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CYTOKINE IMMUNOREGULATION IN B-CLL H FERNANDES AND E RAVECHE INTRODUCTION
    The sustained accumulation of mature CD5+ B-cells in the G0 phase of the cell cycle is a hallmark of B-cell Chronic Lymphocytic Leukemia (B-CLL) —an indolent hematological malignancy. Of the many comparisons between the CD5+ B-cell in B-CLL (1) and its normal counterpart also known as the B-1 cell, the inability of the former to progress past the G1 phase in the cell cycle has been well documented. The relative non-responsiveness of the B-CLL B-1 cell to exogenous growth factors/cytokines has been in part attributed to a defective signal transduction due to a marked reduction of functional Na+/H+ antiporter units (2). In addition, low levels of protein tyrosine kinase activity, defective Ca++ responses and altered patterns of protein tyrosine phosphorylation, all point towards abnormal growth responses in these malig-nant B-1 cells (reviewed in 3, 4). More recently it has been shown that the cells in early stages of CLL when compared with corresponding controls, have lower levels of telomerase, the riboprotein associated with immortality/malignancy (5). The contribution of either endogenous factors and/or secondary growth signals that alter the proliferation of B-CLL cells have led to a better understanding of this biologically heterogeneous neoplasm. These observations in conjunction with current treatment strategies, may enable clinicians to customize more effective individual treatments based on the stage of B-CLL differentiation.

    The events of growth and differentiation in the B-cell cascade is regulated by complex homeostatic mechanisms, including interactions between cells by signals provided by cell to cell contact and/or soluble hormone like factors called cytokines. Cytokines can work either alone or in combination with other cytokines that are induced or present in the microenvironment. In vitro
cytokines can upregulate expression of surface molecules that can in turn facilitate cellular interactions. They function in either an autocrine or paracrine manner and can also act as potentiators or suppressors of the biological activity of a cell. Alternatively, as may be the case in B-CLL, cytokines may function just to maintain the viability of a cell, thereby protecting it from cell death or apoptosis. The fact that B-CLL cells die rapidly in vitro contrasts with their sometimes infinite life span in vivo thereby suggesting that the cytokine network plays a pivotal role in the regulation of this malignancy. In this regard cytokines could be responsible for the development of B-CLL either by promoting cell proliferation or by prolonging cell viability, thus providing B-CLL cells with a survival advantage over cell death. We have therefore attempted to analyze the role of cytokines in B-CLL based on their functional capabilities in vitro and in vivo.
ENDOGENOUS CYTOKINES IN B-CLL
    Studies on cytokine production in B-CLL cells have some inherent problems that lead to a biased interpretation of results. First, the biologic diversity of this malignancy makes it difficult to generalize any observation to the entire B-CLL population. It is likely that cytokines produced by B-CLL cells are further dependent on the level of differentiation and the state of activation of the cell as well as the type of activating agent. Second, the vast majority of the cytokine data represents the mRNA and not the protein level, and therefore may not necessarily reflect the translated product. In addition the analysis of serum cytokine protein levels are indicative of the reactivity of the entire hematopoietic system in addition to the malignant B-1 cells. Thirdly, a number of studies on the analysis of cytokines do not use purified B-CLL populations and that makes the interpretation of results a problem.

    However a number of cytokine and/or their genes have been known to be constitutively expressed, in varying amounts by purified B-CLL cells of which TNF-a, IL-1b, IL-4, IL-6, IL-7, IL-8, IL-10 and IL-13 seem to be the most consistent (3, 4, 6-9). TNF-a has been implicated as an autocrine growth factor and has been detected both at the message as well at the protein level (10). IL-6, a B-cell cytokine also has been shown to immunomodulate B-CLL cells (11). de Celle et al., (12) have reported that IL-8 is consistently and constitu-tively expressed in B-CLL cells and not in other normal B-cells. Also Long et al., (8) have indicated that IL-7 is a cytokine that is consistently present in B-CLL cells. Comparative studies between bone marrow stromal cells from B-CLL patients and normal controls did not reveal any significant differences in production of IL-6, IL-10 and the colony stimulating factors (CSF) (13). However such reports are scattered and have not been totally reproduced by others. Such disputed inconsistencies have been attributed to the clonal heterogeneity that surrounds B-CLL. We and others have shown that IL-10 is also expressed in B-CLL cells and could possibly have an immunoregulatory role (14-16).
CYTOKINE RECEPTORS ON B-CLL CELLS
    A number of cytokines are known to affect the growth and differentiation of B-CLL cells in either a positive or negative manner, either individually by synergizing with other cytokines or more commonly in conjunction with an activating agent. In order for a cytokine to interact with a cell, specific receptors must be expressed by the cell itself. However, the presence of a specific
cytokine receptor on B-CLL cells need not necessarily implicate the cytokine as a functional mediator of growth and differentiation.

    The IL-2 receptor (IL-2R) is expressed on a variable fraction of B-CLL clones, and the p55 subunit of the IL-2 receptor has been detected in the serum of most B-CLL patients (17, 18). Receptor binding assays using radio-labelled IL-2 showed the presence of both high and low affinity receptors (19). Monoclonal antibodies to the IL-2R can block the IL-2 induced proliferation of B-CLL cells (20). IL-2 receptors on the surface of the B-CLL cell are regulated by TNF-a (21). However, since CD5+, CD20+ B-cell populations from normal donors (six of seven) were also found to be express the IL-2R p55-a chain at the RNA level (18) it is unlikely that the IL-2R is related to neoplastic transformation in B-CLL.

    Receptors for other cytokines have also been detected on both the B-CLL cell as well as their corresponding controls tested (see also Chapter 13). TNF-a receptor was shown to be present on B-CLL cells in vitro, but not in vivo (22). The presence of the IL-7 receptors were correlated directly with IL-7 induced proliferation in B-CLL and other leukemias (23). On the other hand
experiments on the effect of IL-13 on B-CLL cells suggest that these cells may lack a functional IL-13 R even though the cytokine can promote DNA synthesis (24).
CYTOKINE INDUCED PROLIFERATION OF B-CLL CELLS
    The in vitro survival and growth of cells depends on the availability of external factors or signals that provide the necessary stimulus leading to cell division. The concept of cytokine induced proliferation of B-CLL is old but still controversial and riddled with problems (reviewed in 4, 25-29; see also Chapter 14). Some of the earlier studies were done on populations of PBL derived from B-CLL patients and did not rule out the contaminating though minor subset of non-malignant host cells. The availability of recombinant cytokines has aided in the analysis of cytokine networking in B-CLL. In general however, the concept of a refractory B-CLL cell arrested in the Go phase of the cell cycle has been revised since studies have shown that B-CLL cells can undergo differentiation in response to certain external stimuli. For the most part however, B-CLL cells, when compared to normal cells, show a minimal proliferative response to recombinant cytokines such as IL-2. This response is significantly enhanced when the cells are first stimulated with either mitogens or other stimulating agents. In the final analysis of proliferation, one needs to consider uptake of 3H thymidine versus increase in cell numbers as the real effect. B-CLL cells can incorporate the labeled thymidine without undergoing mitosis.

    As in normal PBLs, DNA synthesis and sometimes increased Ig secretion, have been noted by several investigators after B-CLL cells are preactivated with anti IgM, anti CD40 or Staphylococcus A Cowan (SAC) and subse-quently stimulated with IL-2 reviewed in (4). IL-2 induced proliferation was significantly enhanced in the presence of soluble CD23 (30). Similarly both IFN-a as well as IFN-g were shown to augment the proliferative response of B-CLL cells to IL-2. IL-4 has no direct effect on B-CLL cells, however it strongly inhibits the IL-2 driven DNA synthesis of B-CLL cells (24). Cell cycle analysis revealed that IL-4 inhibited the Go-G1 and G1-S phase transition of B-CLL cells. TNF-a by itself had negligible effects on B-CLL cells in vitro, however both recombinant TNF-a and TNF-b were capable of increasing DNA synthesis in SAC activated B-CLL cells (31). Maximal DNA synthesis with corresponding increases in mRNA and protein in response to IL-7 was seen in purified CD5+ CD19+ B-CLL cells over a wide dose range (23). Besides regulation of surface markers, cytokines have been shown to play a significant role in both induction as well as protection of B-CLL cells from apoptosis.
CYTOKINES IN APOPTOSIS OF B-CLL CELLS
    Collins et al., in 1989 (32) observed that when in culture about 20% of B-CLL cells undergo spontaneous programmed cell death or apoptosis. It was subsequently shown that anti-IgM even though it was stimulatory to normal B-cells, induced apoptosis in B-CLL cells (33). This abnormal response was attributed to defective signaling in the leukemic cells (34). Glucocorticoid inhibitors like methylprednisolone and Ca2+ ionophore also caused DNA fragmentation in B-CLL cells. The mechanism of action was via a defective sustained Ca2+ increase observed in B-CLL (35). Cytokines are capable of apoptotic modulation in B-CLL cells. The survival of B-CLL cells may be influenced by IFN-gamma, as it was shown that the cytokine is capable of increasing the viability of leukemic cells in culture by inhibition of apoptosis (36). IFN-alpha is also able to rescue the leukemic cells from spontaneous apoptosis and it does this by upregulating bcl-2 gene expression (37). The same group has extended similar observations to IL-1, IL-4 and IL-6 (38). The viability of B-CLL cells was maintained in cultures where B-CLL cells were grown on endothelial monolayers. This viability was concurrent with the expression of IL-7 mRNA. On the other hand the effect of IL-10 on B-CLL cells has been varied. There have been reports that IL-10 induced apoptosis in B-CLL cells (15) but in more recent studies the addition of IL-10 did not induce apoptosis (39). We however were unable to observe any apoptosis inducing effects of IL-10 in the 6 B-CLL patients that we studied (14, 40). Similarly, other authors have observed IL-10 to prevent leukemic cells from cell death, even though the cytokine did not upregulate bcl-2 (41). The availability of cell lines in the study of malignancy has helped to understand the molecular and cellular events in disease progression.
ANALYSIS OF CYTOKINES IN B-CLL CELL LINES
    The Epstein Barr Virus (EBV) can infect and immortalize normal B-cells in vitro. The infected cells express nuclear and membrane proteins that can induce the expression of bcl-2 thus possibly protecting the cell from apoptosis (42). B-CLL cells do not grow well in culture inspite of the fact that they express bcl-2 and immortalization of these cells with EBV is extremely difficult even though they express the EBV receptor (43). During their short life span in vitro EBV infected B-CLL cells are able to express the EBV encoded nuclear antigens (44). Using a combination of EBV infection with costimulatory agents IL-2, SAC and MP6-thioredoxin, Hansen et al., were able to establish B-CLL cell lines (42). The EBV infected cell lines have comparable phenotypes to their normal EBV transformed counterparts. Recently Diaw et al., (45) studied the cytokine expression in heterohybrids that were produced by fusing CD5+ B-CLL with the non secreting X-63 murine myeloma. These heterohybrids however did not secrete or express TNF-alpha and IL-6 when compared to their parental B-CLL cells. A major concern in the study of transformed cell lines is the alterations that may occur in the process. Autologous normal EBV derived lines could serve as a control for such alteration. In B-CLL in particular, one has to keep in mind the heterogeneous nature of the malignancy as the process of immortilization could select for a small part of the entire B-CLL population.
ANTISENSE OLIGONUCLEOTIDES IN B-CLL     Antisense Oligonucleotides (ASO) targeted at specific genes have been used successfully in the functional disruption of the particular gene and the therapeutic potential of this avenue of gene attack appears promising. A number of studies where ASO are used to manipulate cytokine gene expression have shown inhibition of cell proliferation (reviewed in 25). Animal models for B-CLL have been developed (46, 47) and similar analysis of the involvement of cytokines in the growth of B-1 malignancies was found (48). We have shown that the spontaneous B-1 malignant clones that develops in NZB mice constitutively produces both message and protein for IL-10 (49). Subsequently we found that IL-10 was indeed a growth factor that was crucial for survival and potentiation of these B-1 cells. Using an antisense oligonucleotide specific for the 5' region of the murine IL-10 gene, we were able to specifically inhibit the growth of malignant B-1 cells in time dependent fashion (40) (Figure 1).

After 48 hours of contact with the antisense IL-10, there was a 50% inhibition in cell viability in the malignant B-1 cells. Oligonucleotides in the sense orientation also specific for the IL-10 gene did not show any such effect. The pathway of growth inhibition was via apoptosis that was induced by the antisense IL-10. In order to assess the dependency of the malignant B-1 cells on IL-10 in vivo, we administered antisense IL-10 via an osmotic pump that was implanted on the back of the NZB mice. The animals were subsequently challenged with 10x10 6 malignant B-1 cells obtained from a cultured cell line that had originated from a spontaneous B-1 tumor in an NZB mouse. When compared to the appropriate controls we found that control animals succumbed to the malignancy after 30-36 days, whereas 2/3 animals treated with antisense IL-10 still survive at the time of this writing which is more than 60 days after the transfer of malignant B-1 cells (Raveche-unpublished results) (Fig. 2).

In human malignancies antisense cytokines have been shown to inhibit cell growth. Antisense IL-6 can inhibit the proliferation of Kaposi's sarcoma cell lines derived from patients with AIDS (50), melanoma cell lines (51) and ovarian cancer cells (52). In addition to cytokines, bcl-2 has also been the target of antisense therapy (53). More recently the growth of myeloid leukemic cells in SCID mice was inhibited in vivo by targeting two cooperating oncogenes bcr-abl and c-myc (54).

Antisense cytokines have been studied in B-CLL. A few cytokines have been implied to serve as autocrine growth factors for B-CLL cells. TNF-a in vitro increases viability, DNA synthesis as well as expression of protooncogenes myc, fos and jun. It also upregulates endogenous mRNA levels, thus suggesting an autocrine loop for TNF in these cells (55). We wanted to look for a possible autocrine role for IL-10 in B-CLL cells. In a short preliminary study (14), we treated PBL from B-CLL patients with antisense IL-10. 3/6 patients tested showed a significant inhibition of growth to antisense IL-10 (Fig. 3).

When analyzed together with the levels of IL-10 in the same patients, it was evident that 5/6 patients showed a correlation between the detectable IL-10 message level and the inhibition caused by antisense IL-10 (Fig 4). Taken together, the results indicate that B-CLL cells that have message for IL-10 could possibly be targeted for antisense therapy with the cytokine.

    Due to the heterogeneity, it is difficult to assign a generalized cytokine profile to B-CLL, either constitutive or induced. However, it may be conceivable to target any one or probably multiple cytokines that are detected at the RNA level of the malignant cell. OTHER EFFECTS OF CYTOKINES ON B-CLL CELLS
    Incubation of B-CLL cells with cytokines like IFN-alpha, IFN-gamma and IL-4 can upregulate the expression of Leukocyte adhesion molecule-1 (LAM-1). Likewise, stimulation of B-CLL cells with cytokines like IFN-alpha IFN-gamma and IL-2 results in upregulation of CD23, a pre B-cell marker (56). B-CLL lymphocytes frequently express two forms of CD23 that differ only in their cytoplasmic domain, and are differentially regulated by cytokines. IL-2 and TNF-alpha are stimulatory to B-CLL cells and also upregulate type B CD23, whereas IL-4 and IFN-gamma, upregulate type A mRNA of CD23 even though they do not cause direct proliferation of B-CLL cells (57). It has also recently been shown that in addition to IL-4, IL-13 can also upregulate CD23 on anti CD40 activated B-CLL cells (24). IL-4 is also capable of inhibiting the PMA induced hyper-expression of CD5 on B-CLL cells (58). In recent observations Ranheim, Cantwell and Kipps have demonstrated that B-CLL cells coexpress membrane bound and soluble CD27 along with its ligand CD70. The crosslinking of CD27 on T cells provides a costimulatory signal that acts with the T cell receptor to induce proliferation. The presence of soluble CD27 in B-CLL cells may prevent stimulation of T cells, thus impairing their ability to function as effective antigen presenting cells. Furthermore the Th1 cytokines like IFN-gamma can downregulate CD27 whereas Th2 cytokines, IL-4 and IL-10 upregulate the surface antigen on B-CLL cells (59). The exact implication of these surface markers in the oncogenesis of B-CLL is yet to be determined.
SUMMARY
    Due to the heterogenous nature of B-CLL it is difficult not only to analyze but also to assign a defined role for cytokine involvement in differentiation and disease progression. Cytokines appear to be involved in the proliferation of the neoplastic cells in vitro thus showing that B-CLL cells are not irreversibly arrested in the Go phase of the cell cycle. In addition, the upregulation of cell surface markers on cytokine stimulated cells point towards the interactions between B-CLL cells and other cells of the immune system. Some cytokines appear to work in an autocrine manner and act as growth factors for the malignant cells. Interferon a is thought to induce lymphocytosis by interrupting these autocrine loops. It is interesting to note that studies dealing with antisense to B-CLL expressed cytokines appear to work in favor of the host by inhibiting the growth of B-CLL cells. Cytokines can sometimes antagonize each other to prevent cell proliferation and perhaps induce apoptosis. It is worth noting that in general cytokines can maintain the viability of B-CLL cells in culture, by preventing the spontaneous apoptosis that is commonly associated with these cells. Cytokines may therefore serve as viability factors that protect the B-CLL cells and keep them in a resting or low activation state in vivo. Therefore specific disruption of cytokine genes in B-CLL with antisense oligonucleotides that induce apoptosis, may have potential therapeutic potential in the disease.
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