL-6 and regulation of IL-6 gene expression in CLL cells are still largely unknown, we should also consider mutational aspects or potential genetic polymorphism as an explanation for variable IL-6 production. Possible genetic or functional alterations in cytokine expression may partly increase the growth potential and prolong the life of leukaemic cells, leading to increased leukaemic cell mass and organ manifestations. With regard to IL-6 physiology (i.e. normal B-cell development and megakaryocyte maturation), defective capacity to produce IL-6 could be partially responsible for some of the side-effects observed in more advanced forms of CLL (e.g. hypogammaglobulinaemia and thrombocytopenia). Therefore the molecular mechanism and functional genetics concerning diminished IL-6 production is worth further examination.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

This study was supported by grants from the Medical Research Fund of Tampere University Hospital, from the Finnish Foundation for Cancer Research and from the Academy of Finland. We thank Leena Pankko for her technical assistance.

The Tampere CLL Group: Risto Aine, Kyösti Ala-Kaila, Tuomo Honkanen, Mikko Hurme, Jussi Jouppila, Olli-Pekka Kallioniemi, Elli Koivunen, Marja Lehtinen, Tuula Lehtinen, Jukka Lumio, Petri Oivanen, Kalevi Oksanen, Hannu Peltovaara, Immo Rantala, Erkki Seppälä, Sanna Siitonen, Kalle Simola, Marjatta Sinisalo, Juhani Vilpo, Leena Vilpo, Tampere University Hospital, Hatanpää Hospital, Seinäjoki Central Hospital, Kanta-Häme Central Hospital, Päijät-Häme Central Hospital.

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  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
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