Clustering of fibroblasts induces proinflammatory chemokine secretion promoting leukocyte migration.

Fibroblasts can acquire an immunoregulatory phenotype and they play an important role in triggering and upholding inflammation. Yet, the mechanism of this immunoactivation remains unknown. Previously we showed that spheroid formation by human fibroblasts leads to nemosis: activation through upregulation of cyclooxygenase-2, production of growth factors, and proteolysis. We now show that clustering of fibroblasts to spheroids leads to a significant induction of chemotactic cytokines able to attract various leukocyte subtypes. The mRNA contents of several chemokines (CCL2-5, CXCL1-3, and CXCL8) were 6-169-fold higher in fibroblast spheroids than in monolayer controls 36 h after spheroid formation. Similarly, CCL3, CCL5 and CXCL8 levels in spheroid medium were significantly higher than in monolayer medium. Conditioned fibroblast spheroid medium induced chemotaxis of primary human neutrophils and monocyte-like THP-1 cells, and the effects were significantly inhibited by antibodies against CXCL8 and the chemokine receptor CCR1, respectively. The decreased levels of IkappaB alpha and presence of DNA-binding nuclear factor-kappaB (NF-kappaB) after spheroid formation indicate NF-kappaB activity. In conclusion, clustering of fibroblasts provides an experimental model to study their activation and is sufficient to induce substantial proinflammatory chemokine secretion functionally promoting leukocyte migration, and the mechanism seems to involve the NF-kappaB signalling pathway.