B-cell chronic lymphocytic leukemia (B-CLL) is a common form of leukemia affecting mostly elderly individuals. The course of the disease is usually unremarkable, but because it may proceed with impaired immune defense, B-CLL might be complicated with infections and even death. The leukemic microenvironment containing a number of immune cells, mainly lymphocytes and macrophages capable to produce various molecules including inflammatory cytokines, plays an important role in the development and outcome of the disease. We studied the capacity of Epstein-Barr virus (EBV)-transformed B-cell chronic lymphocytic leukemia (B-CLL) cell line (EHEB) cells, an EBV-transformed line established from a B-CLL patient, to affect the production of inflammatory cytokines by human peripheral blood mononuclear cells (PBMC).
PBMC isolated from peripheral blood of healthy donors were incubated either with EHEB cells or with their supernatants and the production of the following cytokines: tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, interferon (IFN)-γ, IL-1ra, and IL-10 were detected using the enzyme-linked immunosorbent assay method.
Direct contact of PBMC incubated with EHEB cells induced a marked increase of TNFα, IL-1β, IL-6, IFNγ, and IL-10 release by the immune cells. Yet, incubation of PBMC with EHEB cells' supernatant resulted in a mild production of the same cytokines.
The noticeable increased production of inflammatory cytokines by PBMC following direct contact with EHEB cells and to a lesser degree with their supernatants implies the existence of an immune dialogue between these two types of cells. The results support the concept that not only leukemic cells, but also peripheral blood mononuclears could serve as a therapeutic target for B-CLL.
B-cell lymphoproliferative disorders and particularly B-cell chronic lymphocytic leukemia (B-CLL) are the most wide-spread hematologic malignancies. B-CLL affects mainly elderly patients and it is infrequently accompanied by clinical symptoms. In the majority of cases the course of the disease is uneventful and with no need for specific treatment. However, because the B-CLL is characterized by continuous accumulation of pathological B lymphocytes and a decrease of normal immune cells, the immune system in these patients is compromised; a process leading to infections and increased morbidity and mortality. The microenvironment in B-CLL contains activated T cells, dendritic cells, macrophages, as well as endothelial cells, endowing it to play a principal role in the cross-talk with leukemic cells, to affect their development and viability, and serving therefore as a potential therapeutic target.[2,3] Similarly to normal B cells, B-CLL cells express a number of surface cluster of differentiation (CD) antigens, including B-cell receptor, CD20, and CD5, the latter being also T-cell associated. Although there are a number of factors that could affect the normal function of the immune system in B-CLL, it appears monocytes and their offspring, the macrophages present in the leukemic microenvironment, act as essential offenders. CLL-associated macrophages not only promote leukemic cell survival but also restrain T-cell antitumor responses and endorse drug resistance.[5–7] The absolute number of monocytes in patients with chronic lymphocytic leukemia was found to be higher in comparison to healthy controls. Furthermore, it has been reported that elevated absolute monocyte count in the circulation of a large group of patients with CLL was associated with bad prognosis. Removing leukemic associated macrophages and even reducing their number exerted a beneficial effect on mice survival. It has been reported that CLL-associated monocytes/macrophages may react with leukemic cells by both cell-to-cell interaction and by their ability to produce cytokines.[6,10,11] Following these observations, we posed the question if it is possible to immunomodulate human peripheral blood mononuclear cells (PBMC) by direct contact with B-CLL cells or with their supernatants. For this purpose, we incubated human PBMC with either Epstein-Barr virus (EBV)-transformed B-CLL cell line (EHEB) cells, a line established from the peripheral blood of a patient with B-CLL prior treatment by EBV transformation or with their supernatants and the secretion of a number of inflammatory cytokines produced by the immune cells was examined.
MATERIALS AND METHODS
The study was approved by the Ethics Committee of Rabin Medical Center. Blood bank donors gave written informed consent, including their agreement as for blood components, not needed for therapeutic purposes, to be used for medical research. PBMC were separated from venous blood by Lymphoprep-1077 (Axis-Shield PoC AS, Oslo, Norway) gradient centrifugation. The cells were washed twice in phosphate buffered saline (PBS) and suspended in RPMI-1640 medium (Biological Industries, Beith Haemek, Israel) containing 1% penicillin, streptomycin, and nystatin, 10% fetal bovine serum (FBS), and designated as complete medium (CM).
EHEB cells, a B chronic lymphocytic leukemia cell line (DSMC German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany), was grown in CM containing RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics (penicillin, streptomycin, and nystatin; Biological Industries). The cells were grown in T-75 culture flasks at 37°C in a humidified atmosphere containing 5% CO2.
We incubated 0.5 mL of PBMC (4 × 106/mL of CM) with 0.5 mL of CM or with equal volume of various concentrations of EHEB cells at 2.5 × 104/mL, 1 × 105/mL, and 4 × 105/mL (resulting in a PBMC:EHEB ratio of 100:1, 25:1, and 10:1). The cultures were incubated for 24 hours at 37°C in a humidified atmosphere containing 5% CO2. At the end of the incubation period, the cells were removed by centrifugation at 250g for 10 minutes, the supernatants were collected, and kept at −70°C until assayed for cytokines content.
Preparation of EHEB Cells' Supernatants and Their Effect on PBMC Cytokine Production
One milliliter of EHEB cells suspended in CM at 1 × 105/mL and 4 × 105/mL was added to each of 24-well plates and incubated for 24 hours at 37°C in a humidified atmosphere containing 5% CO2. At the end of the incubation period, the cells were removed by centrifugation at 250g for 10 minutes and the supernatants were used to examine their effect on cytokine secretion. An equal volume of the various supernatants was added to 0.5 mL of PBMC (4 × 106/mL of CM) and the plates were incubated for 24 hours at the above-mentioned conditions. At the end of the incubation period, the cells were removed by centrifugation at 250g for 10 minutes, the supernatants were collected, and kept at −70°C until assayed for cytokines content.
Cytokine Content in the Supernatants
The concentration of the following cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, interferon (IFN)-γ, IL-1ra, and IL-10 in the supernatants, was tested using enzyme-linked immunosorbent assay (ELISA) kits specific for human cytokines (Biosource International, Camarillo, CA, USA) as detailed in the guideline provided by the manufacturer. The detection levels of these kits were 15 pg/mL for IL-6 and 30 pg/mL for all of the others.
A linear mixed model with repeated measures and assumption of compound symmetry (CS) was used to assess the effect of various numbers of EHEB cells on cytokine secretion by nonstimulated or stimulated PBMC. SAS version 9.4 (SAS Institute, Cary, NC, USA) were applied for this analysis. Paired t-test was used to compare between the level of cytokines produced after incubation with different quantities of EHEB cells and found in control cultures. Probability values of p < .05 were considered as significant. The results are expressed as mean ± SEM.
Cytokine Production by PBMC Incubated with EHEB Cells
Incubation of PBMC with increasing number of EHEB cells caused a dose-dependent stimulation of TNFα release (F = 24.85, p < 0.001). At 1.25 × 104, 5 × 104, and 2 × 105 of EHEB cells/2 × 106 of PBMC, the production of TNFα was 13.6, 20, and 20 times higher (p < 0.005) as compared with that produced by PBMC alone. There was no significant difference in TNFα production between PBMC (2 × 106) incubated with 5 × 104 or with 2 × 105 of EHEB cells (Fig. 1).
Incubation of PBMC with EHEB cells induced a stimulated IL-6 production (F = 12.27, p = 0.0016). In the presence of the three concentrations of EHEB cells, there was three times enhancement in IL-6 production (p < 0.05); however, no significant difference was found at the various concentrations (Fig. 1).
A dosed-dependent stimulation of IL-1β production was observed when PBMC (2 × 106) were incubated for 24 hours with 1.25 × 104 and 2 × 105 EHEB cells (F = 77.7, p < 0.0001). IL-1β secretion was 8, 12.75, and 13.6 times higher (p < 0.01) at 1.25 × 104, 5 × 104, and 2 × 105 of EHEB cells, respectively. The difference in IL-1β secretion in the presence of the two higher numbers of EHEB cells was not statistically significant (Fig. 2).
The production of IFNγ by PBMC incubated with increasing number of EHEB cells was significantly enhanced (F = 16.9, p = 0.0005). At 5 × 104 and 2 × 105 of EHEB cells/2 × 106 of PBMC the secretion of IFNγ was 2.5 (p < 0.05) and 3.6 (p < 0.001) times higher than that produced by PBMC incubated without EHEB cells, with no significant difference between these two concentrations (Fig. 2).
The secretion of IL-10 by PBMC was increased upon 24 hours of incubation with EHEB cell at the three concentrations tested (F = 47.6, p < 0.0001). IL-10 production was 11.7, 10.6, and 9.9 times higher at 1.25 × 104, 5 × 104, and 2 × 105 of EHEB cells/2 × 106 of PBMC, respectively (p < 0.001) as compared with control cultures of PBMC incubated without EHEB cells. There was no significant difference in the levels of IL-10 found in cultures of PBMC incubated with the various concentrations of EHEB cells (Fig. 3).
There was no significant difference in the amount of IL-1ra produced by PBMC incubated for 24 hours without or with EHEB cells at concentration as indicated above (F = 0.64, p = 0.61) (Fig. 3).
Effect of EHEB Supernatants on Cytokine Secretion by PBMC
Supernatants collected from EHEB cells incubated for 24 hours at 5 × 104/mL or 2 × 105/mL contained very low concentrations of all cytokines tested, except for IL-6 that was found to be increased (i.e., 1.59 and 1.73 ng/mL, respectively) (Table 1).
Supernatant derived from EHEB cells caused 4 times increased secretion of TNFα (p < 0.01 for both concentrations) as compared with control cultures incubated without EHEB supernatants (Fig. 1).
The production of IL-6 by PBMC was enhanced by 1.8 (p < 0.005) times upon 24 hours of incubation with supernatants derived from EHEB cells as described above (Fig. 1).
IL-1β (Fig. 2)
Twenty-four hours of incubation of PBMC with 50% of supernatants derived from 1 × 105 and 4 × 105/mL EHEB cells caused 5.8 (p < 0.02) and 6.6 (p < 0.007) times increased production of IL-1β, respectively, as compared with that produced by PBMC incubated without EHEB supernatants (Fig. 2).
The secretion of IL-10 by PBMC incubated with both EHEB supernatants at conditions as indicated above caused 7.7 times increase in IL-10 secretion (p < 0.001) as compared with that produced by cells incubated without EHEB supernatants (Fig. 3).
IFNγ and IL-1ra
The function of macrophages participating in the immune defense of the organism has been profoundly investigated. A detailed review of the role of macrophages in innate and adoptive immunity and their ability to navigate the process of inflammation has been published by Arango Duque and Descoteaux. The capacity of macrophages to produce inflammatory cytokines is one of their mighty armaments against pathogenic intruders and control of immunomodulation. In this sense, macrophages closely collaborate with other cytokine producing cells, mainly T and B lymphocytes. The role of cytokines in the course of B-CLL merits attention. Garley et al. have found a decreased expression of IL-6 receptor (sIL-6Rα) in lymphocytes of B-CLL individuals and a reduced expression of IL-1β and -6 in their neutrophils. According to Alhakeem et al. patients with CLL have higher IL-10 plasma levels compared with healthy controls because of enhanced B-cell antigen receptor (BCR) cross linking. In our hands, EHEB and PBMC cells incubated separately produced small and insignificant amounts of all cytokines, hereby examined respectively, except for higher IL-6 production by both type of cells. On the other hand, when PBMC and EHEB cells were incubated jointly, the production of TNFα, IL-1β, IL-6, IFNγ, and IL-10 was markedly elevated, whereas that of IL-1ra remained unchanged. Incubation of PBMC with EHEB supernatants produced similar effect, albeit to a considerably lower extent. These observations indicate the existence of an immune cross-talk between PBMC and EHEB cells expressed predominantly by a direct cell-to-cell contact and to a lesser degree by interaction between immune cells and malignant cells' supernatants. According to Qu et al. targeting cytokines released in the inflammatory microenvironment via cross-talk between immune and malignant cells may serve as a therapeutic modality in a number of malignant conditions. Certain cytokines, such as IL-2, -6, -10, -12, -15, and -21, were found to be able to improve the survival time of B-CLL cells by extending the period of their apoptotic death. A comparison of the capacity of leukemic and normal B lymphocytes to produce IL-6 and -10 revealed that the gene expression of these cytokines was significantly lower in cells from patients with CLL. Furthermore, the number of cytokine producing T cells in patients with progressive disease was higher than in those being in nonprogressive state.[18,19] Fillip et al. have developed an in vitro model based on a co-culture of CLL and nonneoplastic lymphocytes designated as “nurse-like cells” (NLC). They have observed that NLC prolonged leukemic cells' viability by stimulating the expression of antiapoptotic genes in the malignant cells. Research has proven that T cells in patients with CLL express abnormal function enhancing the downhill course of the disease. The dysfunction of T cells in those individuals may be expressed by abnormal cytokine production, mainly increased IL-2 and in some cases IL-4 which is an antiapoptotic cytokine. The secretion of TNFα and IFNγ was not affected. On the other hand, it was found that the circulating TNFα levels of patients with CLL were higher than those detected in sera of healthy individuals. Moreover, B-CLL cells were able to release various amounts of TNFα in vitro that increased markedly after stimulation of the cells with IFNγ, phytohemagglutinin, and phorbol-myristate-acetate.
The findings of the present work raise the question as for the mechanism by which EHEB cells stimulate PBMC for cytokine production. It is conceivable that EHEB cells promote PBMC to produce inflammatory cytokines by activating receptors located on the cell membrane after a direct cell-to-cell contact. This possibility is supported by previous observations that incubation of PBMC with rituximab, an anti-CD20 antibody, resulted in inhibited IL-2, IFNγ, and IL-10 production by phorbol-myristate-acetate/lipopolysaccharide-stimulated PBMC, and lack of an effect on IL-1β and -6 release. Studies have shown that B cells from CD20-depleted mice expressed decreased cytokine production. Keeping in mind that EHEB cells are EBV-transformed lymphoblastoid cell lines, one may argue the changes in cytokine production hereby observed are induced by the virus itself. However, it has been shown that EBV infection of T-cell lymphoma lines promotes secretion of TNFα, but does not affect TNFα, IFNγ, and IL-1α expression in B-cell lymphoma lines. In not EBV-infected T and B cells the secretion of TNFα was markedly lower compared with infected cells. The marked upregulation of IL-10 and -6 observed in the present study is in accordance with the findings by Miyauchi et al., who have reported that in addition to the induced production of these cytokines, there was increased expression of TNFα, IFNγ, and IL-1α in EBV-transformed B-cell lines expressed in a cell dependent manner. Yokoi et al. have found an increased production of IL-10 and -6 after EBV infection of macrophages and B cells in B-cell malignancies. While the authors interpreted their results being induced by EBV itself, we believe in our study EHEB cells promoted the production of these cytokines by both cell-to-cell interaction and via factors released to the supernatants. This presumption is based on the fact that 24 hours of incubation of EHEB cells at the concentrations used revealed very small amounts of all cytokines tested, except IL-6; whereas incubation of EHEB cells or their supernatant with PBMC caused an impressive increase in inflammatory cytokines secretion. However, the possibility EBV is involved in this effect cannot be ruled out. It has been reported that highly purified B-CLL cells do not produce IL-8 spontaneously, but its release emerged after minor contamination with monocytes, indicating that monocytes were the source of the interleukin. On the other hand Miyauchi et al. have indicated IL-8 is induced in B-cell lines after EBV transformation. In vitro examination of the immune relationship between PBMC and malignant cells in general and EHEB cells in particular may be a limitation of the study, because the cytokine release in the living organism is affected by a variety of additional factors, such as time of interaction between immune and leukemic cells, number of M1-classically activated monocyte differentiated to those M2-alternatively activated resulting with a change in the type of inflammatory cytokine produced, and mostly by the general health status of the patient.
The results of the present study underline the important role that macrophages exert through the course of chronic lymphocytic leukemia, as it has been shown in mice in which macrophage depletion via TNFα pathway and targeting CD20 cells reduced the expansion of leukemic cells and prolonged the survival time of the animals.[9,28] These observations may serve as an additional tool to the therapeutic armamentarium for lymphocytic malignancies.
The results of the present study indicate that EHEB cells induce cytokine production by PBMC as follows: (1) via direct contact between the cells resulting in a substantial release of inflammatory cytokines by the immune cells, and (2) to a lesser degree, as a consequence of factors secreted by EHEB cells. These findings support the statement by Galleti et al. that targeting the capacity of B cells and macrophages to produce inflammatory cytokines offers an additional therapeutic approach for treatment of aggressive cases of B-CLL.
The authors thank Ms. Tzippy Shochat, MSc, Statistical Consultant, Rabin Medical Center, Beilinson Hospital, for performing the statistical calculations.
Source of Support: None, Conflict of Interest: None.