Interleukin-32 Induce Production of IL-6 in Multiple Myeloma Bone Marrow Stromal Cells

Background: Multiple myeloma (MM) is closely associated with inflammation. Patients with auto-immune disease、history of infection and other inflammatory disease have higher incidence of MM. IL-6 is the most important inflammatory factor in MM which plays a key role in the proliferation and progression. We previously demonstrated that MM cells were modified by bone marrow stromal cells (BMSCs) that formulate a inflammatory microenvironment in bone marrow (BM) and secret IL-6. How the inflammation makes BMSCs secret IL-6, however, remained undocumented. Our subsequent study compared the differential secretion of peripheral blood (PB) between MM patients and normal people by cytokine array, and showed that interleukin 32(IL-32) is highly expressed in MM patients. IL-32, also named natural killer 4(NK-4), is a newly found inflammatory factor. It was reported in solid tumors IL-32 is a pro-inflammatory factor which triggers a massive amplification of inflammatory process resulting in the change of other inflammatory factor including IL-6,IL-10,TNF-α. In this study, we examined BMSCs cytokines in MM BM and found that IL-32 was a functional factor in the process of inflammation in MM BM microenvironment.

Results: First, to test our previous study, we detected IL-32 in BM supernatant and PB supernatant in both MM patients (n=45) and healthy controls (n=13) by ELISA. Result showed that in both BM and PB, MM patients have higher expression of IL-32 compared to healthy controls (P<0.05). Next, total BM cells(both CD138⁺ and CD138^- cells) from MM patients were assayed by qRT-PCR for gene expression analysis.IL-32 were highly expressed in MM BM cells and the CD138⁺ cells (P<0.05). We also detected IL-32 in MM cell lines (RPMI 8226,OPM-2) and BMSCs isolated from MM patients by qRT-PCR, Western blot, and ELISA, and found that IL-32 was highly expressed in MM cell lines than BMSCs. In contrast, proteinase 3(PR3, receptor of IL-32) was highly expressed in BMSCs compared to MM cell lines.

Second, we stimulated the MM BMSCs with recombinant IL-32α, and found that the secretion of IL-6,CCL3 (MIP1-α), CCL4(MIP-1β) were significantly increased and CCL-5(RANTES)and IL-10 were decreased (P<0.05). Further, Western blot was applied to detect the inflammation molecular pathway in BMSCs. JAK-STAT pathway and NF-κB pathway were activated, and the phosphorylation of STAT3 was increased. After we knock down the PR3 in BMSCs, these changes were reduced. We repeated these experiments in BMSCs isolated from 15 different MM patients, the phenomenon mentioned above showed in 11 patients. The recombinant IL-32α was also used to stimulate 8226 and OPM-2 cells, but these two kinds of MM cells didn't secret IL-6, and no significant change in cell proliferation or cell apoptosis.

Finally, our group co-cultured the MM BMSCs with 8226 and OPM-2 cells. The secretion of IL-6 and the phosphorylation of JAK-STAT pathway in BMSCs were also increased. Knockdown of IL-32 in 8226 and OPM-2 cells weakened these changes.MM Cell proliferation and cell cycles after co-culture with MM BMSCs are under investigation.

Conclusion: our findings suggest that IL-32 is mainly secreted by MM cells. It may not directly promote the MM cells to grow. However, IL-32 promote the MM BMSCs to secret more cytokines including IL-6,CCL3,CCL4 by activating the JAK-STAT3 pathway, which lead to a amplification of inflammation in BM environment, resulting in the cell proliferation .