Heparin's anti-inflammatory effects require glucosamine 6-O-sulfation and are mediated by blockade of L- and P-selectins

Abstract

Heparin has been used clinically as an anticoagulant and antithrombotic agent for over 60 years. Here we show that the potent anti-inflammatory property of heparin results primarily from blockade of P-selectin and L-selectin. Unfractionated heparin and chemically modified analogs were tested as inhibitors of selectin binding to immobilized sialyl Lewis(X) and of cell adhesion to immobilized selectins or thrombin-activated endothelial cells. Compared with unfractionated heparin, the modified heparinoids had inhibitory activity in this general order: over-O-sulfated heparin > heparin > 2-O,3-O-desulfated > or = N-desulfated/N-acetylated heparin > or = carboxyl-reduced heparin > or= N-,2-O,3-O-desulfated heparin >> 6-O-desulfated heparin. The heparinoids also showed similar differences in their ability to inhibit thioglycollate-induced peritonitis and oxazolone-induced delayed-type hypersensitivity. Mice deficient in P- or L-selectins showed impaired inflammation, which could be further reduced by heparin. However, heparin had no additional effect in mice deficient in both P- and L-selectins. We conclude that (a) heparin's anti-inflammatory effects are mainly mediated by blocking P- and L-selectin-initiated cell adhesion; (b) the sulfate groups at C6 on the glucosamine residues play a critical role in selectin inhibition; and (c) some non-anticoagulant forms of heparin retain anti-inflammatory activity. Such analogs may prove useful as therapeutically effective inhibitors of inflammation.

Figure 1

Representative disaccharide units of heparin and the various heparinoids. Each disaccharide illustrates a characteristic unit in the indicated preparation and does not represent the overall structure of the chains. X = H or SO₃^–.

Figure 2

Inhibition of selectin-Ig binding to immobilized PAA-SLe^X. Inhibition curves were generated using OS-heparin (open circles), heparin (open squares), NDS-heparin (asterisks), 2/3DS-heparin (open triangles), N/2/3DS-heparin (filled triangles), 6DS-heparin (filled squares), and CR-heparin (filled circles) (see Methods). Inclusion of a mAb to P-selectin, E-selectin, or EDTA blocked binding by more than 90%. Each point represents the average of duplicate determinations, and the data are representative of three or four separate experiments.

Figure 3

Effects of heparinoids on selectin-dependent cell adhesion. Adhesion of U937 cells to immobilized selectin-Ig chimeras was measured (see Methods). Inclusion of a mAb to P-selectin, E-selectin, or EDTA blocked binding by more than 90%. Each point represents the average of duplicate determinations, and the data are representative of three or four separate experiments.

Figure 4

Inhibition of U937 cell adhesion to activated human lung microvascular endothelial cells by modified heparins. Inclusion of a mAb to P-selectin or EDTA blocked binding by more than 90%. Each point represents the average of duplicate determinations, and the data are representative of three or four separate experiments.

Figure 5

Analysis of heparin and 6DS-heparin. Samples of heparin and 6DS-heparin were analyzed by ¹³C-NMR (a and b) and nitrous acid degradation to disaccharides (c and d) (see Methods). (a and b) The positions of peaks 1 and 2 correspond to C6 of 6-O-sulfated and unsubstituted glucosamine units, respectively. (c and d) Disaccharides generated by nitrous acid deamination of heparin and 6DS-heparin, respectively. 1, aMan_R (anhydromannitol), GlcA-aMan_R, and IdoA-aMan_R; 2, IdoA-(2OSO₃)-aMan_R; 3, GlcA(2OSO₃)-aMan_R and GlcA-aMan_R(6OSO₃); 4, IdoA-aMan_R(6OSO₃); 5, GlcA-aMan_R(3OSO₃)(6OSO₃); 6, IdoA(2OSO₃)-aMan_R(6OSO₃); 7, GlcA(2OSO₃)-aMan_R(6OSO₃).

Figure 6

Inhibition of thioglycollate-induced peritoneal inflammation. (a) Heparin and the various analogs at 0.5 (gray bars) or 1.25 mg per mouse (black bars) were injected intravenously 5 minutes after thioglycollate was injected intraperitoneally. The number of Gr-1–positive granulocytes in the peritoneal cavity was quantitated after 3 hours (see Methods). *Significant difference in neutrophil counts in the control mice that received standard heparin versus those injected with the indicated heparin derivatives. (b) Wild-type C57BL/6 and selectin-deficient mice were injected with heparin (0.5 mg) and thioglycollate. *Significant difference in neutrophil counts in mice treated with heparin (black bars) versus control mice that received only saline (gray bars). Each bar represents the average value ± SD; n = 5–10.

Figure 7

Inhibition of oxazolone-induced ear swelling in sensitized mice. Mice were sensitized to oxazolone and rechallenged by topical treatment of one ear (see Methods). Swelling was measured by ear thickness, and the value obtained from the vehicle-treated control ear was subtracted from those obtained from oxazolone-treated mice. (a) Animals received a single intravenous injection of heparin (1 mg), heparin derivative (1 mg), or saline within 30 minutes after antigen challenge. *Significant difference in ear swelling in the control mice injected with saline (gray bar) versus those that were injected with the heparin derivatives (black bars). (b) Sensitized wild-type and selectin-deficient mice were injected with heparin (1 mg) and rechallenged with antigen as in a. Each bar represents the average value ± SD; n = 5–8. *Significant difference in ear swelling in mice treated with heparin (black bars) versus control mice that received only saline (gray bars).