INFECTION AND IMMUNITY, JUlY 1974, p. 128-135Copyright © 1974 American Society for Microbiology

Vol. 10, No. 1Printed in U.S.A.

Elastase of Pseudomonas aeruginosa: Inactivation ofComplement Components and Complement-Derived

Chemotactic and Phagocytic FactorsDUANE R. SCHULTZ AND KENT D. MILLER

Divisions of Immunology and Laboratory Medicine, Department of Medicine, University of Miami School ofMedicine, Miami, Florida 33152

Received for publication 4 February 1974

A purified elastase from Pseudomonas aeruginosa was highly destructive forfluid-phase and cell-bound Cl and C3 and fluid-phase C5, C8, and C9.Inactivation of C4, C2, C6, and C7 by the enzyme varied from 0 to 67%. Lowconcentrations of elastase generated, then inactivated, a chemotactic factor fromhuman C5 but not from C3. Higher enzyme concentrations inactivated the C5chemotactic activity at a faster rate. Elastase treatment of sensitized pseudomo-nads containing cell-bound C3 reduced the phagocytic indexes of polymorphonu-clear leukocytes. The data support the proposed chemopathogenic role of theelastase in generation of the characteristic non-inflammatory Pseudomonasvasculitis.

The vasculitis in man caused by some strainsof Pseudomonas aeruginosa is characterized bybacillary infiltration of arterial walls and disin-tegration of the external elastic laminae andhemorrhage, but little or no intramural inflam-matory response (4, 7, 14, 18, 22). Patientshaving a predilection for these lesions are thosewith severe thermal injuries and leukemia (22).Unpublished work by our laboratories, using

homogeneous Pseudomonas elastase prepara-tions, demonstrated enzymatic inactivation ofthe hemolytic activity of whole guinea pigcomplement and all the chemotactic activitiesof whole human serum. These observationssuggested a possible chemopathogenic functionof the microbial elastase.

Since complement (C) is one of the principalserum protein effector systems of the inflamma-tory response (2, 28), we investigated furtherthe effects of Pseudomonas elastase on the Csystem and on C-derived chemotactic andphagocytic factors. (The nomenclature usedhere conforms with that agreed upon by theWorld Health Organization.)

(This paper was presented, in part, at theComplement Workshop, Baltimore, Md., Janu-ary 1971. J. Immunol. [abstract] 107:324.)

MATERIALS AND METHODSAbbreviations and defmitions. The following defi-

nitions and abbreviations are used: C, complement;DGVB2+, glucose gelatin-Veronal buffer with Ca2+and Mg2+; EAC, sheep erythrocytes (E) sensitized

with rabbit antibody (A) to which complement (C)has been added; BAC1423, pseudomonads (B) sen-sitized with rabbit antibody (A) to which Cl, C4, C2,and Cl have been added; PI, phagocyte index.

Serum. Blood was drawn aseptically from healthyadult donors by venipunture and was allowed to clotat room temperature. Serum was separated by cen-trifugation, divided into small portions, and immedi-ately frozen at -70 C until used.

Buffers. Glucose-gelatin-Veronal buffer containing0.00015 M Ca2- and 0.0005 M Mg2+, relative saltconcentration 0.075 M, pH 7.5, was presented asdescribed previously (25). Gey solution with 2% bo-vine or human albumin was prepared by publishedmethods (1).Pseudomonas elastase. Elastase was isolated from

P. aeruginosa strain NYS 64-332 as previously de-scribed (21). Before enzyme production, colonies werepooled from heart blood isolates of two rats that died16 days after receiving scalding burns that wereinfected with the parent strain (23). The enzymeisolated from these organisms was immunologicallyidentical to that from the parent strain and containedthe same specific activity, 1,600 caseinolytic units permg of protein (21). Although enzyme activity wasmost rapidly measured in caseinolytic assays, it wasalso determined in diffusion plates containing either1% efastin powder or a-casein (Sigma Chemical Co.,St. Louis, Mo.) in 1.5% Noble agar (Difco Laborato-ries, Detroit, Mich.) and 0.03 M tris(hydroxy-methyl)aminomethane buffer, pH 8.0. In the experi-ments to follow, only enzyme preparations foundhomogenous by disc electrophoresis (5) were em-ployed.Complement reagents and tests. Functionally

purified human or guinea pig C components, whole

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guinea pig C, sheep erythrocytes (E) sensitized withrabbit antibody (A), and the stable cellular interme-diates EA, EAC1, EAC4, EAC14, and EAC1-7 weremade by published techniques (25).The functional hemolytic activities of the nine C

components were determined in tubes by stoichiomet-ric hemolytic assays (25) unless indicated otherwise.

Purified C3 and monospecific antiserum. HumanC3 (,BlC/,BlA) was prepared by an unpublished tech-nique of one of us (D.R.S.). Normal human serumwas diluted 1: 15 with ice-cold distilled water andadjusted to pH 6.0 with 1 M HCl, and ethylenedi-aminetetraacetate was added to a final concentrationof 0.02 M. Incubation for 6 h at 4 C resulted in aeuglobulin precipitate that was centrifuged, washed,and dissolved in 0.005 M sodium phosphate buffercontaining 0.07 M NaCl and 0.002 M ethylenedia-minetetraacetate, pH 7.5. After it was applied to adiethylaminoethyl-cellulose (Whatman DE-52, Eng-land) column equilibrated with the phosphate buffer,a linear NaCl gradient (0.070 to 0.20 M) yielded frac-tions rich in C3 at approximately 0.10 to 0.12 M. Theywere concentrated by ultrafiltration (Amicon Corp.,Lexington, Mass.) and applied to a hydroxylapatite(Bio-Rad, Richmond, Calif.) column equilibratedwith 0.05 M Na2HPO4 and KH2PO4, pH 6.9. The C3was eluted with a linear sodium-potassium phosphategradient of 0.05 to 0.2 M (peak activity for C3 was12.5 umhos x 10w). The C3-rich fractions were con-centrated by ultrafiltration and applied to a Geon-Pevikon block for electrophoresis. The block wasprepared by mixing equal parts of Geon 427 (B. F.Goodrich, Cleveland, Ohio) and Pevikon C-870 (Mer-cer Chemical Corp., New York, N.Y.) in a tris(hy-droxymethyl)aminomethane-citrate buffer, pH 8.5.Sodium borate buffer, pH 8.7, was used in the.troughs. After electrophoresis for 12 h at 4 C (ca. 4V/cm), the C3 was found in the ,8-region. In each step,C3 was assayed by double diffusion in Ouchterlonyplates (16) with a monospecific goat antiserum to#lC/#lA and by functional hemolytic assays. Theyield of C3 varied from 10 to 30%. A single precipitinband was observed in immunoelectrophoresis withmost preparations, using a goat antiserum to poolednormal human serum. In addition, the C3 preparationfor this study was used to immunize a goat. After fourinjections of 0.25 mg each with complete Freundadjuvant (intramuscular) over the course of 6 months,a single band was demonstrated with normal humanserum in double diffusion plates. The band formed acomplete line of identity with a C3 antiserum pur-chased from Beringwerke (Marburg-Lahn, Germany).

Antisera to C3 were either produced in goats bystandard procedures (9) or purchased from Bering-werke.

Immunoelectrophoresis. The micro-method ofScheidegger was used (19).

Phagocytosis experiments. Sterile materials andtechnique were used in all these experiments. Falconplastic disposable pipettes and polypropylene tubeswere employed.The elastase-producing strain, P. aeruginosa (NYS

64-332), was grown in Trypticase (BBL) soy broth for6 h, washed in Gey solution containing 2% human

albumin, and adjusted to a 7 x 1o8/ml suspension(from a standard plot of optical density at 420 nmversus plate count).

Antiserum to formalinized elastase and Pseudomo-nas cell constituents was prepared in rabbits employ-ing complete Freund adjuvant.

Five milliliters of the washed pseudomonads (B)was added to 0.3 ml of specific antibody (A), incu-bated for 15 min at 37 C, washed once with Geysolution, and readjusted to 7 x 10s/cells/ml (BA).

Rabbit polymorphonuclear leukocytes (PMN) wereobtained from glycogen-induced peritoneal exudates(26). The neutrophils were suspended in Gey solutioncontaining 2% human albumin.The phagocytic index (PI) is defined as the average

number of intracellular pseudomonads per rabbitPMN after a specified time at 37 C. The PI wascalculated from five separate counts of the intracellu-lar bacteria in 100 PMNs.

Chemotactic assays. Modified Boyden chamberswere fitted with membrane filters (Millipore Corp.,Bedford, Mass.) of 1.2-gm pore size (26). RabbitPMNs were obtained from glycogen-induced perito-neal exudates prepared as above.

RESULTSInactivation of serum C by pseudomonas

elastase. To 1-ml portions of normal humanserum were added, respectively, 115 ,ug of elas-tase in 0.7 ml of DGVB2+ and DGVB2+ withoutenzyme. The tubes were incubated at 37 C. At5-min intervals, 0.1-ml portions were removedfrom each mixture and diluted in tubes contain-ing 1.9 ml of ice-cold DGVB2+. After addition of0.5 ml of EA (1 x 108 cells/ml) to each dilutedsample, all tubes were incubated at 37 C for 30min. Ice-cold saline (5 ml) was then added toall tubes to stop hemolysis. The supernatantfluids were separated from cells by centrifuga-tion, and the oxyhemoglobin was read spectro-photometrically at 415 nm.The results in Fig. 1 demonstrate no loss of

hemolytic activity in the serum-buffer control,but activity decreased progressively with timein the serum-elastase mixture. At 22 min ofenzyme action 50% hemolysis was achieved, thevalue being reduced to 5% after 40 min.

Inactivation of individual serum C compo-nents by pseudomonas elastase. Elastase (0.2ml containing 275 Mg of enzyme) and DGVB2+(0.2 ml) were added, respectively, to 2-ml por-tions of normal human serum diluted 1:10 inDGVB2+. The tubes were incubated for 10 minat 37 C. Four milliliters of ice-cold DGVB2+ wasthen added to all tubes, and the nine residual Ccomponents were titered by hemolytic assays.The results in Table 1 indicate that the elastaseeither directly or indirectly caused 93.5 to 97%inactivation of the nine C components. Theexperiment was repeated with different human

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100 i -t_4

8c_ Human Serum Buffer

0 Human Serum Elastase

40

0 5 10 15 20 25 30 35 40

Time in minutes

FIG. 1. Effect of 68 ,g of pseudomonas elastase orbuffer per ml on the complement activity in normalhuman serum at 37 C. Samples were removed at5-min intervals and tested for hemolytic activity withsensitized erythrocytes.

TABLE 1. Effect ofpseudomonas elastase or buffer onthe complement components in normal human serum

after incubation for 10 min at 37 C

CH50 U/ml after incubation

Complement of human serum inComplement c% Lossacomponent Elastase Buffer

(125 jig/mi) Bfe

C1 480 492,000 99.9C4 480 123,000 99.6C2 960 15,000 93.6C3 <30 15,000 >99.6C5 480 61,000 99.2C6 480 123,000 99.6C7 <30 123,000 99.9C8 15,000 492,000 97.0C9 8,000 123,000 93.5

a % Loss = 1 - [(CH50 U/ml in elastase)/(CH50U/ml in buffer) ] x 100.

sera, test reagents, and red cell intermediates,with essentially the same results.

Inactivation of free and cell-bound C com-ponents by pseudomonas elastase. Enzymeeffects on each of the nine functionally purifiedhuman C component were examined in thefollowing way: 0.125-ml portions of each compo-nent were pipetted into four separate wells of amicrotiter plate. The first two wells each re-ceived 0.025 ml of elastase (2.5 gg/ml), while0.025 ml of DGVB2+ was added to the secondtwo wells. The plates were incubated for 10 minat 37 C. Serial twofold dilutions of each wellwere then carried out with DGVB2+, and theremaining reagents and cellular intermediateswere added as described in published reports(25). The hemolytic titers were subsequentlyestimated visually. Following the initial incuba-tion, all of the mixtures containing elastase

were tested for proteolytic activity on casein-agar plates and found active.Data in Table 2 indicate that, in the function-

ally purified form, Cl and C3 were most suscep-tible to elastase action, 94 and 99% of thehemolytic activities being destroyed, respec-tively. From 49 to 86% of the hemolytic activi-ties of the other C components were also de-stroyed. However, the enzyme had no apparenteffect on purified C4 and C7. This experimentwas repeated with different lots of the function-ally pure C components, and the results weresimilar to those in Table 2.

In two experiments, a solution of functionallypurified human Cl was added to 0.05 M acetyltyrosine ethyl ester. Samples were then reactedwith 10 gg of elastase per ml before and afteractivation of the Cl-esterase with 12 pg oftrypsin per ml. Hydrolysis of the ester, deter-mined by titration of the acid formed, was thesame, 0.72 mmol of H+ per min per ml of Cl, inboth mixtures. Thus, the elastase had no effecton the C1-esterase or its precursor. The elastasedid not catalyze hydrolysis of acetyl tyrosineethyl ester.

In other experiments, the stable cellular in-termediates, EA, EACi guinea Pig' EACI-(guinea pig 4human EAC4 human and

(human)IEACI-7 ( were prepared by publishedmethods (25) and adjusted to cell concentra-tions of 108/ml. Elastase (final concentration,2.7 jig/ml) or control DGVB2+ were added tosamples of each cellular intermediate. Theywere incubated for 20 min at 30 C, washedtwice with DGVB2-, and readjusted to 108cells/ml. The elastase-treated and untreated

TABLE 2. Effect ofpseudomonas elastase or buffer onindividual, functionally purified human complement

components after incubation for 10 min at 37 C

CH50 U/ml after incubation.of individual human

Complement components in % LoSSacomponent

Eiastase Bfe(125 jg/ml) Buffer

C1 32,800 524,300 94C4 4,100 4,100 0C2 2,000 6,000 67C3 4 1,000 >99C5 1,000 4,100 76C6 2,000 4,100 49C7 4,100 4,100 0C8 16,400 121,000 86C9 500 2,000 75

a% Loss = 1 - [(CH50 U/ml in elastase)/(CH50U/ml in buffer) ] x 100.

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cells were used for the following tests: (i) EA totitrate C in fresh human serum, (ii) EAC1 totitrate functionally pure human C4, (iii) EAC14to titrate functionally pure human C2, and (iv)EAC1-7 to titrate functionally pure guinea pigC8. The results demonstrated that the onlycellular intermediate affected by the elastasetreatment was EACL. Seventy-five percent ofthe activity for human C4 was destroyed aftertreatment of the EAC1 cells with the enzyme.The effect of elastase on sheep E receptor

sites for specific antibody was also investigated.The results indicated that prior enzymic treat-ment of E (cell suspension of 109/ml; elastaseconcentration, 2.7 ,g/ml) had no effect on thecell reactions with specific 7S rabbit antibody toform EA. A serum C titer of 465 CH50 U/ml wasobtained with the enzyme-treated cells as com-pared with 417 obtained with the untreatedcells.Attempts were also made to lyse unsensitized

E by addition of elastase to a mixture of ninefunctionally pure human C components. Dupli-cate 1-ml samples of E (5 x 107/ml) were added

to 0.1-ml samples of each of the following: Cl(10,000 U/ml), C4 (1,000 U/ml), C2 (100 U/ml),C3 (50 U/ml), C5 (50 U/ml), C6 (50 U/ml), C7(50 U/ml), C8 (500 U/ml), and C9 (500 U/ml).The mixtures were incubated for 2 min at 37 Cand then 0.1 ml of elastase (final concentration,2.7 Ag/ml) or DGVB2+ was added. Followingincubation for 30 min at 37 C, no hemolysis ofEoccurred in either mixture.

Effect of pseudomonas elastase on highlypurified C3 protein. To 1-ml portions of highlypurified C3 (2.5 mg/ml) was added either 0.5 mlof elastase (110 gg/ml) or DGVB2+. The mix-tures were then incubated at 37 C for 30 min.

Immunoelectrophoresis of the C3-buffer con-trol run with a specific antiserum to normalhuman serum showed a single precipitin arc be-hind the antigen well (Fig. 2). After C3 wastreated with elastase, the precipitin arc waswider, and the C3 protein migrated more an-odally than the C3-buffer control.

Effect of pseudomonas elastase on C3 inaged human serum. Pooled normal humanserum was incubated at 37 C until a partial

* Buffer 0C3

IIIMEIEhIPUfhEI_-Antiserum to human serum

''s'"''X'''rAA'-A<f<;' "5' $4',II

A Elastase C3+

"UUvPePwqUIAntiserum to human serum-..I

+e- :: ::.. .... ..

...

_ .. .ffi .. ..ffi .. ..

FIG. 2. Effect of 73 ,gg of pseudomonas elastase or buffer per ml on purified C3 (2.5 mg/ml) for 30 min at37 C. Antigen well: C3 treated with buffer or elastase. Antibody trough: goat anti-normal human serumantiserum.

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conversion of jl1C to f,1A could be demonstratedby immunoelectrophoresis. One milliliter of thisserum was then incubated with either 0.2 ml ofelastase (125 sg/ml) or an equivalent volume ofDGVB2+ for 30 min at 37 C. The hemolyticactivities of C3 were measured, and each mix-ture was examined by immunoelectrophoresisusing a rabbit antiserum to the ,l1C/,lA deter-minants of human C3. The elastase caused areduction of the C3 titer from 4,100 to 260, a lossof 94%. In Fig. 3, the immunoelectrophoreticanalysis demonstrates that elastase caused thecomplete conversion of the f31C to the f,lAdeterminant as well as to an additional frag-ment that migrated in the alpha region.

Effect of pseudomonas elastase and SBTIon serum C3. One milliliter of undiluted nor-mal human serum was added to each of fourtubes. One-half milliliter of elastase (115 Ag/ml)was added to one tube, 0.5 ml of elastasecontaining soybean trypsin inhibitor (SBTI)(2.1 mg/ml) to the second, 0.5 ml of SBTI to thethird, and 0.5 ml ofDGVB2+ to the fourth. After30 min at 37 C, the C3 hemolytic activity wasdetermined. The elastase reduced the C3 titer

from 20,500 to 1,300, a loss of 94%. The samereduction of C3 activity occurred with theelastase-SBTI mixture, whereas SBTI alonehad no effect.Generation by pseudomonas elastase of

chemotactic activity from C5 but not C3. Inpreliminary experiments using various condi-tions and reagent concentrations, pseudomonaselastase was added either to functionally purehuman C3 or C5. After incubation for 1 h at37 C, the chemotactic activity was measured inBoyden chambers. Since activity was generatedonly from C5, this component was used forfurther experiments.

Functionally pure C5 (0.4 ml; 1,000 U/ml)was incubated with 0.4 ml of elastase (27,g/ml)or buffer. Samples were removed periodicallyfor determination of the chemotactic activity.In two different experiments shown in Table 3,maximal activity was generated at approxi-mately 60 min compared with the buffer con-trol, but the activity decreased during 60 addi-tional min of incubation. Chemotactic activitywas reduced before 60 min when the enzymeconcentration was increased to 32 gg/ml.

...~~

nti-C3antbo

FIG. 3. Effect of 104 lAg of pseudomonas elastase or buffer per ml on C3 in aged human serum for 30 min at37 C. Antigen well: aged human serum treated with buffer or elastase. Antibody trough: rabbit anti-human C3antiserum.

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TABLE 3. Time course in generation by Pseudomonaselastase of chemotactic activity from human C5

Incubation mixture

Time Expt la Expt 2b(min) C5+

C5+ elastase C5+ C5+bufferc (14 ,g/ml) buffer elastase

15 43d 72 48 9030 30 115 43 12360 54 140 44 141120 56 60 52 55

a 3.2 x 104 PMN leukocytes/ml.b 5.3 x 104 PMN leukocytes/ml.Gey solution + 2% human albumin.

d Total number of PMN leukocytes/10 oil immer-sion fields (lOOx).

Inactivation of phagocytosis activities.Two-tenths milliliter each of human Cl (50,000U/ml), C4 (730 U/ml), C2 (350 U/ml), and C3(120 U/ml), 1 ml of BA (7 x 108/ml), and either0.3 ml of Gey solution (Mixture A) or elastase(115 Ag/ml) (Mixture C) were combined andincubated at 37 C for 30 min. The cells in eachtube were then washed twice and resuspendedin 1 ml of fresh Gey solution. Also included were1.8 ml of BAC1423 cells, which were formed andwashed prior to incubation with 0.3 ml ofelastase (Mixture B). A small sample of eachmixture was added to a rabbit antiserum tohuman ,BlC/#lA to determine whether the BAcontained cell-bound C3. Finally, 1 ml of rabbitPMNs (7.3 x 106/ml) in Gey solution was addedto each tube. The mixtures were incubated at37 C for 20 min. Thin films were then stainedwith Wright stain, and the PIs were determined.The results of two experiments (Table 4)

show that the BA in Mixture A agglutinatedwith the anti-#lC/,BlA antiserum, indicatingthat C3 was cell bound. The PIs were, respec-tively, 13.4 and 16.2. Preformed BAC1423(Mixture B) treated with elastase also ag-glutinated with ,lC/hlA antibody-, but the PIwas reduced to 3.3 and 3.4, respectively. InMixture C, where the Cl, C4, C2 and C3 weremixed with elastase and BA at the same time,the cells did not agglutinate with antibody, andthe PIs were 2.4 and 3.0, respectively.

In a variation of this experiment, the rabbitPMNs were treated with elastase and thor-oughly washed before adding them to theBAC1423. The PI was 12.1, indicating thatelastase had no apparent effect on the ability ofthe PMN leukocytes to phagocytize the sensi-tized bacteria.

DISCUSSIONIn the present study, normal human serum

was treated with an amount of highly purifiedP. aeruginosa elastase in excess of the serumelastase-inhibiting capacity. The activity of theinhibitor varied in the different sera used forthese experiments. Over 90% of all nine Ccomponents were inactivated. Whereas thoseeffects might be due to activation and subse-quent consumption of one or more of the Ccomponents, the attempts at activation of thenine functionally purified C proteins did notproduce hemolysis of unsensitized sheep E.The studies also demonstrate that the mi-

crobial elastase is a potent inactivator of func-tionally purified Cl, C3, C5, C8, and C9 in thefluid phase, whereas the other components arediminished to lesser extents (neither C4 nor C7were inactivated at enzyme concentrations of1.25 gg/ml) The activities of cell-bound Cl andC3 were also destroyed by the enzyme. In otherexperiments, SBTI did not inhibit the inactiva-tion of C3 by elastase. This confirms the reportby Morihara et al. (15) who showed that SBTIdid not suppress either the elastolytic or proteo-lytic activities of the enzyme. Since SBTIinhibits plasmin, these experiments also showthat elastase action is directly on the C compo-nents and not mediated through the activationof plasminogen (6).The observations, elastase inactivation of

both free and cell-bound Cl and C3, involve thetwo major pathways for C activation. The Csequence may be initiated by activation ofeither Cl, the "classic" pathway (20), or C3, the"alternate" pathway (17). Both pathways resultin release of phlogistic factors such as C-derivedchemotactic materials (10) and vascularpermeability factors (3, 11). Lysosomal pro-teases and other derivatives released from PMNleukocyte granules increase or prolong the in-flammatory reaction and tissue necrosis.The enzymic inactivation of the hemolytic

TABLE 4. Effect ofpseudomonas elastase (16.4 ug/ml)on fluid-phase or cell-bound C3 (BAC1423)a

Incubation Agglutination PismIxtureio with an ti-C33mixture withantibody Expt 1 Expt 2

A + 13.4 16.2B + 3.3 3.4C _ 2.4 3.0

aAfter each mixture was incubated for 30 min at37 C, agglutination was determined with an anti-serum to C3, and the PI was determined following theaddition of PMN leukocytes.

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activity of purified C1 with no effect on thetrypsin-induced C 1-esterase or its precursorsuggests that the elastase attacks the Clq orClr portion of the Cl complex (12).Phagocytosis of sensitized bacteria or erythro-

cytes by PMN leukocytes is enhanced when C3is bound to the immune complex (8). In ourexperiments, after Cl, C4, C2, and C3 wereadded to sensitized P. aeruginosa, the complexwas agglutinated by an antiserum to C3. Treat-ment of the BAC1423 with elastase did notdiminish the agglutination reactions, but the PIwas greatly reduced. It would appear from thesedata that either sufficient cell-bound C3 mole-cules in the unaltered state remained for theagglutination (but not the phagocytosis reac-tion) or the portion of the C3 molecule necessaryfor agglutination remained intact while theactive center of phagocytosis was destroyed.

In other experiments, the C3 protein in nor-mal human serum and highly purified C3 wereconverted to a faster molecular species byelastase action, as shown by immunoelectro-phoresis. In addition, a fragment of serum C3was shown to migrate in the alpha region. TheC3 was not bound to sensitized pseudomonads ifelastase was added to Cl, C4, C2, and C3together with the bacteria. This probably re-flects a direct action on the C components, sincethe enzyme had no effect on PMN leukocytes,erythrocytes, or antibody. Therefore, inactiva-tion and breakdown of C3 were demonstratedboth by functional assays and by immunoelec-trophoresis with specific C3 antibody.

In a recent paper by Ward et al. (26), it wasshown that proteinases from Serratia marces-cens and f3-hemolytic Streptococcus cleavedleukotactic fragments from human C3 and C5.Although each proteinase generated activityfrom both components, there was substrateselectivity in serum. The streptococcal protein-ase generated a C5-dependent chemotactic fac-tor, whereas the Serratia enzyme preferred C3as a substrate. In our study, we were not able togenerate chemotactic activity from functionallypurified C3 using a variety of conditions andconcentrations, but no attempt was made togenerate the factor from native C3 in serum. Itis not known from our data whether the C3chemotactic factor was generated and quicklydestroyed by elastase or not generated at all.However, elastase-induced chemotactic activitywas generated from C5. Kinetic studies showedthat increasing chemotactic activity was formedfrom C5 in 60 min, but diminished after thattime period. The magnitude of the chemotacticresponse produced by the enzyme was not great,although it was approximately threefold higher

than the control values after 60 min. Since thechemotactic activity was reduced before 60 minwith an increased enzyme concentration, elas-tase may partially destroy the C5 factor as soonas it is fragmented from the parent molecule.Ward and Hill (27) also showed that prolongedperiods of incubation of human C5 with anenzyme from lysosomal granules reduced theamount of activity.

Various Pseudomonas strains have beenshown to elaborate hemolysin and other extra-cellular enzymes (13), some of which producehemorrhagic tissue necrosis in experimentalanimals. However, unlike many gram-negativepathogens, endotoxin from P. aeruginosa doesnot appear to be an important factor in thepathogenesis of septic lesions (24). Since severeP. aeruginosa infections are often characterizedby a lack of an inflammatory response by thehost, our data appear to indicate that elastasemay be implicated. The enzyme not only inacti-vated fluid-phase C components, it also im-paired other events that are important in theinflammatory response. It generated, and thendestroyed, the chemotactic fragment from C5,and it reduced the ability of PMN leukocytes tophagocytize sensitized pseudomonads becauseof its action on cell-bound C3.

ACKNOWLEDGMENTS

The paper was supported, in part, by The John A. Hart-ford Foundation and by Public Health Service grant no.AM-09001-09 from the National Institute of Athritis, Metabo-lism, and Digestive Diseases.We appreciate the excellent technical assistance of Pa-

tricia Arnold.

LITERATURE CITED

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2. Austen, K. F. 1971. Inborn and acquired abnormalities ofthe complement system of man. Hopkins Med. J.57:74.

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4. Curtin, J. A., R. G. Petersdorf, and I. L. Bennett. 1961.Pseudomonas bacteremia: review of 91 cases. Ann.Intern. Med. 54:1077-1107.

5. Davis, B. J. 1964. Disc electrophoresis. II. Method andapplication to human serum proteins. Ann. N.Y. Acad.Sci. 121:404-427.

6. Donaldson, V. H., and F. S. Rosen. 1964. Action ofcomplement in hereditary angioneurotic edema: therole of Cl esterase. J. Clin. Invest. 43:2204-2213.

7. Forkner, C. E., Jr., E. Frie, III, J. H. Edgcomb, and J. P.Utz. 1958. Pseudomonas septicemia: observations ontwenty-three cases. Amer. J. Med. 25:877-889.

8. Gigli, I., and R. A. Nelson, Jr. 1968. Complement-dependent immune phagocytosis. I. Requirements forCl, C4, C2, C3. Exp. Cell Res. 51:45-67.

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