Journal of Clinical InvestigationVol. 44, No. 4, 1965

Relationship between Particle Size and Biological Activityof E. coli Boivin Endotoxin *

HERMANNBEER,t THEOPHIL STAEHELIN, HERNDONDOUGLAS, ANDABRAHAMI. BRAUDEt

(From the Department of Medicine, School of Medicine, University of Pittsburgh,Pittsburgh, Pa.)

Many investigators have tried unsuccessfullyto demonstrate that a specific chemical structureis responsible for the toxic action of bacterialendotoxins. Practically all of the protein (orpeptide) and lipid constituents of the endotoxinmolecule may be removed without affecting sig-nificantly its biological activities, and the carbo-hydrate moiety loses toxicity after it is depolymer-ized by even mild acid hydrolysis (1-4). Recentstudies have also shown that toxicity does notdepend upon the presence of the polysaccharideside chains that give the endotoxin its O-antigenicspecificity (5).

Other observations indicate that the size ofthe molecule, or molecular aggregate, is a moreprominent determinant of toxicity than the chem-ical composition (6-7). Variations in particlesize were noted by Schramm, Westphal, andLuderitz (8) in studies involving electron micros-copy, sedimentation in the ultracentrifuge, andviscosity measurements. They demonstrated thatendotoxins in solution are polydisperse, and theaverage particle weight was estimated at 24 mil-lion. The larger particles are formed by aggre-gation of identical subunits with a molecularweight of about 1 million.

The purpose of the present work was to getmore quantitative information about the polydis-persity of a Boivin endotoxin by sucrose densitygradient centrifugation of endotoxin labeled uni-

* Submitted for publication August 5, 1964; acceptedDecember 10, 1964.

Supported in part by U. S. Army medical contract no.DA-49-007-MD-872, grant H-3220 from the U. S. PublicHealth Service, and a grant from the Health Researchand Services Foundation.

t Recipient of financial assistance from the Universityof Zurich.

tAddress requests for reprints to: Dr. A. I. Braude,University of Pittsburgh School of Medicine, Pittsburgh,Pa. 15213.

formly with C14 and to compare the biologicalactivity in particles of different size. We foundthe highest lethal activity in those of an inter-mediate size, thus providing further evidence thatsize was a determinant of biological activity.When this concept was challenged, however, bydissociating the aggregates with sodium dodecylsulfate (SDS) into smaller subunits, their toxicmanifestations were unchanged. Particle size isthereby ruled out as a significant determinant oftoxicity.

MethodsReagents. Uniformly labeled glucose-C",1 Na2Cr'04 in

sterile, pyrogen-free solution (Rachromate),' pyrogen-free distilled water,2 0.9% NaCl,' 5% glucose,2 and so-dium dodecyl sulfate (SDS) ,3 used without furtherpurification, were obtained commercially.

Decontamination. Pyrogens were eliminated fromequipment by heating at 1700 C for at least 2 hours andfrom heat labile materials by immersing in 2i N NaOHfor at least 2 hours. The NaOHwas removed by rins-ing with pyrogen-free distilled water. Sucrose gradientswere prepared with pyrogen-free distilled water and su-crose solutions that were sterilized by Seitz filtration andshown to be free of pyrogens.

Preparation of endotoxins. Endotoxin was preparedby Boivin's method from a nonpiliated and nonflagellatedstrain of Escherichia coli 0: 113 that was originally iso-lated from the blood of a patient. It was stored as afrozen stock culture in trypticase soy broth, and smoothcolonies were obtained on subculture for preparation ofendotoxin in a synthetic medium containing glucose andNH4+ as sole sources of carbon and nitrogen as previ-ously described (9). It was labeled with Na2Cr'04 ac-cording to a standard procedure in this laboratory (10).After dialysis against distilled water, the Cr5-labeledendotoxin was twice precipitated with ethanol to elimi-nate any free chromium.

Endotoxin was labeled biosynthetically with C14 bygrowing E. coli in 125 ml of synthetic medium contain-

' Nuclear-Chicago Corp., Des Plaines, Ill.2Abbott Laboratories, North Chicago, Ill.8 Fisher Scientific Co., Fair Lawn, N. J.

592

RELATIONSHIP OF PARTICLE SIZE TO ACTIVITY OF E. COLI ENDOTOXIN

ing uniformly C"-labeled glucose instead of cold glu-cose. The labeled cells were mixed with a mass of un-labeled cells, and the endotoxin was prepared as usual.Since under these conditions all carbon atoms in theproducts of bacterial metabolism had statistically thesame label, the amount of endotoxin in a sample couldbe calculated from the radioactivity.

In batch E-68-C" a total of 50 ,uc C" of uniformly la-beled glucose was used; in batch E-69-C", 150 ,uc.Specific activity of endotoxin E-68-C" was 4,500 cpm permg, and that of E-69-C" was 28,000 cpm per Ag.

Antiserum. An immune serum was prepared in rabbitswith cells of E. coli 0: 113 that had been boiled for 21hours. The animals were injected intravenously everyother day for 3 weeks and bled 5 days after the lastinjection.

Sucrose density gradient. Linear sucrose gradientsfrom 1.0 M to 0.2 M were prepared in a special mixingchamber by adding distilled water to 1 M sucrose solu-tion with continuous stirring by a magnetic stirrer. Thesucrose solution of decreasing molarity was carefullylayered into a centrifuge tube with a polyvinyl tubeconnected with the bottom of the mixing chamber to a

volume of 28.5 ml. The test sample, containing 1 mg ofendotoxin dissolved in 1 ml 5% glucose, was layered over

the top through a tuberculin syringe.Ultracentrifugation was performed in a Spinco model

L ultracentrifuge with an SW25.1 swinging bucket rotorfor 17 hours at 25,000 rpm and a running temperatureof + 50 C. After centrifugation the bottom of the cen-

trifuge tube was perforated, and 28 to 30 fractions of 1

ml were collected in graduated test tubes.Spectrophotometric analysis. Ultraviolet (UV) -ab-

sorption was determined in a Beckman DB spectropho-tometer provided with a Photovolt automatic recorder torecord scanned UV spectra.

Radioactivity measurements. Radioactivity of C"4-la-beled samples was determined by suspending samples inthixotropic gel and counting in a Packard Tri-Carbliquid scintillation counter. Cr' radioactivity was meas-

ured in a liquid well scintillation counter.4Gel filtration. Sephadex gel of different porosities was

used. Small columns were prepared in disposable plasticor glass syringes with a bed volume of 10 to 70 ml. Theflow rate was adjusted as desired by using needles ofdifferent gauge. Water and saline were used inter-changeably as a vehicle for the samples and for elution.Fractions of 1 or 2 ml were collected.

Lethality. Adrenalectomized mice were used to providea system sensitive enough for assaying the lethality ofthe minute amounts of endotoxin in fractions of the su-

crose gradients. Specific pathogen-free mice (SPF) ,6weighing 15 to 25 g, were challenged 48 hours afteradrenalectomy (6). (Conventional mice were unsatis-factory because latent ectromelia infection was activatedin them by the surgery.) The SPF mice died between8 and 48 hours and most between 12 and 24 hours after

4Nuclear-Chicago Corp., Des Plaines, Ill.5Manor Farms, Staatsburg, N. Y.

injection of endotoxin. Samples of adjacent fractionsfrom the sucrose gradient were pooled and diluted in nor-mal saline, and 0.25-ml volumes were injected intrave-nously into 10 to 15 adrenalectomized mice per dilution.The amount of endotoxin injected was determined fromits radioactivity. Calculations of the LD0o and of therelative potencies with 95%1 confidence limits were doneby the probit analysis of Finney (11) The data wereprocessed in a computer LGP 30 for which a probit analy-sis program was written.6

Pyrogenicity. The test preparations were injected intothe marginal ear vein of carefully conditioned albinorabbits weighing 3 to 5 kg. Rectal temperatures weretaken with a Sargent thermistor apparatus hourly for8-hour periods before and after injection of the samplesto be tested and the results expressed in square centi-meters of fever according to the method of Keene, Sil-berman, and Landy (12). The fractions from sucrosegradient were diluted serially tenfold and 0.25-ml por-tions injected. The dose injected was calculated fromthe radioactivity of the C1' label. The relative pyrogenicpotency of the sucrose gradient fractions as compared tothe parent endotoxin was calculated with 95% confidencelimits by the parallel line assay (13). These data werealso processed in the computer with a parallel line assayprogram.6

Antibody titration by the hemagglutination procedure.The ability of the test materials to stimulate antibodyformation was determined in the rabbits that were usedfor the pyrogen assay. Seven and 14 days later the titerof serum antibody was determined by hemagglutinationof red cells sensitized with alkali-treated endotoxin.The technique for the hemagglutination test has beendescribed elsewhere (14).

Antigen analysis by precipitin reactions in agar. Pre-cipitin reactions in agar were carried out by the method ofOuchterlony (15), as modified by Halbert, Swick, andSoner (16). The antiserum was placed into the centerwell and the different antigens into the outer wells.The plates were kept for 7 days at + 40 C.

ResultsSucrose density gradient analysis of C"4-labeled

E. coli 0:113 endotoxin

Figure 1 shows the distribution of radioactivityand optical density at 254 mu in a typical sucrosegradient centrifugation of the endotoxin batchE-68-C14. The largest particles were collectedin the fractions from the bottom of the gradient,and the smallest were contained in the top frac-tions. No pellet was formed with any of the

ff We are very much indebted to Dr. D. Kodlin, De-partment of Biostatistics, Graduate School of PublicHealth, University of Pittsburgh Medical School, and hisco-workers who wrote the computer programs and helpedus with the evaluation of the results of the bioassays.

593

H. BEER, T. STAEHELIN, H. DOUGLAS,AND A. I. BRAUDE

z,E i) * ._ 3001.0

7 _ 2002~~~~~~~~~~00

200

0 05 10 15 20 25 30

FRACTION NUMBER(EFFLUENT VOLUME, ml)

FIG. 1. DISTRIBUTION OF RADIOACTIVITY AND OPTICAL

DENSITY IN SUCROSE DENSITYGRADIENT OF C14-LABELEDBOIviN ENDOTOXIN.

radioactive Boivin endotoxins of E. coli 0:113we have tested so far under these conditions ofcentrifugation. Radioactivity was almost evenlydistributed from the bottom of the tube up to

9.0

8.0

2.0-

E

6.0-

5.0

4a 0z

0 3.0-

2.0

1.0

0

0 10 20 30 40 50

FRACTION NUMBER(EFFLUENT VOLUME, ml)

FIG. 2. GEL. FILTRATION OF E-68 ENDOTOXINON SEPHA-

DEx G-25. Bed volume, 50 ml; fraction volume, ml;

flow rate, .20 ml per hour; sample, 3.5 mg.

about 23 ml, a finding indicative of a high degreeof polydispersity in the parent endotoxin withvariation of particle size over a wide range. Inthe top fractions, radioactivity and optical densityformed a high peak. The UV-absorption spec-trum of the peak fraction 27 revealed the presenceof- a nucleic acid, presumably RNA. The differ-ence in height of the peaks for radioactivity andoptical density implies another small molecularcompound not absorbing in the UV region. Sinceapproximately 50% of the total radioactivity, or0.5 mg of parent endotoxin, was contained inthe terminal peak, and assuming an absorbancy

5000

4500 A0.5 % SDS : s

4000 s*@ 0

^3500B

Cf3000*

-2500 a

2000 |0

Ix 1500a NO

1000 / SDS

500

5 1 0 15 20 25 30FRACTION NUMBER(EFFLUENT VOLUME, ml)

FIG. 3. DISTRIBUTION OF RADIOACTIVITY IN SUCROSEDENSITY GRADIENT OF C14-LABELED ENDOTOXINBEFOREANDAFTER INCUBATION WITH SODIUM DODECYL SULFATE

(SDS).

of about 25 ODU per mg of nucleotide material,the observed peak at 254 mu of about 6 ODUwould account for only 50% of the radioactivity.This nucleic acid contaminant could easily beseparated from the parent endotoxin by gel filtra-tion on Sephadex G-25. (Figure 2).

The sucrose density gradient centrifugation ofthe same batch of endotoxin gave highly repro-ducible results with virtually identical distributionof radioactivity and optical density. A similardistribution of radioactivity in sucrose gradientswas obtained with C'4-labeled E. coli 0:113 en-dotoxin (E-69-C14) that possessed no nucleicacid contaminants (Figure 3).

594

RELATIONSHIP OF PARTICLE SIZE TO ACTIVITY OF E. COLI ENDOTOXIN

TABLE T

Lethal activity of sucrose density gradient fractions compared to parent endotoxin*

95% confidence Relative 95% confidencePooled fractions LD6o limits potency limits

logParent endotoxin 0.152 0.116-0.198 1.00

1-3 0.092 0.068-0.123 1.66 0.94-2.954-6 0.060 - 0.036-0.104 2.39 1.30-4.407-10 0.056 0.036-0.088 2.73 1.51-4.93

11-13 0.046 0.033-0.065 3.34 1.51-7.0914-16 0.046 0.033-0.062 3.36 1.72-6.5317-20 0.075 0.053-0.106 2.04 1.08-3.8521-24 0.471 0.221-1.00 0.34 0.17-0.6825-28 t t

* Results calculated by probit analysis (11).t The toxicity of these fractions was so low that no end point could be obtained in the bioassy. Only one out of ten

mice was killed by 2.7 jug.

Lethality. The LD50 of the parent endotoxinE-68-0:113-C14 in normal SPF mice is approxi-mately 300 ug, and in adrenalectomized mice0.152 jug, with 95% confidence limits of 0.116 pgand 0.198 jug. The sensitivity of the mice toendotoxin is thus increased about 2,000 timesafter removal of the adrenals.

The distribution of lethality in a sucrose gra-dient centrifugation of the endotoxin is shown inTable I. The fractions with the highest lethalactivity (11 to 16) were found in the midportionof the gradient where the increase over the parentendotoxin was approximately threefold. Both thefractions from the bottom and from the top ofthe gradient were less active. Fractions 1 to 3were not significantly different from the parentendotoxin but were less active than fractions 11to 16. Fractions 4 to 10 and 17 to 20 werestill significantly more toxic than the parent endo-toxin. Fractions 21 to 28 from the upper partof the gradient were considerably less active thanthe parent endotoxin.

From the input of 1 mg endotoxin containingabout 6,500 LD50, we recovered about 5,800 LD50(90%) in gradient fractions 1 to 24.

Pyrogenicity. The dose-febrile response curvefor the parent endotoxin E-68-0:113-C'4, withthe pyrogenic response expressed as square centi-meters of fever, is shown in Figure 4. Each pointon the curve represents the mean fever responseof four to eight rabbits. From a dose as smallas 2.5 ,utg some of the rabbits suffered shock andsubsequent drop- of temperature far below base-line values, so that they were not used for con-struction of the curve. The toxic effects after

injection of 10 ,ug were probably responsible forthe reduced fever response at this dose level.

Single fractions were assayed, and two tofour rabbits were used for each dilution. Thecomputer analysis of the fever indexes by theparallel line assay method (13) showed that frac-tions 1 to 22 were more pyrogenic than theparent endotoxin, with approximate relative po-tencies between 1 and 14. Fractions 23 to 28were less pyrogenic than the parent endotoxin,with approximate relative potencies between 0.25and 0.007.

Fever responses higher than 55 cm2 were notconsidered, and the 28 test preparations showedno evidence of deviation from linearity. Thus the

E

x 40wz

a 30w

O' ,

.001 0.01. 0.1 1.0DOSE, jig

10 100

FIG. 4. RELATIONSHIP BETWEENFEBRILE RESPONSEINRABBITS AND DOSE OF UNTREATEDE. COLI 0 :113 BOIVINENDOTOXIN.

595

H. BEER, T. STAEHELIN, H. DOUGLAS,ANDA. I. BRAUDE

TABLE II

Stimulation of hemagglutinating antibody by a single injection of endotoxin E-68-C14,before and after treatment with 0.5% SDS (sodium dodecyl sulfate)

Untreated endotoxin SDS-treated endotoxin

Average AverageNo. of rise in No. of rise in

Dose animals titer* Range animals titer Range

ug0.001 4 1.0 0-4 4 1.5 0-40.01 4 11.0 4-32 4 0.5 0-20.025 4 1.5 0-40.1 8 2.5 0-4 7 1.5 0-40.5 4 8.5 2-161.0 4 21.0 4-32 4 5.0 0-162.5 4 45.0 4-1285.0 8 32.5 4-64 4 18.5 2-64

10.0 4 80.0 16-256 4 2.0 0-450.0 4 1.0 0-2

* A fourfold rise or higher is significant. Rise in titer = final/initial. If no rise occurred, the result is expressed as0 instead of 1.

area of fever, in the response range used (< 55cm2), is linearly related to log dose.

Five of 28 fractions showed an assay with a

deviation from parallelism of response lines (frac-tion no. 6, 8, 11, 17, 21), i.e., the slope of the logdose-febrile response curve was significantly dif-ferent from the slope for the parent endotoxin.Careful analysis of the data, together with an

analysis of variance for test preparations thatwere assayed on a different batch of rabbits atthe same dose level, revealed that in some in-

stances significantly different results were ob-tained with the same preparation. Hence, theobserved differences in slope were not due to a

fundamental invalidity of the assay system wherestandard preparation (parent endotoxin) and test(fractions) would not behave like -dilutions ofone ingredient (13).

Because of these shortcomings of the pyrogen

assay, we are not reporting the relative potencies

of the 28 fractions and have made no attempt todraw quantitative conclusions. It is safe to say,

however, that the fractions 23 to 28 were lessactive than those from the lower part of thegradient and the parent endotoxin.

A number of fractions from different locationswithin two other gradients (endotoxin batch E-69-C14) were also assayed for pyrogenicity. Theresults were similar to those obtained with thegradient of endotoxin E-68-C14.

Titration of antibodies by the hemagglutinationprocedure. The rise in titer of hemagglutinatingantibodies produced by injection of different dosesof parent endotoxin is shown in Table II, andthat produced by the different fractions of a

sucrose gradient is shown in Table III. Thestimulus to antibody formation was much greaterin fractions from the lower half of the gradientthan from the upper 12 fractions. As in the case

of pyrogenicity, individual variation within occa-

TABLE III

Stimulation of hemagglutinating antibody by fractions of sucrose gradients

Dilution 1:10 Dilution 1:100 Dilution 1:1,000

Average Average AverageFraction No. rise in titer* No. rise in titer No. rise in titer

no. animals (final/initial) Range animals (final/initial) Range animals (final/initial) Range

1-4 8 22.00 0-64 8 3.25 0-8 8 0.50 0-45-8 8 40.50 2-128 12 9.00 2-32 12 2.20 0-49-12 8 16.00 2-64 8 4.00 2-8 8 6.50 0-16

13-16 8 12.25 2-64 8 7.25 0-16 8 4.75 0-817-20 9 4.45 0-16 8 2.00 0-4 8 0.250 0-221-24 12 3.85 0-16 8 2.5 0-8 8 2.75 0-825-28 30 3.75 0-16 20 1.2 0-4 8 0.50 0-2

* A fourfold rise or higher is significant. If no rise occurred, the result is expressed as 0 instead of 1.

596

RELATIONSHIP OF PARTICLE SIZE TO ACTIVITY OF E. COLI ENDOTOXIN

WHOLEBOIVIN ENDOTOXIN

000

0

BOIVIN ENDOTOXIN FRACTIONS 1- 29 FROM SUCROSEGRADIENT

3 9 15

0.0 2 100 00 8 160 0 1~ 14

I1 1ln 0@2°7 17o 136 12 18

21

owto230 4 019

26

025

024 29

FIG. 5. ANTIGEN ANALYSIS IN AGARDOUBLEDIFFUSION OF PARENTENDO-TOXIN AND ENDOTOXIN FRACTIONS OBTAINED BY SUCROSEDENSITY GRADIENTCENTRIFUGATION. Antigen in outer wells, serum in center. In the upperplate only one well contains antigen.

sion and batch-to-batch differences made quanti-tative evaluation of the results impossible.

Antigenic analysis of endotoxin E-68-0 :113-C14in agar double diffusion. A 0.1%o solution ofendotoxin E-68-0:113 displayed three precipita-tion lines against rabbit antiserum prepared byinjection of boiled E. coli.

Undiluted fractions of the sucrose gradient alsoproduced, as expected, the three precipitation linesof the parent endotoxin (Figure 5). The lineof the slow-moving antigenic component waspresent in fractions 1 to 17; in 18 to 23 therewas only one line; then the line of the fast-movingantigen close to the center well appeared withincreasing intensity.

To get a rough idea of the number of antigenicgroups capable of reacting with antiserum, serialtwofold dilutions of each fraction were placed inthe outer wells and antiserum in the center wellof agar diffusion plates. The results are ex-pressed as the reciprocal of the highest dilutionto give one clearly visible precipitation line. Fig-ure 6 indicates that the distribution of serologicalactivity is similar to the distribution of radio-activity.

Sucrose density gradient analysis of Cr5l-labeledE. coli endotoxin

Gel filtration on Sephadex showed that nucleicacid contaminants were not labeled.

The behavior of chromate-labeled endotoxin insucrose gradients was examined with a prepara-

64

z0

LL_0 32

0

W 16

1 5 10 15 20 25 30FRACTION NUMBER(EFFLUENT VOLUME, ml)

FIG. 6. PRECIPITIN TITERS IN AGARDOUBLEDIFFUSIONOF FRACTIONS OBTAINED BY ULTRACENTRIFUGATON OFE. COLI ENDOTOXININ SUCROSEGRADIENT.

597

H. BEER, T. STAEHELIN, H. DOUGLAS,AND A. I. BRAUDE

20

0

xE

I-4

0

0

10 15 20 25 30FRACTION NUMBER(EFFLUENT VOLUME, ml)

FIG. 7. DISTRIBUTION OF RADIOACTIVITY IN SUCROSE

DENSITY GRADIENT OF CHROMATE-LABELEDENDOTOXIN BE-

FORE AND AFTER TREATMENTWITH 0.5% SDS. Note thatchromate distribution before SDS treatment closely paral-lels lethality in Table I.

tion of endotoxin that was originally free of nu-

cleotide contaminants (E. coli 0 :113-E-67-Cr5l).The labeled endotoxin, with a specific activity of125,000 cpm per mg, was dissolved in 5% glucosein a concentration of 1 mg per ml. Figure 7shows the distribution of radioactivity in a typicalgradient of a freshly prepared solution. In con-

trast to our results with the C14-labeled endotoxin,which showed that 50% of the radioactivity isassociated with the small particles in the fractionsat the top, we found that the highest radioactivityfrom Cr51 appeared in the midportion of thegradient in parallel with the toxic material andthat the nontoxic material of low molecular weightwas virtually unlabeled. The unlabeled nontoxicantigen in the top fractions was demonstrated byagar double diffusion.

The distribution pattern of the radioactivity ofchromate-labeled endotoxin in sucrose gradientswas as constant as for C14-labeled endotoxin.

Action of the anionic detergent SDSon endotoxinSDS was used in an attempt to dissociate the

endotoxin particles of different size into more

uniform particles.A) Sucrose density gradient analysis of endo-

toxin in the presence of SDS. Solutions of endo-

toxin (0.2%o) in 5%o glucose were mixed withequal volumes of a 1 %o solution of SDS in water,shaken vigorously, and allowed to stand for atleast 1 hour at room temperature. Longer in-cubation overnight did not change the result. Itwas unnecessary to incorporate SDS into thegradient, since the same distribution was obtainedwith SDS-treated endotoxin whether or not theentire gradient contained 0.5%o SDS.

Figure 3 depicts the distribution in the sucrosegradient of a C"4-labeled E. coli endotoxin (E-68-C14) preincubated with 0.57%o SDS. SDScaused a shift of radioactivity to the top of thegradient.

The influence of SDS on the sucrose gradientanalysis of a Cr51-labeled endotoxin (E-67) isshown in Figure 7. The effect of SDS on thesedimentation pattern of endotoxin carrying thechromate label was more pronounced than on theC14-labeled endotoxin. Most of the radioactivematerial was shifted upward in the gradient witha peak at fraction 24 and a shoulder between frac-tions 19 and 21. Very little material remainedin the lower half of the gradient. The majorityof the chromate-labeled components was thus dis-sociated into two components.

B) Influence of SDS on agar double diffusion.After incubation with 0.5% SDS the outermostprecipitation line of the untreated endotoxin E-67disappeared, and the two remaining lines indicatedthat SDS treatment did not produce a homogene-ous antigen. When the endotoxin was separatedagain from the SDSby precipitation with ethanol,the dissolved precipitate gave the three linescharacteristic of the parent (untreated) endo-toxin.

Identical agar diffusion patterns were producedwith fractions from the -sucrose gradient aftertreatment of either C14_ or chromate-labeled en-dotoxin with SDS, as shown in Figure 8. Noprecipitation lines were detectable in fractions 1to 14. From fractions 15 to 29, three differentlines developed: 1) an outer, hazy, faint line of aslow-moving antigen was discernible from frac-tions 20 to 29, 2) an inner, well-defined, thin lineappeared in fraction 24 and remained visible tofraction 29, and 3) an intermediate line waspresent from fraction 15 to 29 and shifted towardthe center well in the higher fractions, due to anincrease in concentration.

598

RELATIONSHIP OF PARTICLE SIZE TO ACTIVITY OF E. COLI ENDOTOXIN

16 15

0017Q0 014

0018 13

22 21

0 0*23 T 0 ?20

0024 19

28 27

0 029 O id NO26

25FIG. 8. ANTIGEN ANALYSIS IN AGARDOUBLEDIFFUSION

OF ENDOTOXIN FRACTIONS OBTAINED BY SUCROSEDENSITYGRADIENT CENTRIFUGATION AFTER INCUBATION WITH 0.5%SDS. Antigens in outer wells, serum in center well. Noprecipitation lines formed in fractions 1 to 12.

C) Gel filtration of SDS-treated endotoxin.The dissociation of endotoxin observed under theinfluence of SDS was next investigated with gelfiltration on Sephadex. The columns were equili-brated with a 0.5% solution of SDS in H20, andthe elution was carried out with the same solu-tion. Endotoxin E-67 was dissolved in 0.5%SDS in H20 in a final concentration of 0.5%o.On G-25, G-50, and G-75 the endotoxin waseluted in a single, sharp peak after the void vol-ume, as determined by UV absorption and assayfor carbohydrate and protein. On G-100 ashoulder appeared on the right side of the endo-toxin peak, which on G-200 was better separatedand gave rise to a second peak containing carbo-hydrate and protein.

D) Bioassays of SDS-treated endotoxin. 1)Pyrogenicity. Intravenous injection of 5 mg of

SDS in NaCi, the highest dose tested, was harm-less to rabbits and produced no change in body tem-perature. E. coli endotoxin E-68-C14 was incubatedovernight with 0.5% SDS in saline at concentra-tions desired for the pyrogen assay. Four toeight animals were injected at each dose level.The pyrogenic response for SDS-treated endo-toxin was not significantly different from the un-treated endotoxin; the highest response was alsofound at a dose of 5.0 Mug, and 10.0 Mg produceda reduced fever resp6nse.

2) Induction of pyrogen tolerance The resultsof tolerance studies, illustrated in Figure 9,showed that one injection of 0.5 ug SDS-treatedendotoxin conferred tolerance 24 hours later toboth the SDS-treated endotoxin and the parentendotoxin at the same dose level.

3) Shwartzman phenomenon. The productionof the local Shwartzman- phenomenon was testedin adult albino rabbits. Twelve rabbits were pre-pared by an intradermal injection of 0.25 mgendotoxin E-69. Twenty-four hours later, sixof these rabbits were challenged by intravenousinjection of 0.2 mg endotoxin treated with 0.5%SDS in saline. Four of the remaining preparedrabbits received the same dose of untreated endo-

0.5 pg SDS-TREATED 0.5 #g PARENTENDOTOXIN ENDOTOXIN

TI F TI TI

2 5-19 5-20 5-21

U 5-19 5-20 5-21

ir4 _ T3 _ T3 - T3

5-19 5-20

5-20 5-210n2 460246

5-20 5-21

TIME IN HOURS

FIG. 9. PRODUCTION OF PYROGEN TOLERANCE BY SDS-TREATEDENDOTOXININ SIX RABBITS(T5 TOT5).

599

H. BEER, T. STAEHELIN, H. DOUGLAS,AND A. I. BRAUDE

toxin intravenously, and two received 1.25 mgSDS alone, a dose equal to that given to the firstgroup of rabbits.

Rabbits injected with untreated and those in-jected with SDS-treated endotoxin developed anidentical local Shwartzman reaction, whereas rab-bits challenged with SDS alone showed only adelayed inflammatory reaction with no hemor-rhage or necrosis at the site of the preparativeinj ection.

SDS-treated endotoxin could not be used forthe preparative injection, since SDS causes focalnecrosis of the skin upon intradermal injection.

4) Neutropenia. The action on leukocytes ofSDS-treated endotoxin was compared to that ofuntreated endotoxin by injecting 0.5 ptg or 5 lkgintravenously into adult albino rabbits. Fouranimals were used for each dose of SDS-treatedor untreated endotoxin. Leukopenia was marked15 minutes after injection, reached its maximum1 hour after injection, and was followed by neu-trophilia at 4 hours. No significant differencewas observed between untreated and SDS-treatedendotoxin.

SDS alone in a dose equivalent to the amountgiven to the animals that received SDS-treatedendotoxin caused no appreciable change in theleukocyte count during the time observed.

5) Lethality. The lethality of SDS-treatedendotoxin in mice cannot be evaluated because ofthe high toxicity of SDS upon intravenous or in-traperitoneal injection.

The lethal action of SDS-treated endotoxincould be appraised more satisfactorily in rabbits.Six albino rabbits weighing 2.5 to 5 kg wereinjected intravenously with each of a series ofgraded doses of endotoxin that had incubatedovernight at room temperature with 0.5%o SDS.All the deaths occurred within 24 hours afterinjection. The mortality rate for each dose wasas follows: 5.0 mg endotoxin, three of six rab-bits; 2.5 mg endotoxin, two of six rabbits; 1.25mg endotoxin, three of six rabbits; 0.625 mg en-dotoxin, two of six rabbits; 0.312 mg endotoxin,zero of six rabbits. With 2.5 mg untreated endo-toxin, three of six rabbits died.

The lethality of SDS-treated endotoxin in rab-bits is similar to that obtained with untreatedendotoxin (17). In evaluating the results wehave to consider that the lethal action of SDS-

treated endotoxin may be due to the presenceof undissociated endotoxin as was demonstratedin gel-filtration experiments. The results, there-fore, are not conclusive.

6) Antigenicity. The stimulation of hemag-glutinating antibody by SDS-treated endotoxinwas studied in the same animals that were usedfor the pyrogen assay. The result are shown inTable II. They indicate that SDS-treated endo-toxin is a poor antigen in the rabbit. Whereasa dose of 1.0 ,ug or higher of untreated endotoxinstimulated a fourfold or higher rise in hemag-glutinating antibody in 20 of 20 rabbits, onlytwo rabbits of 16 had a higher than fourfoldincrease in titer, and four rabbits had a fourfoldincrease with SDS-treated endotoxin.

Discussion

The finding that toxicity increased with par-ticle size was in keeping with previous impres-sions that toxicity was a function of size. If thispremise were true, it should have been possibleto abolish toxicity by depolymerizing the particlesinto smaller units. The problem was thereforeapproached by examining simultaneously the ef-fects of SDS on particle size, toxicity, and sta-bility of the chromate label of endotoxin. Theresults did not support the original premise, forthey showed that SDS reduced endotoxin to asmall monodisperse particle with a size close tothat of the nontoxic fractions of the parent endo-toxin, but without a loss of toxic activity orchromate label.7 These findings indicated thatsize is not responsible for toxicity and are remi-niscent of those reported by Skarnes and Chedid(19), who isolated from normal mouse serum analpha globulin fraction that seemed to reduce themolecular size of Salmonella enteritidis endotoxinwithout producing a loss of toxicity or chromatelabel. They postulated that reduction in size bya sserum component was a preliminary step todetoxification in the blood stream, since normal

7 The effect of SDS varies with the endotoxin.Oroszlan and Mora (18) found a reduction in particlesize of Serratia marcescens endotoxin and a striking re-duction in its tumor-damaging properties. We obtaineda paradoxical effect with a commercial S. marcescensendotoxin labeled with chromate; there was a shift ofradioactivity towards the bottom of the gradient. Thissuggested to us that larger aggregates were formed bydetergent bridges between the endotoxin particles.

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RELATIONSHIP OF PARTICLE SIZE TO ACTIVITY OF E. COLI ENDOTOXIN

sera could detoxify the endotoxin only if it hadfirst been dissociated into smaller units by thealpha globulin fraction. This detoxification byserum was accompanied by a loss of the chromatelabel from endotoxin. The close association of

chromate ion with the toxic portion of the Boivinextract was demonstrated not only in the suc-

rose gradient, but also under a variety of othercircumstances in vivo and in vitro (6, 7, 19).The binding of chromate may thus provide an im-portant clue to the mechanism of toxicity.

The failure of SDS to affect the lethal, pyro-

genic, neutropenic, or Shwartzman-inducing prop-

erties of endotoxin, or to dislodge the chromatelabel, indicates that the subunits of the macro-

molecule possess full toxicity. Although thesesubunits resemble in size the small nontoxic an-

tigen in the Boivin extract, they are easily recog-

nized as different substances by their toxicity andaffinity for the chromate label.

The association of toxicity with large particlesin the gradient suggests that the toxic subunitsare assembled into large aggregates during syn-

thesis of the cell wall. In addition to the toxicaggregates, it would seem that nontoxic aggre-

gates of variable size are present in the Boivinantigenic complex and reduce toxicity per unitweight on both sides of the peak defined bylethality and chromate labeling. The failure ofSDS-treated endotoxin to stimulate formation ofhemagglutinating antibody suggests that toxicitymay be dissociated from immunogenicity, but notfrom the ability to induce tolerance. Althoughthis dissociation could represent the destructionof a nontoxic contaminating antigen, it mightalso result from the degradation of a toxic anti-gen into subunits too small for antibody stimula-tion.

To solve this problem and others raised bythe present study, we are pursuing the separationof the toxic from the nontoxic fraction of theendotoxin complex and attempting to clarify thenature of the reaction between chromate andendotoxin.

Summary

The relationship between particle size and bio-logical activity of Escherichia coli 0:113 endo-toxin (Boivin) labeled with C14 or Cr5' was

studied within a density gradient containing su-crose in concentrations ranging from 10 to 34%o.Approximately one-half of the Boivin extract col-lected near the top of the gradient as a relativelynontoxic substance capable of precipitating anti-serum in agar but showing little if any capacityto stimulate antibody formation. The remainderof the endotoxin complex was uniformly distrib-uted throughout the gradient with peak toxicityin the midportion of the gradient. Radioactivityfrom chromate-labeled endotoxin seemed intimatelyassociated with toxicity, in contrast to that fromC14, which was uniformly distributed throughoutthe endotoxin complex and produced a high peakat the top of the gradient. Dodecyl sulfate re-duced the endotoxin into small monodisperse par-ticles that appeared near the top of the gradientwith a sharp reduction in ability to stimulate anti-body formation but no demonstrable loss of tox-icity or chromate label. The results indicatethat toxicity is not a function of particle size, de-spite an apparent association between size andtoxicity in endotoxin prepared by the Boivintechnique. This association of toxicity with largeparticles suggests that the toxic subunits areassembled into large aggregates during synthesisof the bacterial cell wall.

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