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• W2161735952 abstract "Leukocytosis refers to an increase in leukocyte count above the normal range in the blood and is a common laboratory finding in patients. In many cases, the mechanisms underlying leukocytosis are not known. In this study, we examined the effects, the structural determinants, and the underlying mechanisms of heparin-induced leukocytosis, a side effect occurring in 0.44% of patients receiving heparin. We observed that heparin induced both lymphocytosis and neutrophilia, and the effects required heparin to be 6-O-sulfated but did not require its anticoagulant activity. Cell mobilization studies revealed that the lymphocytosis was attributable to a combination of blockage of lymphocyte homing and the release of thymocytes from the thymus, whereas the neutrophilia was caused primarily by neutrophil release from the bone marrow and demargination in the vasculature. Mechanistic studies revealed that heparin inhibits L- and P-selectin, as well as the chemokine CXCL12, leading to leukocytosis. Heparin is known to require 6-O-sulfate to inhibit L- and P-selectin function, and in this study we observed that 6-O-sulfate is required for its interaction with CXCL12. We conclude that heparin-induced leukocytosis requires glucosamine 6-O-sulfation and is caused by blockade of L-selectin-, P-selectin-, and CXCL12-mediated leukocyte trafficking. Leukocytosis refers to an increase in leukocyte count above the normal range in the blood and is a common laboratory finding in patients. In many cases, the mechanisms underlying leukocytosis are not known. In this study, we examined the effects, the structural determinants, and the underlying mechanisms of heparin-induced leukocytosis, a side effect occurring in 0.44% of patients receiving heparin. We observed that heparin induced both lymphocytosis and neutrophilia, and the effects required heparin to be 6-O-sulfated but did not require its anticoagulant activity. Cell mobilization studies revealed that the lymphocytosis was attributable to a combination of blockage of lymphocyte homing and the release of thymocytes from the thymus, whereas the neutrophilia was caused primarily by neutrophil release from the bone marrow and demargination in the vasculature. Mechanistic studies revealed that heparin inhibits L- and P-selectin, as well as the chemokine CXCL12, leading to leukocytosis. Heparin is known to require 6-O-sulfate to inhibit L- and P-selectin function, and in this study we observed that 6-O-sulfate is required for its interaction with CXCL12. We conclude that heparin-induced leukocytosis requires glucosamine 6-O-sulfation and is caused by blockade of L-selectin-, P-selectin-, and CXCL12-mediated leukocyte trafficking. Leukocytosis refers to an increase in leukocyte count above the normal range in the blood and is a common laboratory finding in patients. It occurs in response to a wide variety of pathological conditions, including viral, bacterial, and fungal infection, parasitic invasion, cancer, hemorrhage, trauma, myocardial infarction, malignancies, poisoning, and metabolic disturbances (1.Opdenakker G. Fibbe W.E. Van Damme J. The molecular basis of leukocytosis.Immunol. Today. 1998; 19: 182-189Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Based on the type of leukocyte that accumulates, leukocytosis is further subcategorized as neutrophilia, lymphocytosis, monocytosis, eosinophilia, and basophilia (1.Opdenakker G. Fibbe W.E. Van Damme J. The molecular basis of leukocytosis.Immunol. Today. 1998; 19: 182-189Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Clinically, leukocytosis is most commonly caused by neutrophilia, less often caused by lymphocytosis, and rarely is a consequence of monocytosis, eosinophilia, or basophilia (1.Opdenakker G. Fibbe W.E. Van Damme J. The molecular basis of leukocytosis.Immunol. Today. 1998; 19: 182-189Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 2.Abramson N. Melton B. Leukocytosis. Basics of clinical assessment.Am. Fam. Physician. 2000; 62: 2053-2060PubMed Google Scholar). Mechanistic studies have shown that leukocytosis can be attributed to an increase in cell production, an increase in cell release from storage pools, demargination in the vasculature, and/or decreased egress into target tissues. Many molecules that play key roles in inflammation have been implicated in leukocytosis, notably selectins, chemokines, integrins, and proteases (1.Opdenakker G. Fibbe W.E. Van Damme J. The molecular basis of leukocytosis.Immunol. Today. 1998; 19: 182-189Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Interestingly, some medications also induce leukocytosis, including corticosteroids, lithium, β-agonists, and heparin (2.Abramson N. Melton B. Leukocytosis. Basics of clinical assessment.Am. Fam. Physician. 2000; 62: 2053-2060PubMed Google Scholar, 3.Bradfield J.W. Born G.V. Lymphocytosis produced by heparin and other sulfated polysaccharides in mice and rats.Cell. Immunol. 1974; 14: 22-32Crossref PubMed Scopus (33) Google Scholar, 4.Godlowski Z.Z. Prevention of hormona eosinopenia and lymphopenia by inhibition of clotting in blood; preliminary report.Br. Med. J. 1951; 1: 854-855Crossref PubMed Scopus (8) Google Scholar, 5.Sasaki S. Production of lymphocytosis by polysaccharide polysulfates (heparinoids).Nature. 1967; 214: 1041-1042Crossref PubMed Scopus (30) Google Scholar). Heparin, a linear polysaccharide that belongs to the family of glycosaminoglycans, has been used clinically as an anticoagulant/antithrombotic for more than 70 years. It consists of repeating disaccharide units containing a uronic acid and a glucosamine residue that is either N-sulfated, N-acetylated, or occasionally unsubstituted (6.Esko J.D. Selleck S.B. Order out of chaos. Assembly of ligand-binding sites in heparan sulfate.Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1254) Google Scholar). The disaccharides may be further O-sulfated at C6 or C3 of the glucosamine residues and C2 of the uronic acid residues. The potent antithrombotic activity of heparin depends on the specific arrangement of sulfated sugar units and uronic acid epimers, which form a binding site for antithrombin III (6.Esko J.D. Selleck S.B. Order out of chaos. Assembly of ligand-binding sites in heparan sulfate.Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1254) Google Scholar). Heparin also possesses a variety of non-anticoagulant functions such as inhibition of inflammation and tumor metastasis (7.Nelson R.M. Cecconi O. Roberts W.G. Aruffo A. Linhardt R.J. Bevilacqua M.P. Heparin oligosaccharides bind L- and P-selectin and inhibit acute inflammation.Blood. 1993; 82: 3253-3258Crossref PubMed Google Scholar, 8.Borsig L. Wong R. Feramisco J. Nadeau D.R. Varki N.M. Varki A. Heparin and cancer revisited. Mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 3352-3357Crossref PubMed Scopus (602) Google Scholar, 9.Wang L. Brown J.R. Varki A. Esko J.D. Heparin's anti-inflammatory effects require glucosamine 6-O-sulfation and are mediated by blockade of L- and P-selectins.J. Clin. Invest. 2002; 110: 127-136Crossref PubMed Scopus (328) Google Scholar). Heparin was also reported to induce lymphocytosis in mice, rats, cows, and humans (3.Bradfield J.W. Born G.V. Lymphocytosis produced by heparin and other sulfated polysaccharides in mice and rats.Cell. Immunol. 1974; 14: 22-32Crossref PubMed Scopus (33) Google Scholar, 4.Godlowski Z.Z. Prevention of hormona eosinopenia and lymphopenia by inhibition of clotting in blood; preliminary report.Br. Med. J. 1951; 1: 854-855Crossref PubMed Scopus (8) Google Scholar, 5.Sasaki S. Production of lymphocytosis by polysaccharide polysulfates (heparinoids).Nature. 1967; 214: 1041-1042Crossref PubMed Scopus (30) Google Scholar). Importantly, from a clinical perspective, 0.44% of patients develop leukocytosis when taking therapeutic doses of heparin. In the United States more than 12 million patients receive heparin every year (10.Fahey V.A. Heparin-induced thrombocytopenia.J. Vasc. Nurs. 1995; 13: 112-116Crossref PubMed Scopus (27) Google Scholar), resulting in over 50,000 cases per year and indicating that heparin-induced leukocytosis (HIL) 2The abbreviations used are:HILheparin-induced leukocytosisBMbone marrowBWbody weightN-desN-desulfateddes-hepdesulfated heparinCR-hepcarboxyl-reduced heparinCFSEcarboxyfluorescein diacetate succinimidyl esterSPRsurface plasmon resonanceHPChematopoietic progenitor cell. is a frequently seen side effect of the drug in hospitals. However, the structural determinants of heparin that cause HIL and the mechanisms behind the effect remain unknown. heparin-induced leukocytosis bone marrow body weight N-desulfated desulfated heparin carboxyl-reduced heparin carboxyfluorescein diacetate succinimidyl ester surface plasmon resonance hematopoietic progenitor cell. In this study, we observed that heparin induced both lymphocytosis and neutrophilia, and the effect required 6-O-sulfation of the glucosamine residues but did not require its anticoagulant activity. Cell mobilization studies revealed that the lymphocytosis was attributable to the blockage of lymphocyte homing and release of thymocytes, whereas the neutrophilia was mainly caused by neutrophil release from bone marrow (BM) and demargination in the vasculature. Molecular mechanism studies revealed that heparin inhibits L- and P-selectin- and chemokine CXCL12-mediated leukocyte trafficking, resulting in HIL. Porcine intestinal heparin (average mass = 12,000–15,000 Da) was obtained from Scientific Protein Laboratories (Milwaukee, WI). Heparinoids were prepared as reported previously (9.Wang L. Brown J.R. Varki A. Esko J.D. Heparin's anti-inflammatory effects require glucosamine 6-O-sulfation and are mediated by blockade of L- and P-selectins.J. Clin. Invest. 2002; 110: 127-136Crossref PubMed Scopus (328) Google Scholar), including N-desulfated/N-acetylated heparin (N-des), 2-O/3-O-desulfated heparin (2/3-des-hep), N-/2-O/3-O-desulfated heparin (N/2/3-des-hep), 6-O-desulfated heparin (6-des-hep), and carboxyl-reduced heparin (CR-hep) (Fig. 2A). The heparin had an anti-factor Xa activity of 149 units/mg, whereas all heparinoids had no detectable anti-factor Xa activity. The heparin and heparinoids were negative for endotoxin. Wild-type, P-selectin knock-out (P−/−), and L-selectin knock-out (L−/−) mice, and P- and L-selectin double knock-out (PL−/−) mice on a C57BL/6J background were purchased from The Jackson Laboratory (Bar Harbor, ME). The α(1,3)-fucosyltransferase-IV and -VII double knock-out (FucT-IV/VII−/−) mice were kindly provided by Dr. John B. Lowe (11.Weninger W. Ulfman L.H. Cheng G. Souchkova N. Quackenbush E.J. Lowe J.B. von Andrian U.H. Specialized contributions by α(1,3)-fucosyltransferase-IV and FucT-VII during leukocyte rolling in dermal microvessels.Immunity. 2000; 12: 665-676Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) and were bred onto C57BL/6J background. Mice were housed at a specific pathogen-free facility, and the experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Georgia. Previous studies showed that a single bolus injection of sulfated polysaccharide fucoidan (i.v., 1.25–2.5 mg/25 g BW) induced robust leukocytosis in mice and that the leukocytosis peaked at 1.5 h after the fucoidan injection and coincided with reduced CXCL12 concentration in BM (12.Sweeney E.A. Lortat-Jacob H. Priestley G.V. Nakamoto B. Papayannopoulou T. Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice. Involvement in mobilization of stem/progenitor cells.Blood. 2002; 99: 44-51Crossref PubMed Scopus (187) Google Scholar). Glycosaminoglycan mimetics injected intraperitoneally at 1.25 mg/25 g BW similarly induced leukocytosis (13.Albanese P. Caruelle D. Frescaline G. Delbé J. Petit-Cocault L. Huet E. Charnaux N. Uzan G. Papy-Garcia D. Courty J. Glycosaminoglycan mimetics-induced mobilization of hematopoietic progenitors and stem cells into mouse peripheral blood. Structure/function insights.Exp. Hematol. 2009; 37: 1072-1083Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Our pilot study showed that the CXCL12 concentration in BM bottomed out at 90 min after heparin injection (Fig. 6, B and C). Considering that heparin at 0.5–1.25 mg/25 g BW potently blocks selectin-mediated leukocyte trafficking in mice (9.Wang L. Brown J.R. Varki A. Esko J.D. Heparin's anti-inflammatory effects require glucosamine 6-O-sulfation and are mediated by blockade of L- and P-selectins.J. Clin. Invest. 2002; 110: 127-136Crossref PubMed Scopus (328) Google Scholar) and that disruption of CXCL12 signaling in BM and selectin functions may represent the major molecular mechanisms underlying HIL, we rationalized injecting i.v. heparin at 1 mg/25 g BM, a dose close to glycosaminoglycan mimetics and fucoidan administration (12.Sweeney E.A. Lortat-Jacob H. Priestley G.V. Nakamoto B. Papayannopoulou T. Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice. Involvement in mobilization of stem/progenitor cells.Blood. 2002; 99: 44-51Crossref PubMed Scopus (187) Google Scholar, 13.Albanese P. Caruelle D. Frescaline G. Delbé J. Petit-Cocault L. Huet E. Charnaux N. Uzan G. Papy-Garcia D. Courty J. Glycosaminoglycan mimetics-induced mobilization of hematopoietic progenitors and stem cells into mouse peripheral blood. Structure/function insights.Exp. Hematol. 2009; 37: 1072-1083Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), and we used 90 min post-heparin injection as the time point for HIL analysis in our studies. In brief, mice at 8–10 weeks old were injected i.v. via tail vein with the heparinoids at 1 mg/25 g BW. Blood samples were taken from the retroorbital plexus during isoflurane anesthesia 90 min after heparinoid injection. Total leukocytes were counted separately by two investigators using hemocytometers. The leukocyte subpopulations were analyzed by flow cytometry (9.Wang L. Brown J.R. Varki A. Esko J.D. Heparin's anti-inflammatory effects require glucosamine 6-O-sulfation and are mediated by blockade of L- and P-selectins.J. Clin. Invest. 2002; 110: 127-136Crossref PubMed Scopus (328) Google Scholar, 14.Wang L. Fuster M. Sriramarao P. Esko J.D. Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses.Nat. Immunol. 2005; 6: 902-910Crossref PubMed Scopus (392) Google Scholar). Neutrophils and monocytes were identified by high expression of Gr-1 and Mac-1, respectively. The percentage of lymphocyte in leukocyte population was quantified based on forward and right angle light scattering in the cytogram. B cells and NK cells were counted following staining with FITC-conjugated rat anti-mouse CD45R/B220 and pan-NK antibodies, respectively. The subpopulations of T cells were determined by quantifying CD4+/CD8−, CD4−/CD8+, and CD4+/CD8+ cells. For some studies, the mice were preinjected with anti-CXC12-neutralizing antibody (12.Sweeney E.A. Lortat-Jacob H. Priestley G.V. Nakamoto B. Papayannopoulou T. Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice. Involvement in mobilization of stem/progenitor cells.Blood. 2002; 99: 44-51Crossref PubMed Scopus (187) Google Scholar) (R&D Systems, 2 mg/kg BW) i.v. 90 min prior to heparin administration. Lymphocyte suspensions from peripheral lymph nodes were prepared in DMEM, and erythrocytes were lysed with a 0.83% NH4Cl solution. After washing with PBS, the cells (5–10 × 106 cells/ml) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, 5 μm) (9.Wang L. Brown J.R. Varki A. Esko J.D. Heparin's anti-inflammatory effects require glucosamine 6-O-sulfation and are mediated by blockade of L- and P-selectins.J. Clin. Invest. 2002; 110: 127-136Crossref PubMed Scopus (328) Google Scholar). Heparin at 1 mg/25 g BW in 100 μl of saline or only saline was injected into the tail vein of the recipient mice. Five min later, 7 × 106 CFSE-labeled cells in 100 μl of saline were similarly injected. Ninety minutes after heparin administration, the mice were sacrificed. Peripheral blood and single cell suspensions prepared from peripheral (cervical and axillary) and mesenteric lymph nodes were counted, and the percentage of CFSE+ cells was determined by flow cytometry. The chests of anesthetized mice were opened; 10 μl of FITC (Sigma) at 600 μg/ml in PBS was injected directly into each thymic lobe with a 28-gauge needle, and then the chests were closed with surgical clips in the overlying skin (15.Scollay R.G. Butcher E.C. Weissman I.L. Thymus cell migration. Quantitative aspects of cellular traffic from the thymus to the periphery in mice.Eur. J. Immunol. 1980; 10: 210-218Crossref PubMed Scopus (490) Google Scholar). This method labeled 30% of the thymocytes in the thymus. Thirty minutes after intrathymic injection, mice received heparin at 1 mg/25 g BW or only PBS via tail vein injection. Ninety minutes later, the FITC+ cells in the circulation were quantified by flow cytometry. The total of thymocytes released from the thymus was calculated as the (number of FITC+ cells)/(30%). Mice were injected with 0.6 mg of BrdU i.p. twice, separated by 12 h (16.Horwitz B.H. Mizgerd J.P. Scott M.L. Doerschuk C.M. Mechanisms of granulocytosis in the absence of CD18.Blood. 2001; 97: 1578-1583Crossref PubMed Scopus (32) Google Scholar, 17.Basu S. Hodgson G. Katz M. Dunn A.R. Evaluation of role of G-CSF in the production, survival, and release of neutrophils from bone marrow into circulation.Blood. 2002; 100: 854-861Crossref PubMed Scopus (215) Google Scholar, 18.Suratt B.T. Petty J.M. Young S.K. Malcolm K.C. Lieber J.G. Nick J.A. Gonzalo J.A. Henson P.M. Worthen G.S. Role of the CXCR4/SDF-1 chemokine axis in circulating neutrophil homeostasis.Blood. 2004; 104: 565-571Crossref PubMed Scopus (192) Google Scholar). At 24 h after the first injection, 35% Gr-1+-BM cells were labeled with BrdU, and the mice were injected with heparin at 1 mg/25 g BW in 100 μl of saline or saline only via tail vein. Ninety min later, blood was collected, and total leukocytes were counted. The Gr-1+/BrdU+ cells were quantified by flow cytometry, and the neutrophils released from the BM were calculated as (Gr-1+/BrdU+ cells)/35%. Mice were injected intraperitoneally with 0.6 mg of BrdU daily for 7 days at which point all neutrophils in circulation were BrdU+ (19.Nakagawa M. Terashima T. D'yachkova Y. Bondy G.P. Hogg J.C. van Eeden S.F. Glucocorticoid-induced granulocytosis. Contribution of marrow release and demargination of intravascular granulocytes.Circulation. 1998; 98: 2307-2313Crossref PubMed Scopus (193) Google Scholar). Fresh blood was collected into citrate-containing tubes, washed with PBS, and then transfused to the recipient mice at 150 μl/mouse via tail vein. Thirty min after the transfusion, heparin at 1 mg/25 g BW in 100 μl of saline or saline only was injected similarly. Ninety min after the heparin/saline administration, the Gr-1+ and BrdU+/Gr-1+ cells in the peripheral blood were quantified by leukocyte count and flow cytometry. CXCL12α and CXCL12β levels in mouse plasma and BM were determined by ELISA using monoclonal antibodies specific for CXCL12β (BAF351) or CXCL12α (BAF310) with capture antibody MAB350 and recommended protocols from R&D Systems (12.Sweeney E.A. Lortat-Jacob H. Priestley G.V. Nakamoto B. Papayannopoulou T. Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice. Involvement in mobilization of stem/progenitor cells.Blood. 2002; 99: 44-51Crossref PubMed Scopus (187) Google Scholar). BM samples were prepared by flushing the content of two femurs directly into 0.4 ml of sample buffer (0.1% BSA, 0.05% Tween 20 in 20 mm Trizma (Tris base), 150 mm NaCl, pH 7.3) and centrifuged, and the supernatants were collected and used for CXCL12 concentration determination. Transendothelial cell migration was performed using trans-wells containing 3-μm pore-polycarbonate filters to separate the upper and lower chambers in 24-well plates (Costar, Cambridge, MA) (20.Netelenbos T. Zuijderduijn S. Van Den Born J. Kessler F.L. Zweegman S. Huijgens P.C. Dräger A.M. Proteoglycans guide SDF-1-induced migration of hematopoietic progenitor cells.J. Leukocyte Biol. 2002; 72: 353-362Crossref PubMed Google Scholar, 21.Netelenbos T. van den Born J. Kessler F.L. Zweegman S. Merle P.A. van Oostveen J.W. Zwaginga J.J. Huijgens P.C. Dräger A.M. Proteoglycans on bone marrow endothelial cells bind and present SDF-1 towards hematopoietic progenitor cells.Leukemia. 2003; 17: 175-184Crossref PubMed Scopus (81) Google Scholar). Immortalized mouse lung endothelial cells (22.Wijelath E. Namekata M. Murray J. Furuyashiki M. Zhang S. Coan D. Wakao M. Harris R.B. Suda Y. Wang L. Sobel M. Multiple mechanisms for exogenous heparin modulation of vascular endothelial growth factor activity.J. Cell. Biochem. 2010; 111: 461-468Crossref PubMed Scopus (32) Google Scholar, 23.Zhou B. Honor L.B. He H. Ma Q. Oh J.H. Butterfield C. Lin R.Z. Melero-Martin J.M. Dolmatova E. Duffy H.S. Gise A. Zhou P. Hu Y.W. Wang G. Zhang B. Wang L. Hall J.L. Moses M.A. McGowan F.X. Pu W.T. Adult mouse epicardium modulates myocardial injury by secreting paracrine factors.J. Clin. Invest. 2011; 121: 1894-1904Crossref PubMed Scopus (390) Google Scholar) were seeded at 1.5 × 104 per well in the upper chamber and became confluent after 3–4 days in culture. The integrity of the endothelium-covered trans-wells was controlled after each migration by May-Grunwald-Giemsa staining and followed by visualization by microscopy. BM cells were freshly prepared in single cell suspensions from mouse femurs and tibias by flushing in PBS. Red blood cells were lysed with 0.83% NH4Cl solution before rinsing. The BM cells were added at 1 × 105 cells per upper compartment. The migration was initiated by supplying CXCL12α (10 ng/ml) in the lower chamber to determine CXCL12-induced chemotaxis. Only the active migrating cells were able to get through the pores and into the lower chamber. After 4 h, cells that migrated into the lower chamber were counted. To determine whether heparin inhibits the CXCL12-induced transendothelial chemotaxis of BM cells, the experiment was similarly carried out with heparin added in the upper and lower chambers prior to initiation of cell migration. In additional experiments to determine the effect of immobilized CXCL12 on random migration of BM cells (chemokinesis), CXCL12α was preincubated at both apical and basal sides of endothelial cell-covered trans-wells for 30 min at RT. Nonbound CXCL12α was washed away by rinsing with PBS, and the transendothelial cell migration experiments were carried out as described above. Primary human umbilical vein endothelial cells plated at 104 cells/well in 96-well tissue culture plates were grown in EMB-2 media supplemented with growth factors and 2% FBS according to the supplier's recommendations (Lonza Inc., Walkersville, MD) until confluency. The cells were then fixed with 4% paraformaldehyde for 10 min at RT and washed in PBS. After fixation, some wells were incubated with a mixture of heparinases I–III (20–50 milliunits/ml of each enzyme) for 1 h at RT. Nonspecific binding sites were blocked with 5% BSA in PBS, and the cells were then incubated with CXCL12α at 10 μg/ml in PBS with or without heparin, heparan sulfate, or heparinoid at 20 μg/ml each for 1.5 h at RT. Bound CXCL12α was detected by incubation with a mouse anti-CXCL12 IgG antibody (R&D Systems, Minneapolis, MN) and then with an HRP-conjugated goat anti-mouse IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and finally with the 3,3′,5,5′-tetramethylbenzidine chromogen (Thermo Fisher Scientific, Waltham, MA). The absorbance at 450 nm was read using a microplate reader (Molecular Devices, Inc., Menlo Park, CA) equipped with Softmax software. SPR analyses were conducted using a BIACORE T100 apparatus (Biacore AB, Piscataway, NJ) with biotinylated heparin immobilized on streptavidin-coated chips. Biotinylated heparin was prepared using a two-stage procedure with biotin coupled at the reducing end of heparin (24.Osmond R.I. Kett W.C. Skett S.E. Coombe D.R. Protein-heparin interactions measured by BIAcore 2000 are affected by the method of heparin immobilization.Anal. Biochem. 2002; 310: 199-207Crossref PubMed Scopus (111) Google Scholar, 25.Amara A. Lorthioir O. Valenzuela A. Magerus A. Thelen M. Montes M. Virelizier J.L. Delepierre M. Baleux F. Lortat-Jacob H. Arenzana-Seisdedos F. Stromal cell-derived factor-1α associates with heparan sulfates through the first β-strand of the chemokine.J. Biol. Chem. 1999; 274: 23916-23925Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). The biotinylated heparin carried 1.3 biotin/heparin and was immobilized on streptavidin-coated CM5 chip surface. Briefly, the surface of a CM5 chip was activated by injecting a freshly prepared mixture (1:1) of N-hydroxysuccinimide (50 mm in water) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (200 mm in water) at a flow rate of 5 μl/min, followed by a 21-min injection of streptavidin (400 mg/ml). Any remaining activated carboxylic acid groups were blocked by incubation with 1 m ethanolamine hydrochloride, pH 8.6, for 21 min. Approximately 1000 response units of streptavidin was coupled to the cell surface using this method. Heparin immobilization was achieved by injecting biotinylated heparin at 1 ml/min for 21 min over the streptavidin-coated surface and was optimized to give ∼500 response units. As a reference surface for refractive index and correction of nonspecific binding, a second flow cell was treated in the same manner as above, but without the injection of biotinylated heparin. To determine the interaction of CXCL12α with heparinoids, CXCL12α (125 nm) was premixed with heparin (1 μg/ml) or 6-des-hep (1 μg/ml) in HBS-EP buffer (10 mm Hepes, 150 mm NaCl, 3 mm EDTA, 0.005% v/v surfactant P20) at RT. The mixture was immediately injected at a flow rate of 50 μl/min. CXCL12α (125 nm) was used as a positive control, whereas HBS-EP buffer alone was background control. Data were collected with a contact time of 120 s and a dissociation time of 300 s. The response unit for binding was presented with the background response subtracted. Data were evaluated using BIAevaluation Software 3.1, Biacore. Heparin has been reported to cause rapid and transient lymphocytosis in humans, cows, rats, and mice after i.p or i.v. injection (3.Bradfield J.W. Born G.V. Lymphocytosis produced by heparin and other sulfated polysaccharides in mice and rats.Cell. Immunol. 1974; 14: 22-32Crossref PubMed Scopus (33) Google Scholar, 4.Godlowski Z.Z. Prevention of hormona eosinopenia and lymphopenia by inhibition of clotting in blood; preliminary report.Br. Med. J. 1951; 1: 854-855Crossref PubMed Scopus (8) Google Scholar, 5.Sasaki S. Production of lymphocytosis by polysaccharide polysulfates (heparinoids).Nature. 1967; 214: 1041-1042Crossref PubMed Scopus (30) Google Scholar, 26.Bradfield J.W. Born G.V. Inhibition of lymphocyte recirculation by heparin.Nature. 1969; 222: 1183-1184Crossref PubMed Scopus (21) Google Scholar); however, the effect on subpopulations of leukocytes has not been defined. We examined the subpopulation distribution of circulating leukocytes 90 min after heparin treatment in mice. Compared with saline, heparin increased all major subpopulations of leukocytes in the circulation, including neutrophils (4.27 × 106/ml, 4.4-fold), lymphocytes (6.47 × 106/ml, 2.7-fold), and monocytes (0.37 × 106/ml, 2.1-fold) (Fig. 1). An analysis of the lymphocyte subpopulations showed the same tendency with the following results: B lymphocytes (2.24 × 106/ml, 1.9-fold), CD4+/CD8− (2.91 × 106/ml, 4.6-fold), CD4−/CD8+ (1.53 × 106/ml, 3.9-fold), and CD4+/CD8+ (0.06 × 106/ml, 12.5-fold) (Fig. 1). Lymphocytes and neutrophils were the two major subpopulations of circulating leukocytes and accounted for ∼62 and 33% of HIL cell population, respectively. To determine the structure required for heparin to induce leukocytosis, we treated mice with structurally different heparins (Fig. 2A). Compared with heparin, N-des-hep and 2/3-des-hep induced attenuated responses, whereas N/2/3-des-hep was unable to induce leukocytosis, indicating that HIL depended on the degree of sulfation (Fig. 2, B–D). Intriguingly, 6-des-hep was also unable to induce leukocytosis (Fig. 2, B–D). Heparin carries more N-sulfate than 6-O-sulfate, implying that the inefficacy of 6-des-hep to induce leukocytosis was not due to a reduction in the overall degree of sulfation, but specifically to the lack of 6-O-sulfate, revealing that heparin uniquely requires 6-O-sulfation to induce leukocytosis. CR-hep induced attenuated HIL response too, showing that the presence of the carboxyl moiety is also required for heparin to induce leukocytosis (Fig. 2, B–D). Intriguingly, subpopulation analysis observed that CR-hep had a slightly reduced efficacy for inducing neutrophilia, but its ability to cause lymphocytosis was abrogated (Fig. 2, C and D), highlighting that heparin requires carboxyl moiety to induce lymphocytosis but not neutrophilia. Furthermore, N-des-hep, 2/3-des-hep. and CR-hep all were capable of inducing leukocytosis (Fig. 2, B–D), but they were devoid of anticoagulant activities, indicating that the anticoagulant activity of heparin is not required for HIL. Collectively, these observations indicate that HIL depends on the degree of sulfation and carboxylation of heparin, and there is a specific requirement for 6-O-sulfation, but anticoagulant activity is not required. The leukocyte numbers in the peripheral blood are controlled by hematopoiesis in the BM and thymus," @default.
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• W2161735952 date "2012-02-01" @default.
• W2161735952 modified "2023-09-26" @default.
• W2161735952 title "Heparin-induced Leukocytosis Requires 6-O-Sulfation and Is Caused by Blockade of Selectin- and CXCL12 Protein-mediated Leukocyte Trafficking in Mice" @default.
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• W2161735952 doi "https://doi.org/10.1074/jbc.m111.314716" @default.
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