Monocytes/Macrophages Are the Major Targets of the CCL3 Chemokine Produced by CD38⁺CD49d⁺ Chronic Lymphocytic Leukemia Cells.

CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Recent gene expression profiling studies comparing CLL cases expressing low versus high levels of CD38 and CD49d, identified CCL3 as a gene upregulated by CD38⁺CD49d⁺ CLL. The release of CCL3 by cultured CLL cells was also demonstrated upon CD38 triggering, and CCL3 protein was found in CLL cells from bone marrow biopsies (BMB) of CD38⁺ cases (Zucchetto et al., Cancer Res, 2009; 69:4001-9). Given the role of CCL3 as potent chemoattractant for different cell types, we aimed at identifying the major targets of CCL3, as produced by CD38⁺CD49d⁺ CLL cells.

CLL infiltrates of BMB were characterized by immunohistochemistry (IHC). Expression of the CCL3 receptors CCR1 and CCR5 by PB CLL subpopulations was evaluated by flow cytometry. T lymphocyte and monocyte migrations were performed by in-vitro transwell chemotaxis assays.

IHC analysis of BMB from 16 CLL cases revealed a higher number of infiltrating CD68⁺ cells in the context of CLL-involved areas of BMB from CD38⁺CD49d⁺CCL3⁺, compared to CD38⁻CD49d⁻CCL3⁻ cases (p=0.01). CD3⁺ lymphocytes were interspersed in the CLL aggregates, but with no significant difference between the two subgroups. Evaluation of CCR1 and CCR5 in PB cell subpopulations from 40 CLL cases expressing or not surface CD38 and CD49d, showed the highest mean fluorescence intensity (MFI) levels for both CCR1 (624±60) and CCR5 (64±9) in the monocytic component, irrespective of CD38 and CD49d expression by CLL cells. Conversely, both CLL cells and residual T lymphocytes showed low MFI levels for CCR1 (19±4 and 14±3) and CCR5 (21±2 and 20±2). High CCR1 and CCR5 expression levels were detected in in-vitro differentiated monocytes from purified PB cells of four CD38⁺CD49d⁺ CLL. Accordingly, CCR1 expression was documented in macrophage-like cells in BMB from CD38⁺CD49d⁺ CLL. Next, we evaluated the capability of purified monocytes and T lymphocytes from 10 CLL cases to migrate in response to CCL3. In keeping with the strong expression of CCR1, monocytes migrated toward CCL3 at a concentration of 3 ng/mL (migration index, MI= 8.8±0.9, p=0.03), whereas T lymphocytes required a higher CCL3 concentration (100 ng/mL) to display slight migration capability (MI= 1.6±0.2, p=ns). The increased infiltration of macrophages in BMB from CCL3-producing CD38⁺CD49d⁺ CLL, prompted us to verify the capability of CCL3-stimulated macrophages to induce the expression by endothelial cells (EC) of the CD49d specific ligand VCAM-1. By using two different EC models (HUVEC and ADMEC), we documented a significant up-regulation of VCAM-1 by EC exposed to conditioned media (CM) collected from cultures of macrophages challenged in-vitro with CCL3 (p=0.002). Notably, increased levels of the pro-inflammatory cytokine TNF-α were detected in CCL3-CM (p=0.006), and neutralization of TNF-α by specific antibodies reverted the capability of CCL3-CM to induce VCAM-1 by EC models. In agreement with these in-vitro data, we found a more prominent meshwork of VCAM-1⁺ stromal/endothelial cells in lymphoid infiltrates from CD38⁺CD49d⁺ CLL compared to CD38⁻CD49d⁻ cases (p=0.002), and engagement of CD49d by VCAM-1 was able to significantly delay the spontaneous apoptosis observed in cultured CLL cells.

CD68⁺ monocytes/macrophages are likely the main targets for the CLL3 chemokine produced by CD38⁺CD49d⁺ CLL cells, and are active in determining, through the release of TNF-α and other yet unidentified cytokines, the overexpression of VCAM-1 by endothelial cells. Experiments aimed at investigating further roles of CD68⁺ monocytes/macrophage in CLL are currently matter of study.

No relevant conflicts of interest to declare.