From www.bloodjournal.org by guest on October 21, 2014. For personal use only. 1994 84: 474-482 Assembly of contact-phase factors on the surface of the human neutrophil membrane LM Henderson, CD Figueroa, W Muller-Esterl and KD Bhoola Updated information and services can be found at: http://www.bloodjournal.org/content/84/2/474.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From www.bloodjournal.org by guest on October 21, 2014. For personal use only. Assembly of Contact-Phase Factors on the Surface of the Human Neutrophil Membrane By Lydia M. Henderson, Carlos D. Figueroa, Werner Muller-Esterl, and Kanti D. Bhoola H-kininogen (HK), a major factor involved in contact-phase activation, was recently immunolocalized on the external surface of human neutrophils. Experiments were, therefore, designed to consider the question of whether the complete assembly of contact factors occurs on the outer surface of the neutrophil membrane. By immunolocalization techniques, and using specific antibodies directed against the various contact factors, we now demonstrate that plasma prekallikrein (PK), factor XI (FXI), and factor XI1 (FXII) are present on the exterior face ofthe human neutrophil. Failure to localize HK, PK, or FXI by monoclonal antibodies directed to their reciprocal binding sites, and displacement of PK/FXI by peptide HK31, which mimics the relevant binding site(s) of HK, suggested that prekallikrein and FXI are anchored to the neutrophil membrane through attachment to thekininogen molecule. Probing of the kinin moiety by a specific antibody showed that kininogen molecules bound to theneutrophil cell membrane contain the kinin sequence, which can be released by plasma kallikrein or by tissue kallikrein. Our results led us to the novel conclusion that neutrophils provide a circulating platform for the components of the contact-phase system. 0 1994 by The American Society of Hematology. T rected to the mutual binding sites of both PK and FXI on the HK molecule, and a MoAb to the kinin We show that the complete array of contact factors is sited on the surface of the neutrophil plasma membrane. Our results provide us with an insight into the possible involvement of the neutrophil-borne contact-phase system in the release of the vasoactive peptides, kinins, at the sites of inflammation. HE MAJOR CONSTITUENTS of the contact-phase activation system’ are plasma prekallikrein (PK), factor XI (FXI), factor XI1 (FXII), and H-kininogen (HK; highmolecular weight kininogen).’ They are synthesized and secreted by hepatocytes. In plasma, HK circulates as a complex with PK and FXI, re~pectively.~.~ This particular property is mediated by the light-chain segment of HK.s L-kininogen (LK), the low-molecular weight member of the kininogen family, is devoid of the specific light chain sequences that reside in HK.6 Recently, we immunolocalized HK and PK in human hepatocyte^,^ and HK and LK on human neutrophils.* Furthermore, HK binding to platelets,’”’ endothelial cell^,^^"^ and neutrophils’6 hasbeen demonstrated. These findings prompted the question whether all of the circulating contact-phase proteins were bound to the neutrophil surface, and, if so, what was the spatial relationship between these proteins (PK, FXI, FXII) to the HK molecule, known to be attached to the neutrophil cell membrane. To test the hypothesis that a circulating cell, namely, the neutrophil, might provide on its surface a platform for the assembly of the contact-phase proteins, we undertook a number of experiments using several immunolocalization techniques. Specific antibodies against the contact phase proteins were applied including monoclonal antibodies (MoAbs) diFrom the Departments of Biochemistry and Pharmacology, University of Bristol, UK; Institute of Histology and Pathology,Austral University, Vuldivia, Chile; Department of Pathobiochemistry, Institute of Physiological Chemistry, Universityof Mainz, Germany; and the Department of Experimental and Clinical Pharmacology, Medical School, University of Natal, Durban, South Africa. Submitted May 12, 1993; accepted March 15, 1994. Supported by grants fromthe Medical Research Council, the Arthritis and Rheumatism Council (UK), Fondo National de Desarrollo Cietijico y Technologico Direccion de Investigacion y Desarrollo, Universidud Austral de Chile (Chile), the Deutsche Forschungsgemeinschaft, the Volkswagen Foundation, and the Fonds der Chemischen Industrie (Germany). Address reprint requests to Kanti D. Bhoola, MD, PhD, Department of Experimental and Clinical Pharmacology, Medical School, University of Natal, PO Box 17039, Congella 4013, South Africa. The publication costsof this article were defrayedin part by page chargepayment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1994 by The American Society of Hematology. 0006-4971/94/8402-0003$3.00/0 474 MATERIALS AND METHODS Neutrophil isolation. Human neutrophils were isolated from anticoagulatedwhole blood provided by volunteersatTheMedical School, University of Bristol,England, UK andatTheRegional Hospital, Valdivia, Chile. Eighteen milliliters of blood, collected in 2 mLof105-mmol/Lsodiumcitrate,wasmixedwith 20 mLof dextran (6% wt/vol; average molecular weight, 266 kD and 60 rnL of phosphate-buffered saline (PBS; I O mmol/L sodium phosphate, 2.7 mmoW KCI, 137 mmol/L NaC1, pH 7.4) containing 0.4% wt/ v01 trisodium citrate. The mixture was left to stand for 45 minutes at room temperature to allowred blood cells to sediment. The supernatant was spun through Lymphoprep (Nycomed, Birmingham, UK) at 800s for 20 minutes. Remaining red blood cells were removed by two rounds of hypotonic lysis (erythrocyte lysis buffer: 155 mmol/ L NH,CI, 2.7 mmol/L KHCO,, 3.7 mmol/L EDTA, pH 7.4), and rebuffered to restore the osmolarity. The cells were centrifuged at 50013 for I O minutes and the pellet was washed three times in PBStrisodium citrate buffer. This procedure yielded a cell preparation that contained approximately 98% neutrophils; the remaining 2% of the cellsisolated by thistechniquecomprisedmainlyeosinophils and some monocytes, but no platelets ( < . l %). Failure to observe considerable superoxide production by the isolated neutrophils, and lack of a substantial degranulation of the cells indicated that our isolation procedures did not significantly activate the neutrophils. Theisolatedneutrophilswere floated ontocircularcoverslips in tandem, with one set left unfixed and the other fixed with 4% (wt/ vol) paraformaldehyde in PBS. Binding of kininogens I O isolated neutrophils. The amount and rate of loss of cell surface proteins by washing with buffer during cell isolation procedures depends on the nature and mechanism of attachment of the proteins to the cell surface, and is indicatedby the affinity of the protein forits binding site. In the present experiments, neutrophils were washed at4°C to minimize such a release. To judge the losses of neutrophil-bound proteins [‘251]-labeled HK” or LK (100%)were incubated for 45 minutes at 4°C with neutrophils. The cellswereextensivelywashed with coldHanks’buffer,andthe radioactivitybound to theneutrophilsdetermined by ay-counter (Packard, Dorset, UK). A fraction of 13% (1 8%) of ‘251-HK(LK) remained bound to the cells even after several washes. Cytoplast preparation. Cytoplasts, which are right-side out, single-compartment, enucleated cells” 22 were prepared by incubating Blood, VOI 84, N O 2 (July 15). 1994: pp 474-482 From www.bloodjournal.org by guest on October 21, 2014. For personal use only. CONTACTPHASEFACTORS ON HUMAN NEUTROPHILS 9 Fig 1. Localization of the components of the contact-phase systemon neutrophils. Nonfixed neutrophils were incubatedwith la) anti-HK [I-IMIdilution , 1:l.OOO). (b) anti-PK(AS176,1:1,OOO), (c) anti-FXI(I:l,OOO), or (d) anti-FXII (1:l.OOO). The immunostainedcells were imaged under the confocal optical scanning microscope. The intensity of immunoreactivity is indicated by the color strip: blue, nil; green, minimal; yellow, moderate; red, maximal. The barin each row isequivalent to 4 pm. neutrophilsat37°C for 10minutes in 12.5% ( d v o l ) Ficoll-70 (Sigma Chemical CO, Poole, Dorset,UK) containing 5 pg/mL cytochalasinB(Sigma), 5.5 mmovL glucose,beforeloadingontoa discontinuous Ficoll gradient (cushions of 12.5%,16%,and25% d v o l ) . Using a Ficoll-70 stepgradient, neutrophils were resolved into cytoplasts and karyoplasts. The cytoplasts were harvested from the12.5% and 16%Ficoll interfacefollowingcentrifugation at 81,OOOg for 60 minutes at 37OC in a prewarmed Sorvall centrifuge (Sorvall, Newtown, CT). Isolated cytoplasts were floated onto circular coverslips in tandem, with one set left unfixed, and the other fixed with 4% paraformaldehyde in PBS. Antibodies. Neutrophils were separately incubated with the followingantibodies:affinity-purifiedpolyclonalantibodies to HK, mouse MoAbs to the heavy chain (HKH4), from sheep (I-108);23 light chain (HKLIZ), and PK bindingsite (HKL16) of HK19 rabbit antiserum to PR (AS176); mouse MoAb to the HK binding site of PK (PK6);I8 mouse MoAb to bradykinin (SBK1); mouse MoAb to the HKbinding site of FXI (FEHl);" rabbitantiserum to FXk rabbit antiserumto Fxu,rabbit antiserum to atrial natriuretic peptide Fig 2. Ultrastructural double immunolabeling of HK and PK on (ANF, Peninsula Laboratories, Belmont, CA); rabbit antiserum to neutrophils. Affinity-purified antibodiesto HK (1-106,from sheep) preoxytocin (UCB Bioproducts, Brussels, Belgium); rabbit antiserum viously coupled to 15-nm gold particles(smallarrows), and antiserum to residues 389 to409 of the LK light chain (R7);= rabbit antiserum to PK IAS176. from rabbit) followed by goat anti-rabbit IgG coupled to tissue kallikrein (KDBl)." Note that the antibodies were elicited to 30-nm gold particles (large arrows) were applied (g, granule; N. nucleus. (Original magnification x 85,OOO.) to human antigen sequences throughout. From www.bloodjournal.org by guest on October 21, 2014. For personal use only. 47 6 Immunostaining for confocalmicroscopy. The isolated neutrophils (fixed or nonfixed) and cytoplasts (fixed) were washed three times with PBS (pH 7.4) containing 1% (wt/vol) bovine serum albumin (Sigma), 1% (wt/vol) human IgG (Blood Transfusion Service, Southmead Hospital, Bristol, UK), and 0.2% (wt/vol) sodium azide. Before immunostaining, isolated neutrophils or cytoplasts were incubated with 1% (wt/vol) of human IgG in PBS to exclude the nonspecific binding of antibodies to Fc receptors present on the neutrophil membrane. Sodium azide was included in all the washes to prevent internalization of the antibodies by the nonfixed neutrophils. Incubation with the primary antibody lasted for 3 hours. The cells were washed and then incubated for 30 minutes with fluorescein-isothiocyanate (FITC)-conjugated Fab2 fragments of antispecies IgG (diluted 1:140, vol/vol), (Sigma). The irnmunostained cells were observed with a confocal fluorescence microscope. To visualize intracellularly located antigens, fixed neutrophils were permeabilized by treatment with 0.2% (wt/vol) of Triton X-100 before to the primary antibody application. Confocal scanning laser microscopy. Neutrophils were observed under a confocal fluorescence microscope (Bio-Rad, Hemel Hempstead, Hertfordshire, UK) equipped with an excitatory argon laser. Confocal optical scanning microscopy permits noninvasive sectioning of cells, asit collects the emitted fluorescent light from withinthe focal plane ofthe objective lens. This property of the confocal microscope allows visualization of cell antigens by specific antibodies, within discrete regions of cells or tissue by a noninvasive optical sectioning of intact, fixed and nonfixedcells.2sIn the absence of the primary antibody, no fluorescent image was obtained from the cells. lmmunoelectronmicroscopy. The isolated, washed neutrophils were incubated with affinity-purified sheep anti-HK (1-108, in a dilution of 1:100, voYv01) previously coupled to 15-nm gold partiAfter incubation for 45 minutes at room temperature, the cells were washed with PBS, and then incubated for a further 45 minutes with rabbit anti-PK (AS176, in a dilution of 1:500, vol/vol). The neutrophils were washedwith PBS and incubated for 30 minutes with goat antirabbit IgG labeled with 30-nm gold particles (Amersham; Arlington Heights, IL) in a dilution of 1:4 (vol/vol). The cells were washed threetimes with PBS for 5 minutes each and fixed with 3% (vol/vol) glutaraldehyde in PBS for 1 hour at room temperature. Finally, the cells were postfixed with I%(vol/vol) osmium tetroxide and embedded in epon-araldite as previously described.24Ultrathin sections were counterstained with lead citrate and examined under a Philips EM-300 electron microscope (Philips, Eindhoven, Holland). Displacement of P U F X I from neutrophils. A synthetic peptide of 3 1 residues (HK3 1, positions 565 to 595 of the HK sequence), which covers the entire PK binding site of the HK light chain and overlaps the corresponding binding site for FXI,” was synthesized as previously described.” Isolated neutrophils were incubated for 15 minutes inthe absence or presence of 100 pg/mL of HK31 in PBS, pH 7.4, washed withthe same buffer, and subjected to imrnunostaining. Kinin releasefromneutrophil-boundkininogens. Isolated human neutrophils were resuspended inPBS containing 5 mmol/L glucose, and tissue kallikrein or plasma kallikrein was added to a final concentration of 100 ng/mL. For control, cells were incubated without enzyme. Aliquots from the incubation mixture were taken at 0-, 15-, and 30-minute intervals, The cells were spun rapidly in an Eppendorf centrifuge, fixed, and immunostained with an antibradykinin antibody (SBK 1). Immunocytochemicalcontrols. Controls included replacement of the primary antibody by nonimmune serum or IgG of the same species, also by omission of the primary antibody, and by the use of nonkinin peptide antibodies (anti-oxytocin, anti-ANF). Additional controls were prepared by preabsorption of theprimary antibody with 50 p.g/rnL of the respective purified antigen (HK, LK,PK, HENDERSON ET AL Table 1. Differential Staining of Site-Directed MoAbs Neutrophils Antibodv Nonfixed Fixed FEHI HKH4 HKL12 - f Abbreviations: -, staining. + +S + + no staining; +, weak staining; ++, moderate tissue kallikrein, bradykinin, ANF, oxytocin). For immunoelectron microscopy, omission or replacement of rabbit anti-PK by nonimmune rabbit serum resulted in the absence of a specific gold labeling of the neutrophil membrane (not illustrated). RESULTS Conventional light microscopy is limited in its ability to demonstrate many of the details of cellular structure. We therefore used thenew powerful imaging technique of confocal scanning laser microscopy applying FITC-labeled F(ab)z secondary antibodies, with the pseudocolor gradient from blue(nil)tored(maximal)providinganestimateofthe amount of antigen on the cell. A sheep polyclonal anti-HK antibody directed exclusively against the light chain portion of HK gave positive staining circular pattern of onnonfixedneutrophils(Figla).The staining at or near the midoptical section of the cells was HK on the outer surface of indicative of the presence of the neutrophil membrane. A distinct annular localization of labeling was also obtained when nonfixed neutrophils were incubated with the rabbit polyclonal anti-PK antibody (Fig lb). In the absence of the primary antibody, no fluorescent cells (data not shown), thus image was obtained from the demonstrating the specificity of the immunolocalization of HK and PK on neutrophils. The results are consistent with thefindingthat a fraction(13%)ofexogenouslyapplied radiolabeled HK remained bound to the neutrophils even after several washes with Hanks’ buffer (see Materials and Methods) similar results have been reported for cell adhesion molecules exposed on the surface of the neutrophil^.^^^^* Thenextquestionweaddressedwaswhethertheremaining factors of the contact-phase assembly, namely FXII and FXI, also reside on the neutrophil membrane. Both proteins immunolocalized on the nonfixed neutrophil, indicating attachment of these coagulation factors to the external surface of theneutrophilcellmembrane(FigICandd).On electronmicrographs,immunoreactiveHKlocalizedas a cluster of gold particles restricted to the external surface of the neutrophil cell membrane (not shown). Ultrastructural, double immunolabeling of HK and PK confirmed the coexistence of the enzyme and its substrate sequestered on the external surface of the neutrophil plasma membrane (Fig 2 ) . Because HK and PK circulate in plasma as a complex, the colocalization of both these proteins on the external surface of nonfixed neutrophils prompted the question: is PK a kininogenbridgeor is linkedtotheneutrophilthrough bound directly to a recipient site on the neutrophil membrane? This question was investigated using two site-directed antibodies: (1) antibody HKL16 directed to the PK binding From www.bloodjournal.org by guest on October 21, 2014. For personal use only. CONTACTPHASEFACTORS 477 ON HUMAN NEUTROPHILS I. : , -.; c r- '3d U Fig 3. Contact-phase factors on the surface of neutrophils probed by binding site-directed entibodiw. (a) Nonfixed neutrophils, MoAb antibody HKL16 directed to the PK binding site of HK; (b) nonfixed neutrophils; mouse MoAb PK6 directed to the HK binding site of P& (c) fixed neutrophils, HKLl6; (dl fixed neutrophils, PK6; (e) fixed cytoplasts, HKL16; (R fixed cytoplasts, PK6. Antibodies (from escitas] were epplied in a dilution of 1:lOO. The lntendty of immunoreactivity is indicated by the color strip: blue, nil; green, minimal; yellow, moderate; red, maximal, The bar in each row isequivalent to 4 pm. site of the HK light chain (positions 569 to 595), and (2) antibody PK6 directed at the HK binding region of the heavy chain of PK. Our attempts failed to detect HK with HKL16 (Fig 3a) or PK with PK6 (Fig 3b) on nonfixed neutrophils, whereas the location of HK and PK on the plasma membrane of noniixed cells had been demonstrated with the polyclonal antibodies 1-108 (HK) and AS176 (PK), respectively (see Fig l a and b). Together, these results may suggest that the accessfor HKL16 and PK6 totheirrespectiveepitopes, which represent the mutual binding sitesof HK and PK, is From www.bloodjournal.org by guest on October 21, 2014. For personal use only. 470 HENDERSON ET AL I Fig 4. Displacement of PK and FXI from HK by peptide HK31. Isolated neutrophils were incubated for 15 minutes in the absence (a, c) or presence (b. d)oflWpg/mLofHK3l.Atthoendoftheincubation period, the nonfixed cells were immunostainedwith (a, b) anti-PK(AS176; 1:.lOOO) or (c, d) anti-FXI (l:l,OW), andimagedunder the confocal microscope. The intensity of immunoreactivity is indicated by the color strip: blue, nil; green, minimal; yellow, moderate;red,maximal. The bar in each row is equivalent to 4 Cm. I- Fig 7. Kinin liberation from neutrophil-bound kininogens followed by confocal microscopy.Isolated neutrophils were incubated with (ad) plasma kallikrein or (e-h) tissuekallikrein for 0 (a, e), 15 (b. f),and 30 minutes (c,g). Immunostaining was done with antibradykinin (SBKl, 1:lW) followed by FITGlabeled (Fab); fragments of goat antimouse IgG [1:140), and confocal microscopy. For controls, the first antibody was omitted (d) or replaced by an antibody t o an unrelated antigen (h). The intensity of immunoreactivity is indicated by the color strip: blue, nil; green, minimal; yellow, moderate;red, equivalent t o lpm. 0 maximal. The bar in each row is L m From www.bloodjournal.org by guest on October 21, 2014. For personal use only. CONTACTPHASEFACTORS ON HUMAN NEUTROPHILS 479 I Fig 6. Probing for the kinin moiety of neutrophil-bound kininogens. Nonfixed cells were incubated with (a) MoAb to bradykinin (SBK1,1:1001 or lb) antiserum to oxytocin (1:100l. The immuno stained cells were analyzed by confocal microscopy.The intensity of immunoreactivity is indicated bythe color strip: blue, nil; green, minimal; yellow, moderate; red, maximal. The bar in aach row is equivalent to 4 pm. these site-specific antibodies. Demonstration of the immunoreactivity of both m 1 6 and PK6 on fixed neutrophils (Fig 3c and d) and on fixed cytoplasts (Fig 3e and f) provides further evidence of the membrane localization of HK and PK on the neutrophil and shows that the relevant antigenic Fig 5. Specificity of the cellular localization probed by anti-tissue epitopes are exposed upon fixationof the cells. Cells were incubatedwith (a) antitissue kallikallikrein and anti". To furtheranalyze this phenomenon,weappliedtwo krein lnonfixed cells, KDB1,l:l,OOO), (b) antitirue kallikrein (fixed MoAbs, m 4 and HKL12, which bind to well-defined epipermeabilizedcells, KDB1, l:l,OOO), or (c) anti-u (nonfixed cells,R7. 1:l.OOO). and examined under the confocal microscope. The intensity topes ofHK that are distinct from PK thebinding region, and of immunoreectivii is indicated bythe color strip: blue, nil; green, tested for their differential binding to nonfixed neutrophils. A minimal; yellow, moderate; red, maximal. The bar in each row is weak, though distinct, immunostaining of nonfixed neutroequivalent to 4 pm. phils, and a positive staining after fixation of the cells was observed (Table l), indicating that epitopes other than the blocked on nonfixed neutrophils by the complex formation PWHK binding sites are readily accessible on kininogens between the PK heavy chain and the HK light chain. To bound to nonfixedneutrophils. In a similarapproach,the spatial relationship between FXI and HK was probed with address this question, we first fixed the cells to allow dissociation and random cross-linking of the complex components MoAb FEHl directed to the HK binding site on FXI. Fixed to the cell structures, and probed the individual proteins with or nonfixed neutrophils were incubated with FEHl in a dilu- From www.bloodjournal.org by guest on October 21, 2014. For personal use only. 480 tion 1:100,and imaged on the confocal microscope. No immunostaining was observed on nonfixed neutrophils, indicating that the access of FEHl to the binding site is blocked, whereas after fixation the binding site became accessible to the antibody (Table 1). These results are compatible with the notion that PK and FXI are present on the neutrophil surface in form of complexes with HK, which dissociate under the conditions of our fixation procedure. To further explore the anchor sites of PK and FXI on the neutrophil surface we determined whether either of these proteins could be displaced by synthetic peptide HK3 1, a molecular surrogate of the PK/FXI binding site(s) on HK. Isolated neutrophils were incubated for 15 minutes with or without 100 pg/mL of HK31. At the end of the incubation period, the cells were immunostained with antikallikrein (AS176) or anti-FXI antiserum, and imaged on the confocal microscope (Fig 4a and c, controls). Inclusion of peptide HK3 1 effectively displaced PK and FXI from their HK binding site as evidenced by the almost complete absence of staining (Fig 4b and d). Our experiments with peptides that mimic the binding sites for PK and FXI on HK, and with antibodies directed to these sites support the view that PK and FXI are anchored to the outer surface of the neutrophil via HK. To show the specificity of our approach, we localized an enzyme of the kinin system, tissue kallikrein, which is located in intracellular stores of the neutrophil cell.” In nonfixed cells, with the plasma membrane intact, and the access of antibodies to the inside of the cells blocked, no immunostaining was observed with antitissue kallikrein (Fig 5a). When neutrophils were fixed and permeabilized by the use of a nonionic detergent (0.2%Triton X-100 in PBS), tissue kallikrein localized within the neutrophil (Fig 5b). In contrast, the preferred substrate of tissue kallikrein, LK was localized on the outer surface of the intact neutrophil membrane using a specific antiserum directed to the unique light chain portion ofLK (Fig 5c). Therefore, access of tissue kallikrein to LK or HK can only occur when the neutrophil either degranulates or secretes tissue kallikrein in response to stimuli. It was also important to determine whether the kininogens on the isolated, but nonstimulated, neutrophils contain their kinin moiety, thereby providing an endogenous substrate for the enzymic action of the kallikreins after neutrophil activation or degranulation. Using a monoclonal antibradykinin antibody (SBKl), we obtained annular localization patterns (Fig 6a), indicative of the presence of the kinin moiety in the kininogen domain D4. In control experiments, antisera against the unrelated peptides, oxytocin or ANF, failed to produce staining (exemplified for anti-oxytocin, Fig 6b). Hence, at least a fraction, if not all of the neutrophil-bound kininogen, is present in the native, kinin-containing form. The ability of plasma kallikrein and tissue kallikrein to release kinin from kininogens on the neutrophil surface was examined by incubating human neutrophils for 30 minutes at 37°C with either tissue kallikrein or plasma kallikrein at a concentration of 100 ng/mL. Aliquots of the cell suspensions were taken at 0-, 1 5 , and 30-minute intervals. The cells were recovered by centrifugation and immunostained with a monoclonal antibradykinin antibody, SBK1. Cells treated HENDERSON ET AL with plasma kallikrein showed a considerable loss of staining by 15 minutes, which seemed to be complete by 30 minutes (Fig 7a, b, and c), whereas the response time to tissue kallikrein was longer with loss of staining commencing at 30 minutes (Fig 7e, f, and g). Antibody controls were done in the absence of the first antibody (Fig 7d) or in the presence of an unrelated first antibody (Fig 7h); controls where cells had been maintained for 30 minutes in buffer only did not show significant changes in the staining patterns compared with the samples withdrawn at zero time (not shown). These results demonstrate that the kinin moietyof neutrophil-bound kininogens is readily accessible for the kallikreins, and at least a major part of the available kinin is released upon exposure of the cells to active kallikreins. The presently available antibody probes for kinins do not distinguish between the precursor protein holding the kinin sequence. Furthermore, in purified systems, plasma kallikrein and tissue kallikrein each cleave the two types of kininogens though at different rates (not shown). Hence, we are unable to discriminate between the kininogen sources that deliver kinins on the neutrophil surface. DISCUSSION This study demonstrates that all the essential components of the contact-phase system assemble on the surface of the neutrophil cell membrane. Our data suggest that HK and FXII bind to recipient sites exposed on the external face of the neutrophil membrane, whereas PK is secured to the cell surface indirectly through its docking protein, HK. Likewise FXI may be tethered through HK to the neutrophil surface. Hence, an array of functional proteins is assembled on the surface of neutrophils, thereby providing a unique circulating platform (solid phase) for the contact activation system. A similar assembly has been reported for cultured human endothelial cells,29implying that immobile platforms for the contact system may exist as well. Our observation of a cell-bound contact system on neutrophils suggests the involvement of a novel mechanism for the formation of kinins from kininogens. The sequence of events for such a phenomenon would include the discrete and circumscribed formation of kinins (bradykinin, kallidin) from HK and/or LK on the surface of the neutrophil either by the FXII-mediated activation of PK, and/or by the release of Sic Fig 8. Assemblyof the contact-phase fectors on the neutrophil surface. 82. bradykinin B2 receptor. Dotted lines indicate the poltuIated contraction of endothelial cells following stimulation of their B2 receptor by kinin. From www.bloodjournal.org by guest on October 21, 2014. For personal use only. CONTACTPHASEFACTORS ONHUMAN NEUTROPHILS weight kininogen on stimulated washed human platelets. Biochemisneutrophil-borne tissue kallikrein in its active form. We protry 23:6863, 1984 pose that the locally released kinin may, in a paracrine man11. Gustafson El, Schutsky D, Knight LC, Schmaier AH: High ner, enhance the passage of the neutrophils into the extracelmolecular weight kininogen binds to unstimulated platelets. J Clin lular space by causing the endothelial cells to retract (Fig Invest 78:310, 1986 8). Such a mechanism permits the transudation of plasma 12. Schmaier AH, Smith PM, Purdon AD, White JG, Colman content by controlling vascular permeability, and the passage RW: High molecular weight kininogen. Localization in the unstimuof circulating neutrophils into the interstitial tissue space lated and activated platelet and activation by a platelet calpain(s). surrounding the site of injury or inflammati~n.~' Our notion Blood 67:119, 1986 is compatible with the long-known effects of kinins in the 13. Schmaier AH,KuoA, Lundberg D, Murray S , Cines DB: microcir~ulation.~' The expression of high molecular weight kininogen on human umbilFurthermore, kinins generated by the neutrophil-borne ical vein endothelial cells. J Biol Chem 263:16327, 1988 contact system might act in an autocrine manner. This hy14. Van Iwaarden F, de Groot PG, Sixma JJ, Berrettini M, Bouma pothesis is consistent withthe finding that activated PK BN: High molecular weight kininogen is present in cultured human causes the release of elastase from neutrophil^.^^ Our data endothelial cells: localization, isolation and characterization. Blood shed a new light on the observation that the response of 71:1268, 1988 neutrophils to a degraded form of kallikrein, p-kallikrein, 15. Van Iwaarden F, de Groot PG, Bouma BN: The binding of lacking the HK binding properties is markedly a t t e n ~ a t e d . ~ ~high molecular weight kininogen to cultured human endothelial cells. In essence, our findings support the previous conclusion that J Biol Chem 263:4698, 1988 attachment to HK is a major requirement for the observed 16. Gustafson EJ, Schmaier AH, Wachtfogel YT, Kaufman N, effect of plasma kallikrein on neutrophil^.^^ Clearly, addiKucich U, Colman RW: Human neutrophils contain and bind high molecular weight kininogen. J Clin Invest 84:28, 1989 tional experiments are needed to provide further evidence 17. Henvald H, Jahnen-Dechent W, Abd Alla S , Hock H, Bouma for these intriguing suggestions. To this end, studies on the BN, Muller-Ester1 W:Mapping of the high molecular weight kininoimmunolocalization of the bradykinin B2 receptor on neutrogen binding site of prekallikrein. Evidence for a discontinuous epiphils and endothelial cells are being undertaken. ACKNOWLEDGMENT We are indebted to Profs B. Chappell and 0. Jones (Bristol) for the use of the Confocal Optical Scanning Microscope, funded by the Wellcome Trust. We thank Drs B. Bouma (Utrecht) for a gift of antibody FEHI, M. Phillips (London) for antibody SBK1, J. Hock (Marburg) for antiserum to FXI, and A. Schmaier (Ann Arbor) for antibody to FXII. We also thank C. Jeal for preparing the color montages. REFERENCES 1. Furie B, Furie BC: The molecular basis of blood coagulation. Cell 53:505, 1988 2. Kaplan AP, Silverberg M: The coagulation-kinin pathway of human plasma blood. Blood 70:1, 1987 3. Mandle R Jr, Colman RW, Kaplan AP: Identification of prekallikrein and HMW kininogen as a circulating complex inhuman plasma. Proc Natl Acad Sci USA 73:4179, 1976 4. Thompson RE, Mandle R Jr, and Kaplan A P Association of factor XI and high molecular weight kininogen in human plasma. J Clin Invest 60:1376, 1977 5. Thompson RE, Mandle R Jr, and Kaplan AP: Studies of binding of prekallikrein and factor XI to high molecular weight kininogen and its light chain. Proc Natl Acad Sci USA 79:4862, 1979 6. Muller-Ester1 W: Kininogens, kinins and kinships. Thromb Haemost 61:2, 1989 7. Henderson LM, Figueroa CD, Muller-Ester1 W, Stain A, Bhoola KD: Immunovisualisation of plasma prekallikrein andHkininogen on human neutrophils and hepatocytes. Agents Actions 3811590, 1992 8. Figueroa CD, Henderson LM, Colman RW, De La Cadena RA, Muller-Ester1W, Bhoola KD: Immunoreactive H- and L-kininogens in human neutrophils. J Physiol (Lond) 425:65P, 1990 9. Schmaier AH, Zuckerberg A, Silverman C, Kuchibhotla J, Tuszynski GP, Colman RW: High molecular weight kininogen: A secreted platelet protein. J Clin Invest 71:1477, 1983 10. Greengard JS, Griffin JH: Receptors for high molecular tope formed by distinct segments of the prekallikrein heavy chain. J Biol Chem 268:14527, 1993 18. Hock J, Vogel R, Linke RP, Muller-Ester1W: High molecular weight kininogen-binding site of prekallikrein probed by monoclonal antibodies. J Biol Chem 265:12005, 1990 19. Kaufmann J, Haasemann M, Modrow S , Muller-Ester1 W: Structural dissection of multidomain kininogens. Fine mapping of the target epitopes of antibodies interfering with their functional properties. J Biol Chem 268:9079, 1993 20. Haasemann M, Buschko J, Faussner A, Roscher AA, Hoebeke J, Burch R, Muller-Ester1 W: Anti-idiotypic antibodies bearing the internal image of a bradykinin epitope: Production, characterization, and interaction with the kinin receptor. J Immunol 147:3882, 1991 21. Roos D, VoetmanAA,MeerhofLJ: Functional activity of enucleated human polymorphonuclear leucocytes. Cell Biol 97:368, 1983 22. Henderson LM, Chappell JB, Jones OTG: The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H' channel. Biochem J 246:325, 1987 23. Figueroa CD, Henderson LM, Kaufmann J, De La Cadena RA, Colman RW, Muller-Ester1 W, Bhoola KD: Immunovisualisation of high (HK) and low (LK) molecular weight kininogens on isolated human neutrophils. Blood 79:754, 1992 24. Figueroa CD, MacIver AG, Bhoola KD: Identification of immunoreactive tissue kallikrein in human polymorphonuclear leucocytes. Br J Haematol 72:321, 1989 25. Shotton D, White N: Confocal scanning microscopy: Threedimensional biological imaging. Trends Biochem Sci 14:435, 1989 26. Roth J: The colloidal gold marker system for light electron microscopic cytochemistry, in Bullock GR, Petrusz P (eds): Immunocytochemistry, v012. London, UK, Academic, 1983, p 218 27. Bevilacqua MP, Stengelin S , Gimbrone MA Jr, Seed B: Endothelial leucocyte adhesion molecule 1: An inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science 243: 1160, 1989 28. Wallis WJ, Hickstein DD, Schwartz BR, June CH, Ochs HD, Beatty PG, Klebanoff SJ, Harlan JM: Monoclonal antibody-defined From www.bloodjournal.org by guest on October 21, 2014. For personal use only. 402 functional epitopes on the adhesion-promoting glycoprotein complex (CDwl8) of human neutrophils. Blood 67:1007, 1986 29. Berrettini M, Schleef RR, Heeb MJ, Hopmeier P, Griffin JH: Assembly and expression of an intrinsic factor IX activator complex on the surface of cultured human endothelial cells. .lBiol Chem 267:19833, 1992 30. Wright DG, Gallin JI: Secretory response of human neutrophils: Exocytosis of specific (secondary) granules by human neutrophils during adherence in vitro and during exudation in vivo. J Immunol I23:285, 1979 HENDERSON ET AL 31. Majno E, Shea SM, Seventhal M: Endothelial contraction induced by histamine type mediators: an electron microscopy study. J Cell Biol 42:647, 1969 32. Wachtfogel YT, Kucich U, James HL, Scott CF, Schapira M, Zimmerman M, Cohen AB, Colman R W Human plasma kallikrein releases neutrophil elastase during blood coagulation. J Clin Invest 72:1672, 1983 33. Colman RW, Wachtfogel YT, Kucich U, Weinbaum G, Hahn S, Pixley RA, Scott CF. de Agostini A, Burger D, Schapira M: Effect of cleavage of the heavy chain of human plasma kallikrein on its functional properties. Blood 65:311, 1985
© Copyright 2024