SUMMARY. Neutrophil recruitment into systemic inflammatory sites in viva is thought to be initiated by selectin-mediated endothelial adherence. The effect of ...
Vol. 43, No. 2, October 1997
BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Pages443-451
FUCOIDAN INHIBITS LEUKOCYTE RECRUITMENT IN A MODEL PERITONEAL INFLAMMATION IN RAT AND BLOCKS INTERACTION OF P-SELECTIN WITH ITS CARBOHYDRATE LIGAND
M.E. Preobrazhenskaya*, A.E. Berman*, V.I. Mikhailov*, N.A. Ushakova*, A.V. Mazurov^, A.V. Semenov ^, A.I. Usov#, N.E. Nifant'ev#, N.Y. Bovin & *Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya, 10, Moscow, 119832; ^Institute of Experimental Cardiology, Cardiology Research Center, Russian Ministry of Health, 3 rd Cherepkovskaya, 15a, Moscow, 121552; #Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospekt, 47, Moscow 117913; and &Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Miklukho-Maklaya, 16/10, 117871 GSP-7. Received July 10, 1997
SUMMARY Neutrophil recruitment into systemic inflammatory sites in viva is thought to be initiated by selectin-mediated endothelial adherence. The effect of fucoidan (natural sulfated polymer of L-fucose) on the selectin dependent PMN migration into rat peritoneum following the induction of inflammation by peptone injection was studied. Peritonitis was characterized by an increase in the total cell number (from 45.3 x 106to 91.6 x 106/rat), and by highly elevated PMN content (from 0.2% to 58%) in the rat peritoneal cavity 3 h after peptone injection. Intravenous administration of fucoidan was found to reduce, in a dose-dependent manner, neutrophil migration into peritoneum. Fucoidan in a dose as low as 0.8 mg per rat caused 96.8% reduction of neutrophil extravasation. The inhibitory effect of fucoidan was also dependent on the time intervals between the peptone and fucoidan injections. The maximal inhibitory effect of fucoidan was observed within the first 15 rain after the induction of peritonitis and it was ~maintained at a level of 80% during 1.5 h. Administration of fucoidan 2.5 h after peptone injection had practically no effect on PMN extravasation. Since P-selectin is known to play a key role at the earlier stages of PMN extravasation, it was suggested that the inhibitory effect of fucoidan was mostly due to its interaction with P-selectin. The in vitro experiments demonstrated the high affinity of fucoidan for both isolated P-selectin and P-selectin in plasma membranes of activated platelets. Key words: inflammation, fucoidan, neutrophils, extravasation, adhesion molecules, P-selectin, platelets, sialyI-Lewisx.
INTRODUCTION Neutrophil leukocyte traffic from blood vessels into sites of inflammation is a multistep process, the first step of which is the transient "rolling" of leukocytes along the endothelial cells of blood vessels. The rolling phenomenon is mediated by a family of adhesion molecules called
The abbreviations used are: PMN, polymorphonuclear leukocytes (neutrophils); SiaLe*, Sialyl LewisX; SiaLe a, Sialyl Lewis". 1039-9712/97/020443-09505.00/0 443
Copyright 9 1997 by A~tdemic Press Australia. All rights o/reproduction in anyfi~rm reserved.
Vol. 43, No. 2, 1997
BIOCHEMISTRYend MOLECULAR BIOLOGY INTERNATIONAL
selectins. There are three types of selectins: L-, P-, and E-selectins. L- selectin is constitutively expressed on the surface of circulating leukocytes; E-selectin expression on the endothel;al cell surface results from cytokine inducible de novo synthesis of this receptor, while Pselectin is stored in the membranes of intracellular granules of platelets and endothelial cells and is rapidly translocated to the cell surface upon their activation (1-5). The exact structure of ligands for setectins is not yet clear. The selectins are known to bind to sialylated lactosaminoglycans, including SiaLe x and its isomer SiaLe a. However, the affinity of selectins for such o!igosaccharides is poor (6). P- and L-selectins can also bind with high affinity to some sulfatides, and sulfated polysaccharides such as heparin, dextran sulfate and fucoidan (7-9). Fucoidan, a sulfated biopolymer of L-fucose, has been found to inhibit some in vivo processes, including interactions of lymphocytes with postcapillary venules of lymphoid organs (10), as well as leukocyte rolling and adhesion in ishemia and reperfusion injury (11), in lung inflammation (12) and experimental meningitis (13, 14). In most cases fucoidan was more effective than the other sulfated glycans. Fucoidan's inhibitory effect in the above-mentioned cases was explained by its interaction with L-selectin. Besides, the specific binding of FITClabeled fucoidan to L-selectin on human PMN has been observed (9)~ In this paper we report that fucoidan inhibits the neutrophil extravasation into inflamed sites at the early stages of peritonitis development and blocks P-selectin-ligand interaction in vitro. These data show that the anti-inflammatory effect of fucoidan is largely dependent on its interaction with P-selectin.
METHODS Reagents. Peptone was from Reakhim (Russia). Fucoidan was isolated from brown marine algae Laminaria saccharina (15) and consists of 40,5% fucose and 36,7% sulfate (16). Antibodies. Murine anti P-selectin monoclonal antibody (mAb) CRC81, which does not block P-selectin-ligand interaction, was obtained as described (17). Affinity purified rabbit polyclonal antibody against P-selectin and against platelet glycoprotein Ilbllla complex were kindly provided by Dr. Michael Berndt, Baker Medical Research Institute, Prahran, Australia. P-selectin purification. Platelet membrane P-selectin was purified by affinity chromatography on a column with immobilized CRC81 mAb as described (18). Water soluble biotinylated neoglycoconjugate. SiaLea~X-PAA-biotwas prepared by attachment of SiaLe a (10% mol.) and SiaLex (10% mol.) ligands (19) and biotin (5% mol.) to polyacrylamide (mol. wt. 30 kDa) as described (20, 21, 22). Cells. Human platelets were prepared from healthy donor blood by consecutive washings as described (23). Washed platelets were used as suspension in Tyrode/HEPES solution (137 mM NaCI, 2.7 mM KCI, 0.36 mM NaH2PO4, 0.1% glucose, 5 mM HEPES, 1 mM CaCI2, 1 mM MgCI2; pH 7.35). Neoglycoconjugate binding assays, a) SiaLea/X-PAA-biot binding to cell-free P-selectin. 96Well plates (Maxisorb, Nunc, Denmark) were coated with mAb CRC81 in PBS (100 t~l, 10 ~g/ml) for 1 h at 37~ The plates were washed three times with PBS containing 0.05% Tween 20 (PBS/Tween), and blocked for 1 h at 37 ~ C with 150 ~1 of 2% BSA in PBS/Tween.
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BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL
P-selectin in PBS, containing 0.2% Triton X-100 (100 tA, 5 #g/m!) was added for 1 h at 37 ~ C, and unbound P-selectin was washed out with PBS/Tween. SiaLe~/X-PAA-biot (2 #g/ml) alone or together with different compounds (Ab's, EDTA, fucoidan) was added in 100 gl PBS containing 1 mM CaCI2 and incubated for 40 min at 20~ The plates were washed five times and peroxidase-labeled streptavidin (100 #l/well of 1:1500) in PBS/Tween was added. Plates were incubated for 45 min at 37 ~ C. After washing, plates were developed with the chromogenic substrate o-phenilenediaminelH202 and monitored at A492; b) SiaLea/X-PAA-biotbinding to P-selectin of activated plateles. Washed platetets were immobilized onto 96-welt plates as described previously (23). Shortly, 107 cells (100 #l) per well in Tyrode/HEPES solution were added onto plates, sedimented by centrifugation at 1000 x g for 5 min and incubated for 30 min at 37 ~ C. Plates were washed three times and blocked for 1 h at 37 ~ C with 150 F,I of 2% BSA in Tyrode/HEPES solution. The subsequent steps were the same as described for immobilized P-selectin assay, except that Tyrode solution was use_d instead of PBS/Tween as ELISA buffer. Peptone-induced acute peritonitis. Female Wistar rats (180-220g) were injected intraperitoneally with 5 ml of saline (control 1) or saline containing 4% peptone. Rats under ether anesthezia were sacrified 3 h after and their peritoneal cavities were laved with 30 m] of PBS containing 60 units/ml of heparin, 0.02% EDTA and 0.03% bovine serum with vigorous massage for 1 rain. Total cell number in the lavage was counted, and cell suspensions were concentrated by centrifugation at 400 x g for 10 min. After 1:1 dilution with bovine serum the smears were prepared and stained by the Pappenheim method. Neutrophils were counted on two parallel slides (300-400 cells on each slide), and the total neutrophil number was calculated from neutrophil per cent and the total cell number. Fucoidan was injected intravenously under anesthezia by two different experimental schemes. By one, the preparation was injected as a single dose in 0.25 ml of sterile 0.9% NaCI at various times after peptone injection. According to a second scheme, the same amount of fucoidan was given as two equal doses 15 and 120 min after peptone injection. The control rats (control 2) were injected with 0.25 ml of sterile 0.9% NaCI at the same intervals.
RESULTS Peptone-induced acute rat peritonitis was characterized by neutrophil extravasation into the peritoneal cavities. The total cell number and the neutrophil content in peritoneal cavities of NaCf-treated rats were 45.3 _+8.2x106 (n=3) and 0.2 _+0.3%, respectively (cont~-ol 1); 3 h after peptone injection these values increased to 91.6 _+31,2x106 (n=l 1) and 58.0 _+0.1%, respectively (control 2). It is to note that the thioglycol!ate broth, commonly used as inflammatory inductor, was less effective in our experiments than was peptone (data not shown). Intravenous fucoidan injection resulted in considerable inhibition of neutrophil extravasation into the peritoneal cavities of rats. The inhibitory effect of fucoidan depended on the dose used and on time intervals between the peptone and fucoidan injections. Virtually complete inhibition (96.8 + 2.9%; p
