A Study To Identify The Expression And The Role Of MIF And Its Receptor CD74in The IVD Degeneration

Backgroud:Low back pain(LBP) is becoming a major health problem in now days. A lot of attentionhas been paid to study the pathogenesis of intervertebral disc degeneration. Recently, somestudies have demonstrated that inflammatory cytokines are closely associated with discdegeneration disease (DDD) and discogenic low back pain. The microenvironment in the discdegeneration tissues is complicated, and there are various types of cells in the disc, such asinfiltrating macrophages, leucocytes etc. These inflammatory cells together with disc cellsthemselves can secrete inflammatory cytokines, such as IL-6, IL-8, TNF-α, IL-1β, IFN-γ andPGE2. Some cytokines have been reported to a major cause of neurogenic pain.Mesenchymal stem cells (MSCs), especially the resident stem cells in the intervertebraldisc (IVD), hold promise for use in regenerative medicine in the treatment of degenerativediseases, such as IVD degeneration. The therapeutic application of MSCs exploits the abilityof MSCs to home to injured or degenerated tissues and facilitate the healing process. Themigration of MSCs is regulated by a variety of cytokines, such as fibroblast growth factor-2(FGF-2), platelet-derived growth factor (PDGF) and MIF. FGF-2and PDGF can facilitate themigration of MSCs to sites of injury; conversely, MIF (Macrophage migration inhibitoryfactor) inhibits MSCs migration into the sites of inflammationMacrophage migration inhibitory factor (MIF) was first described as a soluble factor thatis released by activated T lymphocytes in1966. MIF has been reported to inhibit the randommigration of monocytes and macrophages. Subsequently, significant quantities of MIF werefound within the pituitary gland and monocytes/macrophages besides T-lymphocytes. As animportant proinflammatory cytokine, MIF might counter-regulate glucocorticoid effects byactivating immune/inflammatory cells and promoting the expression of matrixmetalloproteinases, nitric oxide and prostaglandin E2release, or the release ofproinflammatory and inflammatory cytokines, such as TNF-a, IL-1b, IL-2, IL-6, IL-8, IFN-γ. Moreover, each of those proinflammatory and inflammatory cytokines are involved in thepathogenesis of IVD degeneration. However, a potential role for MIF in the pathogenesis ofIVD degeneration has not yet been investigated.CD74is an integral membrane protein and the major histocompatibility comeplex (MHC)class II-associated invariant chain, which is located in the plasma membrane. CD74functionsas a receptor for MIF, and is highly expressed in inflammatory disorders. CD74is importantfor MIF to mediate its biological activities in vivo, and the up-regulation of cell surfacereceptor CD74is a step towards mediating MIF-CD74signal transduction. MIF can bind toCD74and activate signal transduction, leading to the production of proinflammatorycytokines, and the migration regulation of MSCs.We hypothesized that the MIF and CD74are expressed in the degenerated disc tissues,and they are involved in the pathogenesis of IVD degeneration. MIF, as a upstream cytokine,induce inflammatory cytokines release and amplify inflammatory response. Meanwhile, MIFregulates the migration of cartilage endplate derived stem cells(CESCs) by interacting withCD74. Inhibiting the biological function of MIF through ISO-1(specific antagonist of MIF) orCD74blocker might be an efficient way to reduce the inflammatory response in vivo andexert the "homing" function of CESCs.The primary objective of this study is to investigate the expression pattern of MIF andCD74in the degenerated IVD tissues and their role in DDD, to explore the treatment toinhibit the biological function of MIF, and then help the IVD repair and regeneration.Methods and findings:MIF and CD74expression by IVD tissues was examined using immunocytofluorescenceand immunohistochemistry. The MIF mRNA and MIF-positive cells in the Modic I typechanges is significantly higher than that in the Modic II type changes; the MIF mRNA andMIF-positive cells is the highest in the Pfirrmann IV changes in Pfirrmann III, IV and Vchanges, however, Pfirrmann I and II patients are lacked in our study. The NP cells can bestimulated to secret inflammatory cytokines (LPS and TNF-α; P<0.01) and then inflammatorycytokines can induce NP cells secretion (IL-6, IL-8, and PGE2; P<0.01). The NP cellssecretion and CESCs migration are both regulated by the MIF-CD74signal pathway usingELISA, RT-PCR, siRNA and in vitro cell migration assays. ISO-1treatment significantly(P<0.01) reduced the secretion of IL-6, IL-8and PGE2by NP cells, and restored the CESCs migration, however, ISO-1alone did not increase the secretion of these proinflammatorycytokines. Finally, a CD74activating antibody (CD74Ab) was used to examine the effect ofCD74on NP cells secretion and CESCs motility, and it increased the secretion of NP cellsand inhibited the migration of CESCs in a dose-dependent manner.Conclusion:1. this study indicated that the resident stem cells were in the degenerated CEP and theywere derived from mesenchymal tissues but not embryo tissues. CESCs and BM-MSCsshared the similar characteristics in the cell morphology, cell proliferation, cellimmunophenotype, and trilineage indifferentiation.2. MIF and CD74are expressed in the degenerated IVD tissues. The MIF expression inthe Modic I type changes is more the MIF expression in the Modic II type changes; the MIFexpression is the highest in the Pfirrmann IV changes in Pfirrmann III, IV and V changes,however, Pfirrmann I and II patients are lacked in our study.3. NP cells can be stimulated by LPS and TNF-a to secrete inflammatory cytokines in adose-dependent manner.4. NP cells can be stimulated by MIF to secret inflammatory cytokines. We found thatIL-6, IL-8and PGE2were significantly (P<0.01) increased after stimulation with MIF byacting with CD74through P38MAPK signal pathway, however, TNF-α and IL-1β did notincrease significantly.5.ISO-1does not induce the secretion of proinflammatory cytokines and neutralization ofthe pro-inflammatory activity of MIF by ISO-1would be beneficial for control ofinflammatory activity and the degradation of the extracellular matrix induced byinflammatory cytokines.6.CESCs are capable of spontaneous migration. MIF can inhibit the migration of CESCsby interacting with CD74in a number-dependent manner. ISO-1can be used to inhibit thebiological function of MIF and restore the migration of CESCs.