Relative roles of doxycycline and cation chelation in endothelial glycan shedding and adhesion of leukocytes

Leukocyte [white blood cell (WBC)] adhesion and shedding of glycans from the endothelium [endothelial cells (ECs)] in response to the chemoattractant f-Met-Leu-Phe (fMLP) has been shown to be attenuated by topical inhibition of matrix metalloproteases (MMPs) with doxycycline (Doxy). Since Doxy also chelates divalent cations, these responses were studied to elucidate the relative roles of cation chelation and MMP inhibition. WBC-EC adhesion, WBC rolling flux, and WBC rolling velocity were studied in postcapillary venules in the rat mesentery during superfusion with the cation chelator EDTA or Doxy. Shedding and accumulation of glycans on ECs, with and without fMLP, were quantified by the surface concentration of lectin (BS-1)-coated fluorescently labeled microspheres (FLMs) during constant circulating concentration. Without fMLP, low concentrations of EDTA (1-3 mM) increased FLM-EC sequestration due to disruption of the permeability barrier with prolonged exposure. In contrast, with 0.5 μM Doxy alone, FLM adhesion remained constant (i.e., no change in glycan content) on ECs, and WBC adhesion increased with prolonged superfusion. Without fMLP, EDTA did not affect firm WBC-EC adhesion but reduced WBC rolling flux in a dose-dependent manner. With fMLP, EDTA did not inhibit WBC adhesion, whereas Doxy did during the first 20 min of superfusion. Thus, the inhibition by Doxy of glycan (FLM) shedding and WBC adhesion in response to fMLP results from MMP inhibition, in contrast to cation chelation. With either Doxy or the MMP inhibitor GM-6001, WBC rolling velocity decreased by 50%, as in the case with fMLP, suggesting that MMP inhibition reduces sheddase activity, which increases the adhesiveness of rolling WBCs. These events increase the effective leukocrit on the venular wall and increase firm WBC-EC adhesion. Thus, MMP inhibitors have both a proadhesion effect by reducing sheddase activity while exerting an antiadhesion effect by inhibiting glycocalyx shedding and subsequent exposure of adhesion molecules on the EC surface.