CYTOKINE EXPRESSION IN PATIENTS WITH B-CELL CHRONIC LYMPHOCYTIC LEUKAEMIA

Abstract

B-cell chronic lymphocytic leukaemia (B-CLL) results from a clonal accumulation of CD5+ve B-lymphocytes. This expansion of B-lymphocytes is due to increased proliferation and extended survival secondary to decreased apoptosis. Both proliferation and apoptosis are regulated by a group of soluble proteins, termed cytokines, which are secreted by cells and play a key role in the regulation of immunological responses. In view of this, the role of cytokines in the pathogenesis of B-CLL was studied. Interleukin 4 (IL-4) is an anti-apoptotic cytokine which up-regulates bcl-2 in malignant B-lymphocytes. Peripheral blood samples, from untreated B-CLL patients were investigated, together with a group of ten healthy controls. Flow cytometric analysis was used to quantify (i) the expression of intracellular IL-4 by CD19+ and CD3+ lymphocytes, and (ii) IL-4R expression of CD 19+ and CD3+ lymphocytes. The proportion of CD 19+ve lymphocytes expressing intracellular IL-4 was significantly higher in patients with B-CLL than in controls (p=0.03). Similarly, the proportion of CD3+ve lymphocytes expressing intracellular IL-4 was significantly higher in B-CLL patients than controls (p=0.01). No significant difference was found in the overall proportion of CD 19+ve lymphocytes expressing IL-4R between patients and controls. However, the proportion of CD3+ve lymphocytes expressing IL-4R was significantly greater in patients than in controls (p=0.001 ). A number of anti-apoptotic cytokines, including IL-2, IL-4, IL-7, IL-9 and IL-15 share a sub-unit known as the common gamma chain (γc). A nested RT-PCR technique was used to analyse full-length γc receptor RT-PCR products in B and T-lymphocytes from patients with B-CLL and controls. The presence and concentration of full size IL-2, IL-4, IL-7, IL-9, IL- 15, γc, IL-4R α chain, as well as IL-2 delta 2 (IL-2δ2) and IL-4 delta 2 (IL-2δ2) spliced RT-PCR products were measured. IL-2 full-length RT-PCR products (using exon 1-4 amplifiers) were present ta a significantly more present at higher concentration in patient when compared to control B-lymphocyte samples (p=0.04). No significant difference was found between IL-4 full -length RT-PCR products in patients than in controls. IL-7 RT-PCR products were present at higher concentration in patient B- and T-lymphocytes than in controls (p=0.034 and p=0.041 respectively). IL-15 RT-PCR products were present at a lower concentration in patient than in control T-lymphocytes (p=0.001). IL-2 and IL-4 wild RT-PCR products (using exon 1-3 amplifiers) were present at significantly higher concentration in patient B-lymphocytes (p=0.0001 and p=0.026 respectively). The concentration of IL-482 and the IL- 4Rα RT-PCR products were significantly lower in patient than in control T-lymphocytes (p=0.0001 for both). There was a significant difference in the concentration of the IL-2 δ2 RT-PCR products between patient and control B-lymphocytes (p=0.029). In view of the above results, the apoptotic effect of the addition of IL-2 and IL-4 antisense oligonucleotides (ONs) to B-CLL and control cells was investigated. ONs were designed to block wild type IL-2 and IL-4 mRNA transcript expression as well as their spliced variants IL- 2δ2, and IL-4δ2. The percentages of CD19+/Annexin V+, CD3+/Annexin V+ or Propidium iodide (PI)+/Annexin V+ cells, as well as the percentage of CD19+ve and CD3+ve cells expressing intracellular IL-2, was measured by flow cytometry. The results demonstrated that, when B-CLL cells were incubated with IL-2 ONs, the percentage of CD19+/Annexin V+, as well as PI+/Annexin V+, lymphocytes were significantly increased (p=0.002). The addition of IL-4 and control antisense ONs had a similar, but not statistically significant effect. This pro-apoptotic effect was not seen in B-CLL CD3+ve lymphocytes. ELISA analysis showed that addition of JL-2 ONs decreased the level of IL-2 protein secreted by PHA-stimulated B-CLL cells but the addition of IL-4 antisense ONs increased the level of IL-4 protein. Finally, addition of ONs decreased the percentage of CD 19+ve and CD3+ve B-CLL lymphocytes expressing intracellular IL-2 and IL-4. In summary, the results obtained in this study imply that IL-2, IL-4, IL-7 and IL-15 may play a role in the survival of malignant B-lymphocytes in B-CLL.