Modulation of Polymorphonuclear Leukocyte Microbicidal Activity and OxidativeMetabolism by Fibrinogen Degradation Products D and EJames W. Kazura, Jay D. Wenger, Robert A. Salata, Andre Z. Budzynski, and George H. GoldsmithDivisions of Geographic Medicine and Hematology/Oncology, Department of Medicine, University Hospitals of Cleveland, andCase Western Reserve University, Cleveland, Ohio 44106; and Department of Biochemistry,Temple University School of Medicine, Philadelphia, Pennsylvania 19140

AbstractFibrinogen degradation products (FDP) D and E are typicallypresent in blood of patients with disseminated intravascularcoagulation and related conditions in which granulocyte(PMN) defense against bacterial infection may be compro-mised. This study was intended to determine whether FDPmodify PMNfunctions critical to their bactericidal activity.Incubation of human PMNand Escherichia coli with 50-100Mg/ml FDPdid not affect phagocytosis, but reduced by > 90%the cells' ability to inhibit bacterial colony growth comparedwith control PMNincubated with albumin or fibrinogen. FDP(10-100 gg/ml) inhibited PMNO2 release and chemotaxisstimulated by FMLP by 17-50% (P < 0.005) and 41% (P< 0.01), respectively. Fragment E3, and not fragment D1, wasprimarily responsible for inhibition of FMLP-induced PMNO2 release. Phorbol myristate acetate (10 ng/ml), 1-oleoyl-2-acetylglycerol (10-6 M), AA (4.2 X 10- M), and zymosan-ac-tivated serum-stimulated PMNQ2 release were also de-creased 37-63% by FDPcompared with control protein. Thereare at least two mechanisms by which FDPmay impair PMNresponses. With respect to FMLP, FDP (16-100 ,ug/ml) in-hibited specific binding to the cell surface over a ligand con-centration range of 1.4-85 nM [3HlFMLP. In contrast, FDPdid not effect the extent of phorbol ester binding to PMNbutblocked activation of protein kinase C. These data suggest thatelevated plasma FDPinhibit several PMNfunctions critical tothe bactericidal role of these inflammatory cells.

Introduction

Fibrinogen (Fbg)', fibrin, and degradation products of theseplasma proteins (FDP) have important effects on several

This work was presented in part in abstract form (1984. Clin. Res.32:31 1).

Address reprint requests to Dr. James W. Kazura, Department ofMedicine, Division of Geographic Medicine, University Hospitals ofCleveland, Cleveland, OH44106.

Receivedfor publication 22 February 1988 and in revisedform 15February 1989.

1. Abbreviations used in this paper: DIC, disseminated intravascularcoagulation; Fbg, fibrinogen; FDP, fibrinogen degradation products;KGEP, homogenizing buffer consisting of 139 mMpotassium gluta-mate, 20 mMPipes, pH 6.6, 5 mMEGTA, 5 mg/ml BSA, and 50.ug/ml leupeptin; OAG,' l-oleoyl-2-acetylglycerol; PDBU, phorbol-12,13-dibutyrate; PKC, protein kinase C; PMA, phorbol myristateacetate; ZAS, zymosan activated serum.

aspects of inflammation. Perhaps most thoroughly studied istheir role in platelet activation. In many circumstances, occu-pancy of platelet Fbg receptors with intact Fbg precedes plate-let secretion and aggregation which support normal hemostasis(1-3). Elevation in plasma FDP, such as in disseminated intra-vascular coagulation (DIC) or adult respiratory distress syn-drome, may inhibit the interaction between platelet Fbg re-ceptors and Fbg and contribute directly to platelet dysfunctionand bleeding diathesis (4-6). Several studies suggest that FDPmay also have deleterious effects on other cells involved inacute inflammation. Fragment D, a - 100-kD plasmin-gen-erated FDP, induces cytoskeletal alterations and eventual re-traction of venous endothelial cells in vitro (7, 8). Fragment E,a - 50-kD FDP, competes with Fbg for fibroblast Fbg bindingsites and inhibits directed migration of these cells in a Fbgconcentration gradient (9, 10). Low molecular weight Fbg deg-radation products have been shown to decrease lymphocyteblastogenesis (1 1, 12). With regard to PMN, various low mo-lecular weight (< 20 kD) polypeptides derived from Fbg havebeen found to induce chemotaxis (13). Possible PMNchemo-tactic activity of fragment D has also been reported (14, 15).Human fibrinopeptide B has chemotactic activity for PMNinpotency equivalent to that of O~a, leukotriene B4, and FMLP(16). These observations suggest that increases in the plasmaconcentration of various Fbg fragments modify the inflamma-tory response by virtue of their effects on multiple cellularcomponents of blood. Because clinical situations in which DICoccurs are commonly associated with severe bacterial infec-tions and neutropenia (17), we initiated studies to define moreclearly the effects of FDPand related activated coagulant pro-teins on PMNfunctions related to host defense against bac-teria.

Methods

Preparation of Fbg and FDP. Lyophilized human Fbg (Imco, Stock-holm, Sweden) was dissolved in distilled water and dialyzed against0.01 MPBS, pH 7, for 18 h at 4VC. Dialyzed Fbg was stored at -20'Cuntil use. To prepare FDP, Fbg was incubated with plasmin (0.0025mg/ml final concentration) at 370C for 1-3 h. Proteolysis was stoppedby addition of tosyllysyl chloromethyl ketone and phenylalanylarginylchloromethyl ketone (Sigma Chemical Co., St. Louis, MO), followedby dialysis at 4VC against PBSand sodium azide (18). Mixed Dand Efragments were separated from lower molecular weight degradationproducts by passage of the preparations over a Sephadex G150 column(80 X 2.5 cm). FDPwas dialyzed against PBSbefore use. The presenceof D and E fragments was verified by SDS-PAGErun under nonre-ducing conditions. These preparations displayed characteristic bandsof 80-100 (fragments D1,2,3) and 50 kD (fragment E), and minor com-ponents between 50 and 100 kD after staining with Coomassie blue(Fig. 1) (18-20). In studies directed at defining the species of FDPthataffect FMLP-stimulated PMNO° release, fragments DI and E3 wereseparated and purified from plasmin-digested Fbg by Pevikon block

1916 Kazura, Wenger, Salata, Budzynski, and Goldsmith

J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/89/06/1916/09 $2.00Volume 83, June 1989, 1916-1924

100IkD -

5OkD -

Figure 1. SDS-PAGEof FDP. 20 Mg of FDPwere run on a 10% SDS-PAGEand stained with

~ Coomassie blue.

electrophoresis as described (21). The homogeneity and lack of cross-contamination between preparations of fragments DI and E3 were

confirmed by PAGE. Humanplasmin and thrombin were prepared bystandard methods (22). Protein concentrations were measured by themethod of Lowry et al. (23).

Cells. PMNof healthy adults were prepared from peripheral bloodanticoagulated with 1 U/ml heparin by centrifugation through Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ) followed by sedi-mentation in 3%dextran in 0.15 MNaCl (24). Contaminating erythro-cytes were removed by hypotonic lysis. These preparations contained95-98% neutrophils and 2-5% eosinophils as assessed by microscopicinspection of Wright's-Giemsa-stained cytocentrifuge preparations.Viability was > 98% as assessed by trypan blue exclusion.

PMNbactericidal activity. To assess bactericidal activity of PMNin the presence of BSA, Fbg, or FDP, a modification of the methodsdescribed by Steigbigel et al. (25) and Mandell (26) was used. Esche-richia coli cultured in trypticase soy broth were washed in 0.15 MNaCIthen incubated in HBSSand 10% normal human serum at 370C for 30min. After washing three times in HBSS, E. coli density was assessed byabsorbance in a spectrophotometer. E. coli (1 X 10') and PMN(5X 106) were mixed in 0.7 ml HBSScontaining 50% vol/vol heat-inac-tivated FCS/HBSS/gelatin (Difco Laboratories Inc., Detroit, MI). 100

Ml of heat-inactivated (560C for 1 h) normal human serum and 0.1%vol/vol gelatin containing 50-100 Mg/ml BSA, Fbg, or FDPwere thenadded to triplicate tubes. PMN-bacterial mixtures were incubated at370C for 15 min with constant rotation. Extracellular organisms were

killed by addition of 100 U/ml of penicillin and 100 Mg/ml of strepto-mycin (Gibco Laboratories, Grand Island, NY) with continued rota-tion for 2 h. 200-Ml aliquots from each tube were then removed, washedin 0.15 MNaCl, PMNsonicated, and diluted 1:1,000 in HBSS. Tripli-cate I00-Ml aliquots of the diluted sonicates were plated on blood agarand bacterial colonies counted after incubation at 37°C for 24 h.

PMNphagocytosis. 0.5 ml blood was placed on a coverslip andallowed to incubate for 90 min at 37°C in a humidified chamber. Theformed clot was rinsed off the coverslip with 0.9% NaCI and I07 E. coliin 0.5 ml NaCl containing either 100 Mg/ml BSA or FDP. Duplicatecoverslips were incubated with added bacteria for 10, 20, or 30 minbefore washing with saline and fixation with 100% methanol. Phagocy-tosis was quantified in two ways: (a) the proportion of PMNwithingested bacteria was determined by microscopic inspection ofGiemsa-stained smears; and (b) the number of ingested bacteria perPMNwas calculated from the total number of E. coli counted in 100PMN(27).

Superoxide production. Superoxide production was measured bySOD-inhibitable cytochrome c reduction (28). PMNand cytochromesolution (1.2 mgcytochrome c/mi HBSS) were added to glass cuvetteswith a final concentration of 1 X 106 PMN/ml HBSS. The cells were

incubated for 2 min with 10-100 Mg/ml FDP or control BSA

(< 0.005% fatty acids) at 37-C (or other proteins listed in Results).FMLP, phorbol myristate acetate (PMA), AA, l-oleoyl-2-acetylglyc-erol (OAG) diluted first in DMSOthen PBS, zymosan-activated serum(containing C5a; ZAS) (29), or opsonized zymosan (30) (a phagocyticstimulus), were then added. Absorbance at 550 nmof triplicate sam-ples was measured at 1-min intervals unless otherwise indicated.FMLP, OAG, AA, PMA, cytochrome c, and BSAwere purchased fromSigma Chemical Co. In some experiments, stimuli were added to PMNsuspensions 1.5 or 5.5 min after BSA, Fbg, or FDP. Preliminary ex-periments using acetaldehyde-xanthine oxidase to generate reducedoxygen products (31) indicated that the amount of O° detected by thecytochrome c reduction method was similar in buffer containing 100Ag/ml BSA, Fbg, or FDP.

Chemotaxis. A modification of the chemotaxis under agarose assay(32) was used to assess chemotactic responses of PMNto FMLPin thepresence of FDP, Fbg, or BSA. A layer of 0.6% wt/vol agarose waspoured into plastic petri dishes and a circular array of wells cut into thegel using a standard template. PMNand either FDP or BSA (100Ag/ml) were placed in a middle well. FMLP (1 X 10-6 M) or HBSSalone was then added to opposite outer wells. After 2 h incubation at370C in 5%CO2 in air, the agar dishes were fixed with methanol andformalin, dried, and stained with Wright's-Giemsa for microscopicinspection. The chemotactic index was calculated by dividing the lin-ear distance of the leading edge of PMNmoving from the center welltoward the outer well containing FMLPby the linear distance of PMNtraveling toward the control well containing buffer alone.

Measurements of FMLPand phorbol ester binding to PMN. In theinitial series of experiments PMNbinding of FMLPwas measured asdescribed previously (33). Briefly, triplicate aliquots of 5 X 106 PMNin0.5 ml HBSSwere preincubated for 2 min with 100 Mg/ml Fbg, BSA, orFDP at 250C. 1.7 X 10-9 M [3H]FMLP (specific activity 48.3 Ci/mmol; NewEngland Nuclear, Boston, MA) with or without 1,000-foldexcess unlabeled FMLPwas added and incubation continued for 30min. PMNsuspensions were then layered on silicone oil (PC550;Fluka AG, Ronkonkoma, NY) contained in plastic tubes and cellpellets obtained by centrifugation (10,000 g) for 2 min. The bottom ofthe tube containing the cell pellet was cut off, placed in liquid scintil-lation counting fluid, and bound counts per minute determined bybeta spectrometry. Specific binding of FMLPwas calculated by sub-traction of counts per minute bound in the presence of excess unla-beled FMLPfrom counts per minute in the absence of excess FMLP.Additional experiments were performed to examine whether FDPpreferentially altered binding to high or low affinity PMNFMLPre-ceptors. Internalization of chemotactic peptide receptors and FDPbyPMNwas minimized by carrying out 30-min incubations at 4°C inHBSScontaining 10 mMsodium azide and 10 mM2-deoxyglucose(Sigma Chemical Co.) (34). 1.4-85 nM [3H]FMLP (60.0 Ci/mmol;Amersham Corp., Arlington Heights, IL) was incubated with triplicatesamples of 2.5 X 106 PMNin the presence of either 100 jsg/ml BSAorFDP. A 1,000-fold excess of unlabeled FMLPwas added to determinenonspecific binding. Based on previous studies (35, 36), a two-sitesaturable model for FMLPreceptors was assumed. Binding data wereanalyzed using NONLIN, a weighted nonlinear least squares regres-sion computer program (37). To examine the concentration depen-dence of the possible effect of FDPon PMNFMLPbinding, a similarassay was set up with 21 nM [3H]FMLP as input and FDPconcentra-tions ranging from 16 to 100 ug/ml.

Phorbol ester binding to PMNwas quantified by measurements ofspecific binding of [3H]phorbol-12,13-dibutyrate (PDBU) (38). PMN(1 X 106) were suspended in 200 Ml HBSSat 25°C in plastic microtiterwells containing either 100 Mg/ml BSAor 100 Mg/ml FDP. 2 X lI-' M[3H]PDBU (18.9 Ci/mmol; NewEngland Nuclear) in the presence orabsence of 5 X l0-5 Munlabeled PMAwas added and incubationcontinued for 20 min at 37°C. PMNin triplicate wells were thenharvested on to glass filter paper and specific binding of [3H]PDBUdetermined.

Binding of fluid-phase ligands to FDP and BSA. To determinewhether FMLP, phorbol esters, or AAbound differentially to FDPvs.

Fibrinogen Degradation Products Inhibit Neutrophil Function 1917

BSA, 1.7 X 10-9 M[3H]FMLP (48.3 Ci/mmol), 2 X 10-7 M[3H]PDBU(18.9 Ci/mmol), or 8.2 X 10-7 M[13H]AA (191 Ci/mmol; AmershamCorp.) was incubated with BSA or FDP (50 ,ug/ml) in PBS for 1 h at371C. The mixtures were then layered on a G-25 Sephadex columnpreequilibrated with excess BSAand 0.5-ml fractions eluted with PBS.The profile of protein elution was measured with a spectrophotometer(A2sq) and the pattern of ligand elution determined by beta spectrome-try. The patterns of [3H]-ligand coeluting with protein and free ligandwere compared for BSAand FDP.

Effect of FDP on PKCactivation by phorbol esters. The effect ofFDPon PMNPKCactivation was compared with that of BSA usingthe method of TerBush and Holz (39). PMN(107) were incubated inHBSSwith 10-8 MPMAin the presence of either FDP or BSA (100Mug/ml) for 10 min at 370C. The cells were then washed three times inHBSS and resuspended in 0.4 ml cold (40C) homogenizing bufferconsisting of 139 mMpotassium glutamate, 20 mMPipes, pH 6.6, 5mMEGTA, 5 mg/ml BSA, and 50 ug/ml leupeptin (KGEP) as de-scribed (40). The suspensions were sonicated twice (setting of 7 for 10 s;Kontes Co., Vineland, NJ) and subjected to centrifugation at 148,000g for 10 min. The supernatant (containing cytosol) was removed. Thepellet (containing membranes) was washed twice in cold KGEP, resus-pended in KGEPwith trituration, and Triton X-100 added to bothpellet and supernatant to a final concentration of 0.1%. 20 ul of eachfraction was then added to a protein kinase C (PKC) assay solutioncontaining 42 mMpotassium glutamate, 20 mMPipes (pH 6.6), 10mMMgCl2, 10 mMDTT, 0.016% Triton X-100, 50,ug histone/ml, 0.8mgBSA/ml, ±0.8 mMCaCI2, ±167 Mg phosphatidylserine/ml, ±26.7Mg 1,2-diolein/ml per tube. The reaction was started by addition of 30mM[32P]ATP (100,000 cpm; sp act, 23.8 Ci/mmol; New EnglandNuclear) and incubation carried out for 10 min at 28°C. The reactionwas stopped by addition of 5%TCA, 0.25% sodium tungstate, 15 mMNaH2PO4, and 2 mMATP. After addition of BSA and washing, theradioactivity in each of the precipitates from the PMNpellet fraction(membranes) and supernatant fraction (cytosol) was determined. PKCactivity is defined as the difference between activity in Cae and phos-phatidylserine/diglyceride buffer vs. buffer without Ca2' and phos-phatidylserine/diglyceride. Results are expressed as picomoles phos-phate incorporated/1O min per I07 PMN. Comparisons of PKCactivi-ties in cytosolic (supernatant) and membrane (pellet) fractions were

performed as a measure of translocation of PKC from the cytosol tomembrane that occurs concomitantly with phorbol ester-induced cel-lular activation (41).

Results

PMNbactericidal and phagocytic activity in the presence ofFDP. PMNincubated with 100 ,ug/ml FDP had significantlyless (alpha < 0.05, Wilcoxon-rank sum test) bactericidal activ-ity for ingested E. coli compared with autologous cells mixedwith an equivalent protein concentration of Fbg or BSA. Col-ony counts resulting from PMNpreincubated with BSAranged from I to 10 vs. 35 to 177 for those with FDP. FDPat aconcentration of 50 ,g/ml also significantly reduced the abilityof PMNto kill E. coli (Table I). BSA, Fbg, or FDP had noeffect on bacterial colony counts when PMNwere omitted(data not shown). In contrast to the inhibitory effects of FDPon PMNbacterial killing, phagocytosis of E. coli was unaf-fected. In the presence of 100 gg/ml BSA, 7, 45, and 85% ofPMNcontained ingested bacteria after 10, 20, and 30 min,respectively. The respective values for PMNcoincubated with100 ,g/ml FDPwere 6, 44, and 90% (duplicate samples from asingle donor). There was also no difference in the number ofbacteria per PMN. At 10, 20, and 30 min of incubation therespective values for PMN/BSAmixtures were 0.08, 2.85, and16.10 bacteria/cell vs. 0.06, 3.30, and 17.65 bacteria/cell forPMN/FDPmixtures.

Table . Capacity of PMNTo Exert Bactericidal Activityagainst E. coli in the Presence of BSA, Fbg, or FDP

Number of bacterial colonies* (PMNcoincubated with the following proteins)

Cell donor BSA Fbg FDP

1 2* 4 1772 1 2 453 10 ND 454 3 ND 355 3 ND 30

* PMN(5 X 106) and E. coli (- 107) were incubated in presence ofBSAor FDP(100 ,gg/ml for cell donors 1-4 and 50 Mg/ml for donor5) for 15 min, noningested bacteria were killed by addition of peni-cillin and streptomycin, and incubation was continued for 2 h at370C (25, 26). PMNwere then sonicated, aliquots plated on bloodagar, and bacterial colonies counted after incubation for 24 h at 370C.$ Results represent the mean of triplicates.ND, not done.

Effects of plasma coagulant proteins on FMLP-stimulatedPMN°2 release. Incubation of PMNwith FDP (10-100,tg/ml), Fbg (10-200 ,g/ml), plasmin (10 Atg/ml), thrombin (1or 10 U/ml), or BSA (100 ,ug/ml) for 10 min did not result indetectable O2 release. Cell viability as assessed by trypan blueexclusion was > 98% before and after addition of each of theproteins.

When5 X I0-' MFMLPwas added to PMNsuspended in100 gg/ml BSA, 2.5-22.5 nmol O2/10 min was released. Sub-stitution of BSA with an equivalent concentration of FDPresulted in a 49-57% reduction of FMLP-stimulated O2 accu-mulation using PMNof six donors (P < 0.005 by paired t test,Fig. 2). The ranges for triplicate determinations represented bythe mean values in this figure and Fig. 3 vary < 10% from themean in every case. For example, the mean of 22.5 nmol0O/10 min (the highest point under CONT. in Fig. 2) wascalculated from values ranging from 21 to 23 nmol 0/10 mmin.In contrast to FDP, Fbg (100 ,ug/ml), thrombin (1 or 50 U/ml),or plasmin (10 ,g/ml) did not affect the amount of FMLP-in-duced PMNO2 release compared with cells incubated with100 ,ug/ml BSA (range of 5 to 15 nmol 0O/10 min in fiveexperiments comparing coagulant proteins with BSA). Theinhibitory effect of FDPon FMLP-induced °2 release was notobserved when FDPwere removed before addition of the acti-vating ligand. PMNwere preincubated with 100 ,ug/ml FDPfor 10 min at 37°C, washed three times in HBSS, and 5 X 10-7FMLPadded. A total of 9.8, 11.6, and 13.0 nmol 0-/10 min

.E2E

I-0

E

z0

000

.aN0

25r

Figure 2. PMNO2 release induced byFMLPin the presence of 100 Mg/ml BSA(CONT.) or 100 Mg/ml FDP. FMLP(5X Io-' M) was added to triplicate PMNsamples for each cell donor 2 min after BSAor FDP. P < 0.005 for CONT. vs. FDP.

1918 Kazura, Wenger, Salata, Budzynski, and Goldsmith

12.5 rItut Figure 3. Kinetics of

20 jig FMLP-induced PMNE 10.0 /30 jig O° release in the pres-z o50 jg ence of 10-100 pg/ml0 7.5 - 100._ '°°O9 FDP(o) or 100 pig/ml

BSA(W). FMLP (50 5. X 10-' M) was added toX- XPMN2 min after BSAN 2.5-

o or FDP. Results at each/______________________ _ time point represent the

2 4 6 8 10 mean of duplicate cellTIME (minutes) suspensions.

per 106 cells (mean±SE of 11.4±0.9) was released using PMNof three donors. Substitution of BSA for FDP in these experi-ments resulted in release of 10.2, 11.1, and 12.3 nmol O2,respectively (mean±SE of 11.2±0.6, P> 0.05 vs. FDP).

Results of studies examining the effects of various concen-trations of FDP on FMLP-induced PMNO2 release are pre-sented in Fig. 3. Compared with control PMNincubated with100 Aig BSA/ml, concentrations of 10, 20, and 50 ,g FDP/mlinhibited PMNO2 released over 10 min by 17, 35, and 50%,respectively. The maximal rate of O2 release (calculated fromthe steepest portion of the curve representing change in absor-bance vs. time) also decreased progressively in the presence ofincreasing concentrations of FDP (15, 23, 32, 42, and 48%reductions with 10, 20, 30, 50, and 100 ,gg/ml FDP). Furtherinhibition was not observed when 200 ,g/ml FDP was added(data not shown). Similar results were obtained using cellsfrom two other donors. No difference in FMLP-induced PMNO2 release was observed in the presence of equivalent concen-trations of Fbg or BSA.

To determine whether the inhibitory effect of FDP couldbe reversed by increasing the amount of stimulating ligand,PMNwere preincubated for 2 min with 100 ,g BSA/ml (con-trol) or 100 ,ug FDP/ml and increasing amounts of FMLPadded. When 1 X 10-7 MFMLPwas added, 50%inhibition ofPMNO2 release was produced by FDP ( 1±2 nmol/ 10 minfor FDPvs. 22±3 nmol/l0 min in BSAcontrols, mean±SE ofexperiments using PMNof three individuals). A similar degreeof FDP-induced inhibition (62%) occurred when the FMLPconcentration was increased to 5 X l0-7 M(16 nmol 0-/10min with FDPvs. 42 nmol/ 10 min with BSA). Less inhibitionwas observed when the concentration of FMLPwas increasedto 1 X 0O-6 M. In two experiments control PMNreleased58±4 and 60±2 nmol O/l0 mmin vs. 51±1 and 56±4 nmol/10min in the presence of FDP (25 and 16% inhibition, respec-tively).

As a first step in defining the specific proteins in FDPresponsible for suppression of PMNresponses, PMNwerepreincubated with purified fragments DI or E3 2 min beforeaddition of 5 X l0-7 MFMLP. Release of O2 from PMNpreincubated with 10 ,ug/ml BSA (controls) was 20.0±1.0nmol/10 min. In the presence of 10 pig/ml fragment D1 or E3these values were, respectively, 20.0±1.5 and 14.1±2.0 nmolOy/10 min (mean±SD for triplicate determinations of PMNof one donor; P < 0.05 for fragment E3 vs. controls). UsingPMNof a second donor preincubated with 50 j.g/ml BSA orfragment DI, O2 release over 10 min was, respectively,16.1±1.0 and 16.0±2.0 nmol. In contrast, fragment E3 at a

E

z0

-0

0.0a°:

IN

TIME (minutes)

Figure 4. Effect of time ofaddition of FDPor BSAonkinetics of FMLP-stimu-lated PMNO2 release. 5X lo-7 MFMLPwasadded to PMNat time 0.BSA (100 gg/ml) wasadded 1.5 (e) or 5.5 min(o) later. In parallel studies,FDP(100 gg/ml) wasadded 1.5 (A) or 5.5 min(A) later. Results representthe mean of triplicate de-terminations at each timepoint.

concentration of 50 jug/ml suppressed O2 release from PMNofa third donor by 21% (29.1±3.0 in the presence of 50 ,g/mlBSA vs. 22.0±3.0 nmol/10 min, P < 0.05).

Capacity of FDP to reverse FMLP-induced PMNO2 re-lease. To ascertain whether FDP could abrogate O2 produc-tion from PMNpreviously exposed to FMLP, 100 ,g/ml FDPor control BSAwas added to cells 1.5 or 5.5 min after 5 X l0-'MFMLP. Addition of BSA 1.5 (Fig. 4, closed circles) or 5.5min (open circles) after FMLPdid not have a differential effecton the rate or accumulation of O2 generated by PMN(10.5±1.0 nmol O2 over 10 min, mean±SDof triplicate deter-minations). In contrast, addition of FDP 1.5 min after FMLP(closed triangles) resulted in an immediate decrease in the rateand accumulation of O2 compared with addition at 5.5 min(open triangles) (7.5±0.5 nmol/10 min vs. 12.5±1.5 nmol/10 min).

Binding offluid-phase FMLP by FDP. Results reportedthus far are consistent with a mechanism whereby FDPbindfluid-phase FMLP more effectively than BSA, thereby pre-venting interaction of the stimulus with its cellular target. Toexamine this possibility, equivalent concentrations (50 ,4g/ml)of FDP or BSA were incubated with radiolabeled FMLPandthe proportions of protein-bound vs. free ligand compared.100% of added radiolabel coeluted with free ligand for mix-tures of FMLP/BSA and FMLP/FDP. FMLP thus did notappear to bind to either protein.

Effect of FDP on PMNbinding of FMLP. Because ourprevious experiments demonstrated that the effects of FDPonFMLP-induced PMNO2 release occurred when the proteinswere added subsequent to ligand (Fig. 4) and were not second-ary to removal of fluid-phase FMLPby protein binding, wenext evaluated the possibility that FDPinterferes with interac-tion of FMLP with the PMNsurface. In experiments usingcells of four donors, FMLPbinding was 72±12% (mean±SE)lower in the presence of 100 jug/ml FDP compared with anequivalent concentration of BSA(1.7 nMinput of [3H]FMLP)(Table II). There were no differences in the effects of Fbg andBSAon PMNbinding of FMLP(Table II). To define in moredetail the possible interaction of FDP with FMLPreceptors,additional experiments were performed in conditions limitingPMNinternalization of FMLP and coagulant proteins andover a broad concentration range of radiolabeled ligand(1.3-85 nM [3H]FMLP). In the presence of 100 ,g/ml BSA(control protein), PMNwere calculated to have high affinityFMLP receptors with Kd = 2.8 x 10-9 Mand low affinityreceptors with Kd = 1.5 X l0- M(curve generated from tripli-cate determinations of six points) (Fig. 5). Parallel studies con-

Fibrinogen Degradation Products Inhibit Neutrophil Function 1919

Table I. Effect of FDPon PMNBinding of FMLPand PDBU

Specific binding of ligand

Cell donor Ligand BSA(100 p/ml) Fbg (100lug/ml) FDP(100l g/ml)

cpm

1 FMLP* 24,000 25,200 12,022 (50)t2 FMLP 10,980 11,053 5,421 (51)3 FMLP 1,090 ND 245 (88)4 FMLP 2,301 ND 48 (98)5 PDBU1 7,099 ND 7,836 (0)6 PDBU 5,560 ND 6,900 (0)

* [3H]FMLP (1.7 X 10-9 M) was incubated in the presence or ab-sence of 1,000-fold excess unlabeled FMLPwith BSA, Fbg, or FDPto measure specific binding (32). Results represent the mean of tripli-cate determinations for each cell donor.tPercent reduction compared with BSA.[3H]PDBU (2 X 10-7 M) was incubated in the presence or absence

of 250-fold excess unlabeled PMAwith BSAor FDP(100 pg/ml) tomeasure specific binding (38). Results represent the mean of dupli-cate determinations for each cell donor.ND, not done.

ducted with 100 pg/ml FDPshowed that FMLPbinding wasreduced > 90% in both the steep and flat portions of the curvegenerated by the Scatchard plot (Fig. 5). Additional bindingstudies showed that lower concentrations of FDP also inhib-ited PMN-FMLPinteraction. When the total protein concen-tration was kept constant at 100 pg/ml, increasing the propor-tion of FDP:BSA from 16 to 25% resulted in respective reduc-tions of specific FMLPbinding of 44 and 80%compared withsamples containing BSA alone (mean of triplicate samples ateach point, Fig. 6). Greater reduction in specific radiolabeledligand binding was not demonstrable when the FDPconcen-tration was increased to 50 or 100 ug/ml (85% decreases inbinding compared with PMNcoincubated with 100 pg/mlBSA and no FDP) (Fig. 6). The input of [3H]FMLP in theseexperiments was 21 nM.

FDPand other procoagulant protein effects on FMLP-stim-ulated PMNchemotaxis. FMLP-stimulated chemotaxis wasused as an additional functional assay, independent of oxida-tive metabolism, to confirm that FDP interfered with ligandbinding to PMN. In experiments using cells from four donors,

0.0401

0.027-r

0.5 1.5 2.5

FMLPbound (nM)

Figure 5. Scatchard plot of FMLPbinding to PMNin the presenceof 100 mg/ml BSAor FDP. Spe-cific binding FMLPwas measuredin conditions that minimize inter-nalization of ligand as previouslydescribed (34, 35). Each point rep-resents the mean of triplicate de-terminations. Curves were gener-ated according to NONLINcom-puter program with a two-sitemodel of FMLPbinding assumed(36). Closed circles representFMLPbinding in the presence of100 mg/ml BSA. Open circles rep-resent binding in the presence of100 mg/ml FDP.

8.0

27.0

- 6.0I Figure 6. Concentration de-

5.0 pendence of FDP inhibition of\ FMLPbinding to PMN. Spe-

4.0 cific binding of FMLPwasffi \ measured as described with 21x. 30 \ nm [3H]FMLP input (35). The° 2.0 total protein concentration was

constant at 100 pg/ml and the1.0 - proportion of BSA:FDP varied

as indicated on the abscissa.BSA 100 84 75 50 0 Each point represents theFDP 0 16 25 50 100 mean of triplicate determina-

(pg/ml) tions.

FDP reduced directed migration of PMNby 32-48% com-pared with BSA. A chemotactic index of 3.4±0.4 (mean±SE)was obtained for control PMNincubated with BSAvs. 2.0±0.3for those with FDP (P < 0.01) (Table III). The chemotacticindices of PMNincubated with Fbg (100 pg/ml), thrombin (50U/ml), or plasmin (10 pg/ml) did not differ significantly fromcontrols (data not shown for latter two proteins).

FDP modification of PMN02 release induced by addi-tional nonphagocytic and phagocytic stimuli. To examinewhether the inhibitory effect of FDPoccurred under circum-stances involving other nonphagoytic stimuli, studies wereconducted with PMA, OAG, AA, and ZAS. PMAand OAGmay stimulate the respiratory burst primarily through directactivation and translocation of PKC (41), while the mecha-nism by which AA induces PMNO2 release is not well estab-lished (42). ZAS containing C5a presumably stimulates PMNrespiratory burst activity by a pathway similar to that for otherligands having surface PMNreceptors (e.g., FMLP). When 10ng/ml PMAwas added to PMN, 02 release was 20-74% lowerin the presence of 100 pg/ml FDPvs. an equal concentrationof BSA (P < 0.001, Table IV). Unlike the situation withFMLP, however, addition of FDP 1.5 min after PMAdid notinhibit PMNOj release (data not shown). Stimulation of

Table III. FMLP-induced PMNChemotaxis in the Presenceof BSA, Fbg, or FDP

Chemotactic index(Protein [100 pg/m] added to PMN)

PMNdonor BSA Fbg FDP

1 2.1* 2.3 1.1 (48)t2 4.1 3.2 2.4(41)3 3.8 ND 2.6 (32)4 3.7 ND 2.1(43)

Mean±SE 3.4±0.4 ND 2.0±0.3 (41)§

* Results represent the mean of triplicate determinations using a che-motaxis in agarose technique (32).* This value is the percent inhibition compared with PMNincubatedwith BSA.0 The mean value for PMNpreparations incubated with FDPwassignificantly less compared with controls (BSA) with P< 0.01.ND, not done.

1920 Kazura, Wenger, Salata, Budzynski, and Goldsmith

0.081

0067

0.054wwc-

2z0

0.013

0

Table IV. Effect of FDPon PMA-stimulated PMN02 Release

Maximal rate of Oj release

Experiment BSA (100 ,g/ml) FDP(100 tg/ml) % Inhibition by FDP

nmol/min

1 6.8* 1.8 742 3.5 2.2 373 8.8 5.6 364 8.7 7.0 205 8.1 4.9 396 7.0 4.8 31

PMNwere incubated with 100 ,g/ml BSAor FDPat 37°C and 10ng/ml PMAadded 2 min later. Maximal rates were calculated fromthe steepest portion of curve generated by absorbance readings takenat 1-min intervals (28).* Results represent the mean of triplicate determinations.

PMNO2 release induced by OAGwas also inhibited by FDP.PMNpreincubated with 100 sg BSA/ml for 2 min followed byaddition of 10-6 MOAGreleased 8.9±0.4 nmol O/20 min(mean±SD of triplicate determinations), whereas those prein-cubated with 100 jig FDP/ml released 3.3±0.6 nmol (P<0.01). AA (4.2 X IO-' M)-induced O- release was also re-duced by FDP. PMNof two donors stimulated with AA re-leased 20.0 and 19.0 nmol 0/l0 mmin per 106 cells in thepresence of 100 ,ug/ml BSA vs. 8.4 and 8.0 nmol/10 min,respectively, when an equivalent concentration of FDP wasincluded. Addition of FDP to PMN1.5 min after AA did notaffect O2 release, as reported for PMA. Addition of 20 ul ZASto 106 PMNsuspended in 1 ml buffer containing 100 jig/mlFbg resulted in generation of 1.3±0.4 nmol O2 over a 10-minperiod (triplicate samples). When the same individual's PMNwere stimulated simultaneously with ZAS in the presence of10, 50, or 100 ,ug/ml FDP(the remaining protein in the formertwo samples consisted of Fbg), the amount of O2 releaseddecreased to 1.1±0.1, 1.0±0.2, and 0.9±0.1 nmol O2.

In contrast to the inhibitory effects of FDP on PMNO2release induced by these nonphagocytic stimuli, Q2 releaseinduced by phagocytosis of opsonized zymosan was not al-tered. 50 ji opsonized zymosan was added to PMNcoincu-bated with either 100 ,g/ml BSAor FDP. The mean amountsof O2 released over a 30-min period in the presence of BSAvs.FDPwere, respectively, 14.6 vs. 15.8, 2.6 vs. 5.3, and 21.9 vs.17.4 nmol (cells of three individuals, P > 0.05).

Effect of FDPon AA and phorbol ester binding to PMN. Wedetermined first whether FDPbound fluid-phase phorbol esteror AA to a greater extent than BSA. In the case of [3H]PDBU,100% of radiolabel coeluted with free ligand after preincuba-tion with BSAor FDP, as described for FMLP. In contrast, AAbound to both BSA and FDP. Coincubation of [3H]AA withBSA for 1 h resulted in 55.9% of radiolabel being proteinbound and 44.1% unbound. WhenFDPwas substituted, 2.2%of radiolabel bound to protein vs. 97.8% unbound. These dataindicate that BSA binds AA to a greater extent than FDP inour assay conditions. To ascertain whether FDPalso reducedPMNbinding of phorbol esters as reported for FMLP, specificbinding of [3H]PDBU was measured in the presence of BSAorFDP(100 ,g/ml). Similar amounts of PDBUwere specifically

Table V. Effect of FDPon Activation and Translocationof PMNPKC

PKCactivity*

Donor Protein Unstimulated PMAstimulated

pmol P/O0 min per 10 PMN

F.H. BSA 410±31t (75)§ 581±191 (25)FDP 500±27 (65) 490±20 (59)

J.K. BSA 498±53 (67) 613±28§ (35)FDP 491±29 (72) 486±25 (63)

* PMNwere incubated with 100 ,g/ml BSAor FDPand PKCactiv-ity was measured after exposure to buffer alone (unstimulated) orI0O- PMA(39, 40).

This value represents the mean of triplicate determinations.The number within the parentheses represents the percent PKCac-

tivity in the soluble (cytoplasmic) fraction of PMNsonicates sub-jected to centrifugation at 148,000 g for 10 min."P < 0.05 compared with unstimulated PMN.

bound by PMNwhen the assay was performed in the presenceof BSAor FDP(Table II).

PKCactivation.- FDP completely abrogated PKC activa-tion by PMAin intact PMN. When 10-8 MPMAwas added toPMNcoincubated with 100 ,g/ml FDP, there was no changein total PKCactivity or the proportion in soluble fractions ofPMNsonicates (65 and 72% for unstimulated cells). In con-trast, when PMNwere coincubated with 100 ug/ml BSA, PMAcaused a 23-41% increase in total PKCactivity (P < 0.05) andone-third to one-half reduction in the proportion in solublefractions (Table V). Cells not exposed to protein or PMAhadvalues that were identical to those incubated with BSA (e.g.,400±35 pmol P/10 min per I0' PMNwith no protein or PMAvs. 380±40 pmol P/10 min per 107 PMNwith 100 ,g/ml BSA;triplicate samples for each with cells of one donor).

Discussion

Fbg and several proteolytic fragments of this plasma proteinhave important effects on circulating PMN. Low molecularweight (< 20 kD) fragments produced by extensive plasmindigestion are chemotactic for rabbit PMNin vitro and induceegress from the vascular space in vivo (43, 44). FibrinopeptideB, a thrombin-derived cleavage product of Fbg BB chains, isalso chemotactic but does not stimulate degranulation or re-spiratory burst activity (16). The possible alterations in PMNfunction mediated by FDPD and E, the major FDPelevatedin plasma of persons with DIC and predisposing conditionssuch as adult respiratory distress syndrome (45-47) have, how-ever, not been studied. The current results indicate that FDPDand E at concentrations similar to those observed in DIC sig-nificantly impair the capacity of PMNto kill E. coli in vitro.This defect in bactericidal function is apparently related toalterations in intraphagolysosomal function since bacterialphagocytosis and extracellular release of O2 induced by phago-cytic stimulation (opsonized zymosan) were unaffected byFDP. To define the possible basis of the FDP inhibitory effecton PMNbactericidal function, a series of studies using ligandsthat elicit relevant PMNresponses by initially occupying sur-

Fibrinogen Degradation Products Inhibit Neutrophil Function 1921

face-exposed receptors (i.e., FMLPor C5a) or by directly bind-ing and activating PKC (PMA and possibly AA) were con-ducted.

Several aspects of the PMNresponse to FMLPare inhib-ited by FDP but unaffected by thrombin, plasmin, or Fbg.Superoxide production decreases in a dose-dependent mannerupon addition of 10-100 gg/ml of fragments Dand -E. Com-parison of ihe capacities of purified preparations of fragmentsDI and E3 to affect FMLP-stimulated PMNO° productionsuggests that the latter fraction is responsible for the inhibitoryeffect of FDP. These experments have not excluded the possi-bility that higher concentrations of fragment DI than thoseused here (50 pg/ml) might also be inhibitory. As a first step indissectinig the possible mechanism of FDP' inhibition ofFMLP-stimulated PMNO° release, we examined the revers-ibility of the effect. Whereas preincubation of cells with FDPfollowed by washing failed to alter FMLP-induced °2 release,addition of FDP 1.5 min after FMLPresulted in immediatecurtailment of oxyradical accumulation. Since continual oc-cupancy of FMLP receptors is required for optimal stimula-tion of PMN°2 release (48, 49), these results suggest that FDPdisrupts the interaction of FMLPwith its corresponding cellu-lar receptors. The ability of FDP to inhibit FMLP-inducedPMNchernotaxis without binding directly to fluid-phase li-gand is also consistent with a process involving impairment ofcellular receptor-FMLP interaction. To examine this event di-rectly, specific binding of'[3H]FMLP to PMNwas measured.FDPinhibited FMLPbinding (1.7 nMinput) by 50-98%. Theinhibitory effect occurred at FDPconcentrations as low as 16Ag/ml and was maximal at 2 25 pg/ml. Chemotactic bindingstudies perfQrmed over a broad concentration range of[3HJFMLP in conditions that minimize receptor internaliza-tion showed that FDP reduced FMLPbinding at all levels of[3HJ-ligand input. These results suggest that FDPdo not inter-fere preferentially with FMLP interaction with high or lowaffinity PMN'chemotactic peptide receptors. However, moredetailed studies using multiple concentrations of FMLPinputare required to confirm this. It is also not possible on the basisof these data to distinguish among the possible mechanisms bywhich FDP interfere with- FMLPbinding to PMN, e.g., occu-pancy of 'the FMLP binding site(s), steric hindrance, alter-ations in surface receptor mobility, and/or stabilization of thereceptor-ligand complex (50).

The effects of Fbg fragments D and E on PMNoxidativemetabolism are not limited to FMLPstimulation. Productionof °2 induced by PMA, AA, and OAGare also significantlyreduced by FDP. The pathways by which these stimuli inducePMNO2 release are the subject of a great deal of study andhave not been completely elucidated. These molecules are li-pophilic and rapidly interact with the PMNplasma mem-brane. PMAand probably OAGbind directly to PKC(41, 51,52) and induce translocation of the enzyme from the cytosol toplasma membrane (51). This process is concurrent with acti-vation of the enzyme(s) involved in the initial one electronreduction of 2 (41). Although the mechanisms by which AAactivates the respiratory burst have not been well defined, thislipid may alter surface membrane fluidity (53, 54), directlyActivate PKCin some circumstances (55, 56), and is a requiredcofactor for NADPHoxidase activity prepared from brokenresting PMN(57, 58). The current studies indicate that FDPinhibition of respiratory burst activity induced by these stimuliis not attributable to protein binding of fluid-phase ligands or

interference with phorbol ester binding to PMN. Rather, ourobservations are consistent with the -possibility that FDP im-pair activation and translocation of PKCfrom the PMNcyto-solic to membrane fraction. Further studies to examine thisinclude direct measures of PMNmembrane fluidity in thepresence of FDP and determination of whether FDP influ-ences activation of PKCisolated from broken cells.

By analogy with the functional consequences of Fbg inter-action with platelets (2, 5, 6), it might be expected that theobserved effects of FDPon PMNactivities are in part relatedto occupancy of Fbg binding sites. Although there are dataindicating that promyeloid U937 cells possess GPlib/Illa, themolecular complex that represents the platelet Fbg receptor(59), recent reports suggest that circulating human PMNspe-cifically bind Fbg via receptors that are similar or possiblyidentical to those for high molecular weight kininogen (60).Detailed molecular characterization of the interaction of FT)PD and E with PMNand their functional effects on cells willrequire isolation of these receptor sites and use of highly puri-fied preparations of fragments DI and E3. Finally, the role ofPMNproteases in modifying cellular responses to Fbg andFDPneeds to be investigated (61, 62). The relevance of suchmolecules to PMNfunction has been suggested by recent ob-servations of Wachtfogel and co-workers, who showed thatfibronectin degradation products containing cytoadhesivepeptide sequences identical to those in FDP(63) induce releaseof neutrophil elastase (64).

Acknowledoments

The authors wish to thank Pedro de Brito and Sue Hudson for techni-cil assistance and Pat Amato for typing the manuscript.

This work was supported by National Institutes of Health grantsHL-371 17 and HL-34641.

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1924 Kazura, Wenger, Salata, Budzynski, and Goldsmith