Kynurenic Acid Triggers Firm Arrest of Leukocytes to Vascular Endothelium under Flow Conditions

Recent studies have demonstrated that kynurenic acid (KYNA), a compound produced endogenously by the interferon-γ-induced degradation of tryptophan by indoleamine 2,3-dioxygenase, activates the previously orphaned G protein-coupled receptor, GPR35. This receptor is expressed in immune tissues, although its potential function in immunomodulation remains to be explored. We determined that GPR35 was most highly expressed on human peripheral monocytes. In an in vitro vascular flow model, KYNA triggered the firm arrest of monocytes to both fibronectin and ICAM-1, via β1 integrin- and β2 integrin-mediated mechanisms, respectively. Incubation of monocytes with pertussis toxin prior to use in flow experiments significantly reduced the KYNA-induced monocyte adhesion, suggesting that adhesion is triggered by a Gi-mediated process. Furthermore, KYNA-triggered adhesion of monocytic cells was reduced by short hairpin RNA-mediated silencing of GPR35. Although GPR35 is expressed at slightly lower levels on neutrophils, KYNA induced firm adhesion of these cells to an ICAM-1-expressing monolayer as well. KYNA also elicited neutrophil shedding of surface L-selectin, another indicator of leukocyte activation. Taken together, these data suggest that KYNA could be an important early mediator of leukocyte recruitment. Recent studies have demonstrated that kynurenic acid (KYNA), a compound produced endogenously by the interferon-γ-induced degradation of tryptophan by indoleamine 2,3-dioxygenase, activates the previously orphaned G protein-coupled receptor, GPR35. This receptor is expressed in immune tissues, although its potential function in immunomodulation remains to be explored. We determined that GPR35 was most highly expressed on human peripheral monocytes. In an in vitro vascular flow model, KYNA triggered the firm arrest of monocytes to both fibronectin and ICAM-1, via β1 integrin- and β2 integrin-mediated mechanisms, respectively. Incubation of monocytes with pertussis toxin prior to use in flow experiments significantly reduced the KYNA-induced monocyte adhesion, suggesting that adhesion is triggered by a Gi-mediated process. Furthermore, KYNA-triggered adhesion of monocytic cells was reduced by short hairpin RNA-mediated silencing of GPR35. Although GPR35 is expressed at slightly lower levels on neutrophils, KYNA induced firm adhesion of these cells to an ICAM-1-expressing monolayer as well. KYNA also elicited neutrophil shedding of surface L-selectin, another indicator of leukocyte activation. Taken together, these data suggest that KYNA could be an important early mediator of leukocyte recruitment. Leukocyte recruitment into tissue compartments is a tightly regulated process orchestrated by chemokines (1.Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6414) Google Scholar). Chemokines convert leukocyte rolling or tethering on the vascular endothelium to firm arrest via the activation of leukocyte surface integrins (2.Campbell J.J. Hedrick J. Zlotnik A. Siani M.A. Thompson D.A. Butcher E.C. Science. 1998; 279: 381-384Crossref PubMed Scopus (838) Google Scholar, 3.Gerszten R.E. Garcia-Zepeda E.A. Lim Y.C. Yoshida M. Ding H.A. Gimbrone Jr., M.A. Luster A.D. Luscinskas F.W. Rosenzweig A. Nature. 1999; 398: 718-723Crossref PubMed Scopus (1070) Google Scholar). As chemoattractants, chemokines subsequently play an important role in the directional migration of leukocytes through tissues. Chemoattractant receptors are a subtype of G protein-coupled receptors (GPCRs), 3The abbreviations used are: GPCRG protein-coupled receptorIL-8interleukin-8MCP-1monocyte chemoattractant protein-1fMLPformyl-methionyl-leucyl-phenylalanineKYNAkynurenic acidIDOindoleamine 2,3-dioxygenaseAAanthranilic acidPTXpertussis toxinHUVEChuman umbilical vein endothelial cellICAM-1intercellular adhesion molecule-1QPCRquantitative polymerase chain reactionshRNAshort hairpin RNA. one of the largest known families of human proteins. Chemoattractant receptors bind a variety of agonists, including proteins such as interleukin-8 (IL-8, CXCL8) (4.Holmes W.E. Lee J. Kuang W.J. Rice G.C. Wood W.I. Science. 1991; 253: 1278-1280Crossref PubMed Scopus (917) Google Scholar) and monocyte chemoattractant protein-1 (MCP-1, CCL2) (5.Charo I.F. Myers S.J. Herman A. Franci C. Connolly A.J. Coughlin S.R. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 2752-2756Crossref PubMed Scopus (654) Google Scholar), small peptides such as fMLP (6.Koo C. Lefkowitz R.J. Snyderman R. J. Clin. Invest. 1983; 72: 748-753Crossref PubMed Scopus (113) Google Scholar), as well as bioactive lipids including leukotriene B4 (7.Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (856) Google Scholar). As such, chemoattractant receptors mirror the entire family of GPCRs, which can be activated by ligands ranging in size from metabolites to large proteins (8.Lagerström M.C. Schiöth H.B. Nat. Rev. Drug Discov. 2008; 7: 339-357Crossref PubMed Scopus (1138) Google Scholar). G protein-coupled receptor interleukin-8 monocyte chemoattractant protein-1 formyl-methionyl-leucyl-phenylalanine kynurenic acid indoleamine 2,3-dioxygenase anthranilic acid pertussis toxin human umbilical vein endothelial cell intercellular adhesion molecule-1 quantitative polymerase chain reaction short hairpin RNA. Because GPCRs serve as targets for therapeutic intervention, considerable activity has gone into the identification of both putative GPCR genes and the ligands for the resulting receptors (8.Lagerström M.C. Schiöth H.B. Nat. Rev. Drug Discov. 2008; 7: 339-357Crossref PubMed Scopus (1138) Google Scholar). Recently, the tryptophan metabolite kynurenic acid (KYNA) was identified as an agonist for the previously “orphaned” receptor GPR35. KYNA was shown to elicit intracellular release of Ca2+ in Chinese hamster ovary cells in which GPR35 was co-expressed in the context of a chimeric G protein signaling apparatus. HEK93 cells transfected with GPR35 and Gqo proteins accumulated inositol phosphate upon exposure to KYNA. KYNA also induced internalization of GPR35 on HeLa cells, which is commonly seen following the activation of GPRs with agonists such as chemokines (9.Wang J. Simonavicius N. Wu X. Swaminath G. Reagan J. Tian H. Ling L. J. Biol. Chem. 2006; 281: 22021-22028Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). KYNA is produced endogenously as a result of the degradation of tryptophan (Fig. 1). In most tissues, the rate-limiting step in this degradation is the conversion of tryptophan to N-formylkynurenine, a reaction that can be catalyzed by the inducible enzyme indolemine 2,3-dioxygenase (IDO). IDO is induced by interferon-γ, which leads to a substantial increase in the concentration of KYNA and other tryptophan catabolites during inflammatory processes. Previous work has demonstrated that KYNA acts as a neuroprotective agent by antagonizing both N-methyl-d-aspartate and α7-nicotinic receptors (10.Stone T.W. Pharmacol. Rev. 1993; 45: 309-379PubMed Google Scholar, 11.Parsons C.G. Danysz W. Quack G. Hartmann S. Lorenz B. Wollenburg C. Baran L. Przegalinski E. Kostowski W. Krzascik P. Chizh B. Headley P.M. J. Pharmacol. Exp. Ther. 1997; 283: 1264-1275PubMed Google Scholar, 12.Hilmas C. Pereira E.F. Alkondon M. Rassoulpour A. Schwarcz R. Albuquerque E.X. J. Neurosci. 2001; 21: 7463-7473Crossref PubMed Google Scholar). Many peripheral tissues, including the heart and vasculature, are also capable of generating KYNA (13.Baran H. Amann G. Lubec B. Lubec G. Pediatr. Res. 1997; 41: 404-410Crossref PubMed Scopus (28) Google Scholar, 14.Stazka J. Luchowski P. Wielosz M. Kleinrok Z. Urbańska E.M. Eur. J. Pharmacol. 2002; 448: 133-137Crossref PubMed Scopus (42) Google Scholar), although its role in these tissues has not been well defined. Expression analysis indicates that GPR35 is selectively present in immune and intestinal tissues. From a functional perspective, KYNA treatment inhibited the secretion of tumor necrosis factor-α by mononuclear cells treated with lipopolysaccharide (9.Wang J. Simonavicius N. Wu X. Swaminath G. Reagan J. Tian H. Ling L. J. Biol. Chem. 2006; 281: 22021-22028Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). This finding is in general agreement with the prevailing literature suggesting that IDO-mediated tryptophan catabolism appears to play a significant counter-regulatory role in dampening down the activation of the immune system (15.Mellor A.L. Munn D.H. Nat. Rev. Immunol. 2004; 4: 762-774Crossref PubMed Scopus (1875) Google Scholar). However, the potential spectrum of physiological roles for KYNA in immune modulation remains incompletely characterized. Given the reported high level of GPR35 expression on circulating leukocytes, here we tested the hypothesis that KYNA may play a chemokine-like role in modulating leukocyte-endothelial interactions under physiologically relevant flow conditions as seen in the vasculature. We explored the intracellular signaling pathways by which KYNA may be activating leukocytes, as well as the surface integrins modulating these effects. We report the unanticipated finding that KYNA is sufficient to drive early leukocyte adhesion. RPMI 1640 and Dulbecco's phosphate-buffered saline with or without Ca2+ and Mg2+ were obtained from Invitrogen. KYNA, tryptophan, anthranilic acid (AA), and pertussis toxin (PTX) were obtained from Sigma. Human chemokines MCP-1 and IL-8 were obtained from Peprotech (Rocky Hill, NJ). Human umbilical vein endothelial cells (HUVECs), endothelial growth medium-2, and endothelial basal medium-2 were obtained from Lonza (Basel, Switzerland); HUVECs were cultured according to the supplier's directions. To prepare slides for flow adhesion studies, 0.8-cm2 chambers were attached to Permanox slides (Nunc, Rochester, NY) using 2% agarose in phosphate-buffered saline. The interior surface of the resulting well was coated with 0.05% fibronectin (Sigma) in phosphate-buffered saline (with Ca2+ and Mg2+) for 1 h at 37 °C. For monolayers, ∼20,000 freshly trypsinized HUVECs (passages 2–4) were seeded in the fibronectin-coated wells and grown at 37 °C for 18 h prior to transfection with adenoviral ICAM-1 vector as previously described (16.Gerszten R.E. Friedrich E.B. Matsui T. Hung R.R. Li L. Force T. Rosenzweig A. J. Biol. Chem. 2001; 276: 26846-26851Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) using endothelial basal medium-2 as a transfection medium and endothelial growth medium-2 for growth conditions. HUVEC monolayers were used in flow experiments 48 h after transfection. HP2/1, a monoclonal antibody to α4 integrin (CD49d) was obtained from Immunotech (Marseille, France). TS1/18, a monoclonal antibody to human β2 integrin (CD18) was obtained from Endogen (Rockford, IL). Mouse IgG1 isotype control antibody was obtained from Pharmingen. LM609, a blocking antibody to αvβ3 integrin was obtained from Millipore (Billerica, MA). Leukocytes were freshly prepared from healthy human donors. Mononuclear cells were isolated via Ficoll-Hypaque (LSM, Fisher Scientific, Fair Lawn, NJ) density gradient centrifugation and washed in RPMI 1640. CD14+ monocytes were further purified from this subset of cells by depletion of other mononuclear cell types with magnetic-activated cell separation (Monocyte Isolation Kit II, Miltenyi Biotec, Auburn, CA). Neutrophils were isolated by Ficoll-Hypaque gradient, followed by dextran sedimentation and hypotonic lysis of contaminating red blood cells (17.Clark R.A. Nauseef W.M. Coligan J.E. Kruisbeek A.M. Margulies D.H. Shevach E.M. Strober W. Current Protocols in Immunology. John Wiley and Sons, Inc., New York1996: 7.23.2-7.23.4Google Scholar). Quantitative PCR (QPCR) analysis of transcript levels of GPR35 and other chemokine receptors was performed on freshly isolated leukocytes. Total cellular RNA was isolated using RNeasy columns (Qiagen, Valencia, CA), treated with DNase I (Invitrogen), and reverse transcribed to cDNA using TaqMan reagents (Applied Biosystems, Foster, CA). QPCR was performed on an Mx4000 quantitative PCR instrument (Stratagene, La Jolla, CA) using Power SYBR Green PCR reagents (Applied Biosystems). Primers for GPR35 were as previously described (9.Wang J. Simonavicius N. Wu X. Swaminath G. Reagan J. Tian H. Ling L. J. Biol. Chem. 2006; 281: 22021-22028Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar); primers for chemokine receptors were the generous gift of Dr. Terry Means, and were designed as previously described (18.Means T.K. Hayashi F. Smith K.D. Aderem A. Luster A.D. J. Immunol. 2003; 170: 5165-5175Crossref PubMed Scopus (335) Google Scholar). To measure shedding of L-selectin, 3 × 106 neutrophils in a total volume of 200 μl in RPMI 1640 were incubated with test compounds for 30 min at room temperature. Cleaved L-selectin in the supernatant was measured using the Human sL-Selectin ELISA adhesion studies were in a flow using phosphate-buffered saline with Ca2+ and Mg2+ fibronectin-coated or RPMI as from 1 for fibronectin-coated to for the ICAM-1-expressing to be tested were to the leukocyte and the cells on the test surface were at the in at For studies, leukocytes were incubated with the antibody for min on or with for min at 37 prior to into the flow were obtained from the of The at the J. D.A. X. A.M. G. B. B. N. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). were purified using the transfected into cells with a system to J. D.A. X. A.M. G. B. B. N. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, L. P. R. Science. PubMed Scopus Google Scholar). cells at × were with the in and for 30 min at 37 °C and × cells were by the to 3 of was determined by QPCR analysis as described the cell with the of GPR35 was for further of adhesion under flow conditions. data are as For an test was The hypothesis was at To the potential role of KYNA in leukocyte adhesion we measured the level of GPR35 in leukocyte cell as well as in endothelial GPR35 was most highly expressed in with significant expression in neutrophils as well. QPCR experiments expression in endothelial cells (Fig. The level of GPR35 expression on monocytes was that of the monocyte chemokine receptor, although with that of (Fig. of both of these receptors in monocyte adhesion R.E. Garcia-Zepeda E.A. Lim Y.C. Yoshida M. Ding H.A. Gimbrone Jr., M.A. Luster A.D. Luscinskas F.W. Rosenzweig A. Nature. 1999; 398: 718-723Crossref PubMed Scopus (1070) Google Scholar) and also to monocyte in in C. 2008; PubMed Scopus Google Scholar, L. J. M. I.F. Nature. 1998; PubMed Scopus Google Scholar). GPR35 expression that of a chemokine receptor that is highly expressed on and present at levels on monocytes S. T.W. J. Wu L. W. C. J. Exp. PubMed Scopus Google Scholar). To test KYNA was sufficient to β1 integrin-mediated firm arrest under physiological flow we a flow to monocytes a fibronectin-coated a of KYNA treatment in a increase in the of cells as with slightly adhesion induced by the agonist and metabolites to KYNA that not bind GPR35 (9.Wang J. Simonavicius N. Wu X. Swaminath G. Reagan J. Tian H. Ling L. J. Biol. Chem. 2006; 281: 22021-22028Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar), on the adhesion of monocytes (Fig. of the monocytes with HP2/1, an α4 antibody that with significantly reduced the of cells induced by KYNA with an control antibody not result in in KYNA-induced firm arrest (Fig. Many in leukocytes are on the G protein of the G protein Y. Wu Y. H. Wu J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To the of KYNA on monocyte adhesion this we cells with prior to into the flow its by the of the G protein the from with receptors on the surface of the cell Sci. Google Scholar). Incubation of monocytes with prior to use in flow experiments significantly reduced the KYNA-induced adhesion of monocytes to fibronectin (Fig. that this adhesion is triggered by a Gi-mediated process. activated β2 integrins and intercellular adhesion are also sufficient to monocyte arrest under flow conditions R.E. Garcia-Zepeda E.A. Lim Y.C. Yoshida M. Ding H.A. Gimbrone Jr., M.A. Luster A.D. Luscinskas F.W. Rosenzweig A. Nature. 1999; 398: 718-723Crossref PubMed Scopus (1070) Google Scholar). We the adhesion of monocytes to endothelial monolayers ICAM-1 using an adenoviral expression We have previously demonstrated that this system is to R.E. Garcia-Zepeda E.A. Lim Y.C. Yoshida M. Ding H.A. Gimbrone Jr., M.A. Luster A.D. Luscinskas F.W. Rosenzweig A. Nature. 1999; 398: 718-723Crossref PubMed Scopus (1070) Google Scholar). KYNA treatment of monocytes in this system adhesion in a with significant arrest as as treatment with 1 anthranilic acid elicited (Fig. This is significantly lower the concentration to adhesion to fibronectin with that firm arrest of leukocytes on adhesion endothelial monolayers to purified protein on is highly to chemoattractant agonists R.E. Garcia-Zepeda E.A. Lim Y.C. Yoshida M. Ding H.A. Gimbrone Jr., M.A. Luster A.D. Luscinskas F.W. Rosenzweig A. Nature. 1999; 398: 718-723Crossref PubMed Scopus (1070) Google Scholar). To the of the we the leukocytes with the β2 with this not an LM609, the KYNA-induced (Fig. As with adhesion to of monocytes with significantly reduced adhesion to ICAM-1-expressing monolayers, (Fig. Although KYNA-triggered of monocytes on fibronectin was the the reverse was with the ICAM-1 As KYNA and MCP-1 both be to be present under inflammatory other chemokines as we tested the of both agonists adhesion. treatment with both MCP-1 and KYNA in an not on firm arrest not To that the adhesion was to activation of GPR35 by KYNA, we used to cells with reduced expression of GPR35. cells a in GPR35 expression to control cells with an protein (Fig. Green control cells a level of KYNA-triggered firm arrest to that is to that in freshly isolated human leukocytes. cells with reduced GPR35 expression not a significant increase in firm arrest after KYNA treatment (Fig. Because GPR35 was also in human neutrophils, we tested KYNA was sufficient to adhesion of this leukocyte subset as well. Because neutrophil adhesion under flow is on β2 we also neutrophils ICAM-1-expressing a of KYNA was sufficient to significant in neutrophil adhesion (Fig. treatment with 1 not increase adhesion In this system as of the neutrophils with the β2 antibody the of KYNA on leukocyte To that KYNA-induced arrest is a result of and not endothelial we treated the HUVEC monolayers for min with KYNA in and washed the monolayers prior to in the flow As treatment of the monolayers in increase of neutrophil adhesion (Fig. KYNA to be its on the We also the of L-selectin from the surface of neutrophils as an indicator of the of cellular activation T.K. M.A. Butcher E.C. Science. PubMed Scopus Google Scholar). a with of the known activating agent a significant increase in L-selectin was (Fig. with T.K. B. R. Butcher E. J. Clin. Invest. 1991; PubMed Scopus Google Scholar). with KYNA in shedding of L-selectin from neutrophils in a a increase in L-selectin at a concentration of and a significant increase in the at the KYNA tested (Fig. we tested other metabolites from the tryptophan that are known not to GPR35 (9.Wang J. Simonavicius N. Wu X. Swaminath G. Reagan J. Tian H. Ling L. J. Biol. Chem. 2006; 281: 22021-22028Abstract Full Text Full Text PDF PubMed Scopus (479) Google tryptophan and anthranilic a concentration of 1 compound elicited an increase in L-selectin shedding the level (Fig. We that KYNA is capable of activating of leukocytes, firm arrest in these cells through both β1 and β2 integrin-mediated adhesion. This adhesion was which that through a compounds that are known not to bind tryptophan and anthranilic acid (9.Wang J. Simonavicius N. Wu X. Swaminath G. Reagan J. Tian H. Ling L. J. Biol. Chem. 2006; 281: 22021-22028Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar), not activation or adhesion of leukocytes, with a of GPR35 expression using significantly reduced KYNA-induced firm arrest of a monocytic cell to ICAM-1-expressing GPR35 in this process. The of KYNA to adhesion is with other studies unanticipated roles for small in immune chain and are to a receptor that is significantly in monocytes. with these compounds the of lipopolysaccharide treatment on secretion of the by cells J. Wu X. Simonavicius N. Tian H. Ling L. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). The concentration of KYNA in human is L. N. T.W. Clin. Exp. Pharmacol. 2006; PubMed Scopus Google Scholar, Z. A. A. T. T. L. B. Z. J. PubMed Scopus Google Scholar, A. E. J. J. Sci. 2002; PubMed Scopus Google Scholar), which is to leukocyte adhesion in of IDO leads to the degradation of tryptophan and of catabolites including KYNA. As such, the concentration of KYNA could be to the K. M.A. M. J. A. Lee K. A. S. A. Smith M. T. PubMed Scopus Google Scholar, P. A. S.R. T.W. J. Sci. 2002; PubMed Scopus Google Scholar, S.R. T.W. Exp. Biol. 2003; PubMed Scopus Google Scholar), a concentration sufficient to significant adhesion of both monocytes and neutrophils to ICAM-1-expressing endothelial cells in studies, the of endothelial cells have been demonstrated to convert to KYNA, with sufficient KYNA concentration to elicit a leukocyte may be present in the vasculature. studies that KYNA can firm adhesion at as as This is significantly the of previously reported for KYNA activation of human GPR35 on Chinese hamster ovary cells (9.Wang J. Simonavicius N. Wu X. Swaminath G. Reagan J. Tian H. Ling L. J. Biol. Chem. 2006; 281: 22021-22028Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). However, we that the receptor expressed in its context in a cell The of GPRs to chimeric G proteins for Ca2+ may significantly increase the measured for to C. S.J. C. T.W. J. Pharmacol. Exp. Ther. 2001; Google Scholar, J. C. T.W. Eur. J. Pharmacol. 2003; PubMed Scopus Google Scholar); to have been reported C. J. S. B. C. N. R. T.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the arrest of leukocytes on ICAM-1-expressing endothelial cells is a highly R.E. Garcia-Zepeda E.A. Lim Y.C. Yoshida M. Ding H.A. Gimbrone Jr., M.A. Luster A.D. Luscinskas F.W. Rosenzweig A. Nature. 1999; 398: 718-723Crossref PubMed Scopus (1070) Google Scholar), and significant adhesion may be seen with ligands at activation have that KYNA can the secretion of the tumor necrosis factor-α and (9.Wang J. Simonavicius N. Wu X. Swaminath G. Reagan J. Tian H. Ling L. J. Biol. Chem. 2006; 281: 22021-22028Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar, M. M. E. Google Scholar). This suggest a of the immune other types of were not Although is with that tryptophan metabolites after the of IDO in an (15.Mellor A.L. Munn D.H. Nat. Rev. Immunol. 2004; 4: 762-774Crossref PubMed Scopus (1875) Google Scholar), the data here suggest that the may in be Given the of KYNA to elicit early arrest of leukocytes, tryptophan catabolism by IDO may have a followed by a into the of leukocytes to KYNA be to both the and of KYNA on leukocyte in GPR35 expression was by a in KYNA-triggered adhesion of monocytic We were also to that cells in although the cells could be by for functional The spectrum of of GPR35 in vitro and in remains the of is the potential of KYNA on with Because the of of tryptophan catabolites is through the have levels of KYNA. has shown that levels of KYNA this is reduced by with KYNA at the level K. J. M. W. 2001; PubMed Scopus Google Scholar). As with from an of further into the of KYNA in these may be In studies have shown that KYNA is capable of firm arrest of both neutrophils and monocytes under physiological flow conditions in an in vitro to the adhesion process in that are not present in this and studies in are We are for the ICAM-1 in the of Dr. with