NEUTROPHIL CHEMOTAXIS IN RESPONSE TO TGF-BETA ISOFORMS (TGF-BETA-1, TGF-BETA-2, TGF-BETA-3) IS MEDIATED BY FIBRONECTIN

TGF-beta isoforms regulate numerous cellular functions including cell growth and differentiation, the cellular synthesis and secretion of ex tracellular matrix proteins, such as fibronectin (Fn), and the immune response. We have previously shown that TGF-beta 1 is the most potent chemoattractant described for human peripheral blood neutrophils (PMNs ), suggesting that TCF-beta s may play a role in the recruitment of PM Ns during the initial phase of the inflammatory response. In our curre nt studies, we demonstrate that the maximal chemotactic response was a ttained near 40 fM for all mammalian TGF-beta isoforms. However, there was a statistically significant difference in migratory distance of t he PMNs: TGF-beta 2 (556 mu M) > TGF-beta 3 (463 mu M) > TGF-beta 1 (3 80 mu M) (beta 2:beta 3, p less than or equal to 0.010; beta 3:beta 1, p less than or equal to 0.04:beta 2:beta 1, P less than or equal to 0 .0012). A mAb to the cell binding domain (CBD) of Fn inhibited the che motactic response to TGF-beta 1 and TGF-beta 3 by 63% and to TGF-beta 2 by 70%, whereas the response to FMLP, a classic chemoattractant, was only inhibited by 18%. In contrast, a mAb to a C-terminal epitope of Fn did not retard migration (<1.5%). The Arg-gly-Asp-ser tetrapeptide inhibited chemotaxis by approximately the same extent as the anti-CBD (52 to 83%). Furthermore, a mAb against the VLA-5 integrin (VLA-5; Fn receptor) also inhibited TGF-beta-induced chemotaxis. These results in dicate that chemotaxis of PMNs in response to TGF-beta isoforms is med iated by the interaction of the Arg-gly-Asp-ser sequence in the CBD of Fn with an integrin on the PMN cell surface, primarily the VLA-5 inte grin. TGF-beta isoforms also elicited the release of cellular Fn from PMNs; we observed a 2.3-fold increase in Fn (389 to 401 ng/ml) in the supernatants of TGF-beta-stimulated PMNs compared with unstimulated ce lls (173.6 ng/ml). The concentration of TGF-beta required to cause max imal release of Fn from PMNs (4000 fM) is a concentration at which TGF -beta is no longer chemotactic, suggesting that PMNs only use Fn that is constitutively expressed for migration. At higher concentrations of TGF-beta, the Fn released may accumulate basal to the cell, ultimatel y retarding cellular migration and modulating the chemotactic response .