Studying cartilage differentiation, we observed the emergence of inflammation-related proteins suggesting that a common pathway was activated in cartilage differentiation and inflammation. In the present paper, we investigated the expression pathway of the inflammation-related enzyme Cyclooxygenase-2 (COX-2) during differentiation and inflammatory response of the chondrocytic cell line MC615. Cells were cultured either as (i) proliferating prechondrogenic cells expressing type I collagen or (ii) differentiated hyperconfluent cells expressing Sox9 and type II collagen. The p38 and the NF-kB pathways were investigated in standard conditions and after inflammatory agents treatment. NF-kB was constitutively activated in differentiated cells. The activation level of NF-kB in differentiated cells was comparable to the level in proliferating cells treated with the inflammatory agent LPS. In both cases, p65 was bound to the NF-kB consensus sequence of COX-2 promoter. p38, constitutively activated in differentiated cells, was activated in proliferating cells by treatment with LPS or IL-1alpha. In stimulated proliferating cells the two pathways are connected since addition of the p38-specific inhibitor SB203580 inhibited p38 activation, significantly reduced NF-kB activation and repressed COX-2 synthesis indicating that p38 is upstream NF-kB activation and COX-2 synthesis. In differentiated cells, the treatment with the inflammatory agent neither enhance NF-kB activation, nor synthesis of COX-2 while the addition of SB203580 neither repressed activation of p38, nor COX-2 synthesis, suggesting a constitutive activation of a p38/NF-kB/COX2 pathway. Our data indicate that in chondrocytes, COX-2 is expressed via p38 activation/NF-kB recruitment during both differentiation and inflammatory response.

Wound healing is achieved through distinct programmed phases: hemostasis, inflammation, mesenchymal cell proliferation and migration, and tissue remodeling. At the injury site, clot formation and platelet degranulation release cytokines and growth factors and actively participating in the healing process and regulating the migration of inflammatory cells, such as neutrophils, macrophages, and lymphocytes. We previously demonstrated that, in an inflammatory environment, prostaglandin E2 (PGE2) secreted by mesenchymal stem cells (MSCs) promoted the macrophage switch from a proinflammatory to a proresolving phenotype. Using an in vitro model, we here evaluated the role carried out by the two main players of the wound healing process, the platelet degranulation content mimicked by the platelet lysate (PL) and the inflammatory stimulus, on the modulation of mouse bone-marrow-derived MSC paracrine activity. We demonstrated that, in MSCs, PL induced nuclear factor kappaB (NF-κB) activation, expression of COX-2 and mPGE synthase, and PGE2 production; in an inflammatory microenvironment, PL increased the inflammatory response and promoted the secretion of the proinflammatory cytokine IL-6. We assayed on mouse primary macrophages the paracrine activity of MSCs exposed to the different microenvironments and we observed that PL-treated MSC-conditioned medium maintained macrophages in a proinflammatory state. The involved factors were granulocyte macrophage-colony stimulating factor induced by PL in MSCs and TNF-α induced by PL-MSC-conditioned medium in macrophages. Our findings indicate that PL triggers an inflammatory response in MSCs and induces the secretion of factors maintaining macrophages in a proinflammatory state thus enhancing the initial inflammatory response to the injury, a key element in the activation of wound healing.

Platelet lysates (PL), which are derived from platelets, are cocktails of growth factors and cytokines that can promote tissue regeneration. Until today, most studies have focused on growth factor content of platelets rather than on their potential as a reservoir of mediators and cytokines. Taking advantage of an in vitro scratch assay performed under both normal and inflammatory conditions, in the present work, we report that at physiologic concentrations, PL enhanced wound closure rates of NCTC 2544 human keratinocytes. This effect was clearly detectable 6 h after wounding. Moreover, PL induced a strong cell actin cytoskeletal re-organization that persisted up to 24 h. The accelerated wound closure promoted by PL, in either presence or absence of serum, was associated with a high expression of the inflammatory cytokine interleukin-8. Further, after 24 h PL treatment, confluent keratinocytes also expressed low amounts of interleukin-8 and of the antimicrobial peptide neutrophil gelatinase-associated lipocalin, which dramatically increased under inflammatory conditions. These effects were associated with activation of the inflammatory pathways, p38 mitogen-activated protein kinase, and NF-κB. Our findings support the concept that platelet-derived preparations could accelerate regeneration of difficult-to-heal wounds by triggering an inflammatory cascade and having an antimicrobial role.

SUMMARY Galline Ex-FABP was identified as another candidate antibacterial, catecholate siderophore binding lipocalin (siderocalin) based on structural parallels with the family archetype, mammalian Siderocalin. Binding assays show that Ex-FABP retains iron in a siderophore-dependent manner in both hypertrophic and dedifferentiated chondrocytes, where Ex-FABP expression is induced after treatment with proinflammatory agents, and specifically binds ferric complexes of enterobactin, parabactin, bacillibactin and, unexpectedly, monoglucosylated enterobactin, which does not bind to Siderocalin. Growth arrest assays functionally confirm the bacteriostatic effect of Ex-FABP in vitro under iron-limiting conditions. The 1.8Å crystal structure of Ex-FABP explains the expanded specificity, but also surprisingly reveals an extended, multi-chambered cavity extending through the protein and encompassing two separate ligand specificities, one for bacterial siderophores (as in Siderocalin) at one end and one specifically binding co-purified lysophosphatidic acid, a potent cell signaling molecule, at the other end, suggesting Ex-FABP employs dual functionalities to explain its diverse endogenous activities.