SYMPOSIUM ONBACTERIAL ENDOTOXINS1 II. POSSIBLE …bactericidal power of serum for Escherichia coli wasalso elevated after bacterial cell wall (21) or endotoxin administration (9)

SYMPOSIUM ON BACTERIAL ENDOTOXINS1

II. POSSIBLE MECHANISMS WHEREBY ENDOTOXINS EVOKE INCREASEDNONSPECIFIC RESISTANCE TO INFECTION

JAMES L. WHITBY,2 J. GABRIEL MICHAEL,3 MARK W. WOODS, AND MAURICE LANDY

National Cancer Institute, National Institutes of Health, Department of Health, Education, and Welfare,U. S. Public Health Service, Bethesda, Maryland

Experiments in which the administration of sub-stances to the host was followed by a rapid changein host resistance to infection have been describedmany times. The early literature has been wellassembled and reviewed by Philipson (19). How-ever, no unifying concepts of either commonmechanisms of action or common substances pres-ent in administered materials were formulated,so that the phenomena largely remained asisolated and individual examples which had beenelicited in special circumstances. On examinationof the reported findings, among them those ofPfeiffer and Issaeff (18), Teague and McWilliams,(26) and Philipson (19), it is clear that a num-ber of such phenomena may well have been initi-ated by endotoxin.Contemporary work in this field had its begin-

nings in observations of a similar nature. In thefirst descriptions, administration of bacterial cell-wall preparations (20) or whole bacteria (8) wasfound to result in a temporary and rapid increasein the resistance of mice to infection with anti-genically unrelated gram-negative bacteria.Shortly thereafter the active component in thesepreparations was identified as endotoxin (12, 21).At that time it was thought that the alterations

in resistance might be explained as a consequenceof the interaction of substrate (endotoxin) with aserum component concerned with the bactericidalreaction, which resulted in an initial depletionfollowed by a temporary overproduction of thelatter substance. The actual pattern of alteredresistance fitted this view very well in that, forabout 3 hr after receiving large doses of endo-

1 This symposium was held at the Annual Meet-ing of the American Society for Microbiology inChicago, Ill., 24 April 1961, under the sponsorshipof the Division of Medical Bacteriology and Im-munology, with Maurice Landy as convener.

2 Present address: Queen Elizabeth Hospital,Birmingham, England.

3Present address: House of the Good Samari-tan, Boston, Mass.

toxin, mice are more susceptible to experimentalinfection with gram-negative bacteria; subse-quently the situation is reversed rapidly and isfollowed by a period of up to 5 days when en-hanced resistance is demonstrable. However, itwas later found that increased resistance to in-fection produced by endotoxin was also exertedagainst mycobacterial and staphylococcal infec-tions (7) and this resistance differed in that itpersisted for several weeks. Subsequently, the ef-fects were shown to extend to still further bac-terial species and to certain virus infections (25).Moreover, some of the bacterial species were in-sensitive to the bactericidal action of serum.As soon as the phenomenon had been ade-

quately characterized, work went forward toelucidate the mechanisms by which enhancedresistance was mediated. Endotoxin producesmany changes in the host, which could be signifi-cant in altering the state of resistance, but someof these appear to be of special relevance. It hasbeen shown that endotoxin profoundly alters thefunctional capacity of the reticulo-endothelialsystem as measured by the clearance of intra-venously injected carbon particles (3), P32-labeledendotoxin (10), or similarly labeled bacteria (1).With these tests, an initial period of reducedclearance, lasting for several hours, can be shown.This is followed by enhanced reticulo-endothelialactivity characterized by both more rapid clear-ance and greater phagocytic capacity, whichpersists for several days before returning to nor-mal. The functional activity of both polymorpho-nuclear (6) and mononuclear phagocytes (23) hasalso been shown to be enhanced by endotoxin.The cellular changes following administration

of endotoxin were found to be paralleled by altera-tions in the serum. An initial depression of theserum properdin level was followed by increasedvalues which persisted for a few days (12). Thebactericidal power of serum for Escherichia coliwas also elevated after bacterial cell wall (21) orendotoxin administration (9). Serum from mice

437

on March 12, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

http://mmbr.asm.org/

WHITBY, MICHAEL, WOODS, AND LANDY

after endotoxin treatment was found to have en-hanced opsonic power for gram-negative bacterialspecies (11). In addition, increased opsonic powercould be measured by the blood clearance of p32_labeled E. coli (1) and this capacity could bepassively transferred. The accelerated cutaneousreactivity to endotoxins, which followed the intra-venous administration of these agents to rabbits,could be passively transferred and displayed someof the characteristics of the Arthus reaction (14).Moreover, the serum from these animals, in thepresence of complement, was found to possessenhanced ability to lyse red cells coated withendotoxins serologically unrelated to the ad-ministered material.Hence it is clear that after administration of

endotoxin the serum of animals is altered in sucha way that it displays increased activity in anumber of different tests. Most of these tests em-ploy situations in which antibody is known to befunctional, and where an increase in antibodylevel could account for the improved performance.However, the nature of the host response to endo-toxin differs from the classical immune reponsein two important ways. The onset is extremelyrapid so that serum changes are readily detectablewithin hours, and the elicitation of the effect bya single product is followed by activity against awide range of antigenically unrelated gram-nega-tive organisms. Thus, it has been suggested thatendotoxin may stimulate the production by thehost of cross-reacting antibodies which havemultiple specificity (14); or, alternatively, thatgram-negative bacteria possess a common antigenpossibly in the lipid component of the endotoxin(2) against which antibodies are produced. Nosuch common antigen has as yet been demon-strated. There are other possibilities. Some non-specific substance capable of acting like antibodymay appear in increased amount following endo-toxin administration; this had already been firstproposed and later rejected in the case of proper-din. Other nonspecific substances have been de-scribed (25). Alternatively, greater amounts ofsome cofactor may appear in the serum after endo-toxin, which improve the effectiveness of naturalantibodies already present in the serum. Finally,the possibility should be considered that endo-toxin stimulates a general increase in the level ofserum antibodies which are individually specific;in this case the apparent cross reactivity wouldnot be cross reaction at all but would be a conse-quence of multiple antibody release.

It is apparent that administration of endotoxinto animals leads both to cellular and to humoralchanges in the host. The work to be described re-lates to both of these parameters. The changesboth in cells and serum were different from thosewhich had been described previously, and theobjective was to attempt to determine whetherthe changes, both humoral and cellular, were ofsuch a nature that they might reasonably accountfor the increased resistance which follows ad-ministration of endotoxin.

EFFECTS OF ENDOTOXIN ON MONONUCLEARPHAGOCYTES

Previous work on the effects of endotoxin oncell metabolism has been concerned with effectselicited on a variety of cells exposed to endotoxinin vitro. The cells, whether mononuclear or granu-locytes, originated from rabbits, a species inwhich nonspecific resistance has not been studied.Our attention was focused on the peritonealmacrophages of mice inasmuch as these are thephagocytic cells directly involved in the resist-ance of this species to a peritoneally induced ex-perimental infection. These cells can be obtainedin adequate numbers essentially free of poly-morphonuclear leukocytes without prior stimula-tion of the host to induce exudate formation (27).Under appropriate conditions, peritoneal macro-phages have been shown to function more effi-ciently as phagocytes in vivo after endotoxintreatment (11) and we were interested in deter-mining whether general changes in metabolicbehavior of these cells would also follow adminis-tration of endotoxin.4

Glycolysis provides a significant index of themetabolism of cells, particularly of phagocytes.We have measured aerobic and anaerobic gly-colysis of peritoneal macrophages derived frommice at various times after treatment with endo-toxin. For these cells anaerobic glycolysis hasshown the effects more clearly than aerobic gly-colysis, but similar changes were seen in bothparameters. After a dose of endotoxin the rate ofaerobic and anaerobic glycolysis showed an earlyincrease, followed by a period of depression whichin turn was replaced by a period when glycolytic

4The endotoxin preparations employed in thiswork were prepared by the following investigators:D. A. L. Davies, Shigella dysenteriae; 0. Westphal,Escherichia coli strain 08; E. Ribi, Salmonellaenteritidis.

438 [VOL. 25


http://mm

br.asm.org/

Dow

nloaded from


ENDOTOXINS AND NONSPECIFIC RESISTANCE TO INFECTION

LL

0

0.

2 4 8 16 32TIME (hours)

FIG. 1. Glycolytic activity of mouse peritonealmacrophages during the first 24 hr after administra-tion of thug of Salmonella enteritidis endotoxin. Therate of anaerobic glycolysis for normal macrophages(2 X 107 cells per Warburg vessel) averaged 20 Mil ofCO2 per 1 hr.

activity was elevated. The latter stimulationcould persist for many days and did so for at least8 days in animals we have tested.

Figure 1 outlines the glycolytic activity ofperitoneal cells recovered from mice at times upto 24 hr after the animals had received a dose of5,ug of Salmonella enteritidis endotoxin. Cellsharvested 1 hr after endotoxcin administrationshow a stimulation of glycolysis, but at all sub-sequent periods up to 16 hr glycolysis is depressed.At 24 hr and at periods up to 96 hr, as shown inFig. 2, the glycolytic activity i~s again elevatedabove normal. It should be emphasized that thelevel of anaerobic glycolysis displayed by the cellsafter harvest continues at a constant rate in theWarburg flasks, and the cells recovered from thehost do not pass through the progressive changesin activity which are found at various times invivo. It is also noteworthy that macrophages ex-posed to endotoxin in vitro respond only withstimulation and do not show depression (28).

These findings indicate that the depression ofglycolysis which is found shortly after in vivo ad-ministration of endotoxin is induced by hostmechanisms that regulate cell glycolysis and isnot a direct effect of endotoxia. With smallerdoses of endotoxin, the inhibitory phase is eithergreatly reduced or absent, and stimulation ofglycolysis, although it arises more slowly, isestablished at levels well above normal, whereascells from animals receiving larger doses are stillin the negative phase. The actual level of stimu-

cr 150_\

0zLL.0

6 12 24 48 96TIME (hours)

FIG. 2. Glycolytic activity of mouse peritonealmacrophages after 60 jig of Shigella dysenteriae endo-toxin. The rate of anaerobic glycolysis for normalmacrophages (2 X 107 cells per Warburg vessel)averaged 20 Mul of CO2 per 1 hr.

lation following small doses of endotoxin hasvaried, but one example, illustrated in Fig. 3,was extremely marked. In this figure, the gly-colytic rates 11 hr after various doses of endo-toxin are compared.

It is well known that small doses of endotoxindo not induce a state of acute stress in the host,whereas large doses do, and this may well be thecause of the glycolytic depression. After the ef-fects of stress have passed off, the stimulatedstate returns. Up to now, no data have been ob-tained to determine whether the long stimulatoryeffect of high doses of endotoxin is due to the in-duction of some steady metabolic state in thephysiology of these cells, or whether endotoxin assuch persists in tissues.

Other workers have shown that phagocytosis ofinert particles or bacteria is accompanied by in-creased glycolytic activity of polymorphonuclearleukocytes (5, 24). Substances which stimulateglycolysis, including endotoxins (6), have beenshown to promote phagoyctosis by these samecells, whereas substances which depress glycolysisare accompanied by reduced phagocytic activity.This correlation has not proved so easy for us todemonstrate with macrophages, although a bi-phasic response of mouse peritoneal macrophageshas been shown with respect to their ability tophagocytize bacteria in vivo (11). In flasks,macrophages have been found to display poorphagocytic activity for gram-negative bacteria,unless attached to a surface (22). In contrast

4391961


http://mm

br.asm.org/

Dow

nloaded from



NORMAL 50ug 5y9g O-5#g 0-05AgCELLS

FIG. 3. Glycolytic activity of mouse peritonealmacrophages 11 hr after various doses of Salmonellaenteritidis endotoxin. The rate of anaerobic gly-colysis for normal macrophages (2 X 10 cells per

Warburg vessel) averaged 20jul of C02 per 1 hr.

Mackaness (15) found that unattached suspen-

sions of rabbit macrophages readily ingestedstaphylococci in the presence of serum. In thepresent experiments, polystyrene particles, in-cluded in parallel flasks, were not taken up well,but administration in vivo of polystyrene parti-cles, to animals in parallel with animals in whichglycolysis was studied, did induce differences inphagocytic potential. In animals where parallelstudies showed depressed glycolysis by macro-

phages, phagocytosis was less than normal, butincreased glycolysis was associated with a largernumber of cells containing polystyrene particles.Thus, glycolytic behavior in vitro was reflected inthe phagocytic activity of macrophages in vivo.

EFFECTS OF ENDOTOXIN ADMINISTRATIONON SERUM

Endotoxin administration to animals is knownto alter a variety of biological activities exertedby serum. We wished to study changes in anti-body titers in mice at various times after endo-toxin administration to investigate the specificityor lack of it in any alterations that occurred. Un-der normal conditions, mouse serum appears tohave little or no antibody to gram-negative bac-

Pooled normal human serum(bactericidal)

Absorbed with heat killed bac- Mouse serumteria at 4 C (contains

4. antibody)Absorbed human serum: Com-plement source (not bacteri-cidal for absorbing strain)

+ bacteria= bactericidal system

Mouse serum can be titrated for its antibody con-tent as all other necessary factors are present inexcess.

FIG. 4. Bactericidal reaction with mouse serum

teria as measured by agglutination tests. How-ever, antibodies to these organisms, together witha suitable complement source, form a bactericidalsystem, and we have developed a method basedon this system (13) which is illustrated in Fig. 4.Under the test conditions outlined, we have foundthat mouse serum is capable of restoring bacteri-cidal activity to human serum from which anti-body has been absorbed. We have further shownby absorption studies that the contribution ofnormal mouse serum in this test is to supplyspecific antibody. The method will detect between0.001 and 0.0005 ,ug of antibody N as testedagainst an immunochemically calibrated immunerabbit serum, which makes it among the mostsensitive methods of antibody determination atpresent available (4). Table 1 shows that by add-ing progressively smaller quantities of mouseserum to a series of reaction mixtures it is possibleto titrate the antibody content of the mouseserum as a dose response in relation to the numberof bacteria surviving in the reaction mixture after1 hr of incubation. Here two samples of mouseserum have been compared for activity againstSalmonella typhosa strain 0901, one from normaldanimals an the other from animals which hadreceived 50 Mg of Shigella dysenteriae endotoxin24 hr previously. At each serum increment, theserum of endotoxin-treated mice shows greaterbactericidal activity than that of normal mice.When the results obtained from such experi-

ments are plotted as probits of percentage sur-vivors against the log serum dose, the dilution ofserum at which 50% bacterial survival occurscan be read off the slope. Figure 5 shows the plotof two serum samples, one derived from normalmice and the other from a group of mice 1 hr

440 [VOL. 25


http://mm

br.asm.org/

Dow

nloaded from



TABLE 1. Bactericidal activity ojSalmonella typhosa stra

Bacterial sur

SerumNormal mice

ml %

0.01 70.0066 590.005 720.004 870.0033 95

The bacterial inoculum was* Obtained at 24 hr following

dysenteriae endotoxin iv. The (

giving 50% killing was 0.007 i

normal mice and 0.004 ml ftreated mice.

1 hr. afterendotoxin

7

4.)

0

S43

C)

m

6

5

4

befor

I0.0005 0.001 0.4

Log serum

FIG. 5. Effect of endotoxintivity of mouse serum. Endoto.monella enteritidis; inoculum, Iteriae.

after the administration of 50endotoxin. The serum from theanimals shows greater activitythe normal mouse serum. Theare 0.0013 ml and 0.0037 ml foendotoxin-treated and the co

spectively.The differences between indi

of a large order, but by using E

f mouse serum for end point can be titrated as a dose response, andain 0901 because the dose response slope is steep, it has

vival in serum from been possible to distinguish one serum from an-

other in a way which would not have been possibleEndotoxin-treated when measuring agglutination, for instance. After

mice' administering 10 to 50,ug of endotoxin to mice

% we have found that the bactericidal activity of3 their serum as measured in our assay is consist-3 ently raised to an activity of 1.5 to 4.0 times that

23 found in normal mouse serum. Because the phe-46 nomenon of nonspecific resistance can be elicited68 by considerably smaller doses of endotoxin than

106 cells.this, it was important to determine if lower doses

106 cells. would also alter serum bactericidal activity. It

q

50jng of Shigella was recognized, however, that at low doses anquantity of serum Xml in the case of effect might be produced that could not be deter-Dr the endotoxin- mined by available methods.

Many factors could influence the duration andmagnitude of these changes, as has been shown

e endotoxin previously in investigations of increase in naturalresistance. Among these are the properties of theendotoxin itself, the size of dose administered, thestrain of mice used, the time the samples aretaken, and the antigenic relationship of the or-ganism tested in the assay to the endotoxin ad-ministered. Tables 2 to 6 illustrate the changes inantibody levels found in mouse serum at various

a\ times after administration of endotoxin. Not allof these variables were examined. However, two

0 strains of mice were employed, three endotoxinsof different immunological specificities and fromdifferent sources were utilized, and bactericidal

0 assays for antibody against three unrelated en-terobacteriaceae were carried out. Two of thestrains shared common antigens with the endo-

I025 l.005 l01 toxins employed, which enabled a comparisonto be made between homologous and heterologous

dose situations. The major variables examined, how-

on bactericidal ac- ever, were the dose of endotoxin administered andrin, 50 ig of Sal- the interval between injection and bleeding.106 Shigella dysen- Each of the endotoxins used was capable of evok-

ing the effect but quantitative comparisons oftheir potencies were not made. Although there

MAg of S. enteritidis were only two situations when homologous andeendotoxin-treated heterologous responses were compared, the dif-y at all levels than ferences were so striking as to be conclusive. Thee end point values dose effect over an extended range was studiedr the sera from the with a single product which provided information)ntrol animals, re- on the magnitude and duration of the serum

changes in relation to dose.ividual sera are not In the experiment illustrated in Table 2 wherel method where the 50 Mg -of S. enteritidis endotoxin were adminis-

441-1961]


http://mm

br.asm.org/

Dow

nloaded from



TABLE 2. Alterations in level of antibodies for Salmonella typhosa, Shigella dysenteriae, andEscherichia coli in N.I.H. mice receiving Salmonella enteritidis endotoxin

Relative serum antibody levels for test strains

Test strain Hours after 50 jig of S. enteritidis endotoxinBefore

endotoxin*1 3 12 24 48 96 144 192

S. typhosa strain 0901.... 1.0 0.3 0.3 0.6 1.2 - 1.5 9.0S. dysenteriae type 1 ......... 1.0 3.0 2.9 2.9 2.4 2.5 1.3 -

E. coli strain 0127.......... 1.0 2.8 2.9 2.6 2.1 1.8 1.3 0.8 1.3

* Quantity of normal serum effecting a 50% reduction in an inoculum of 106 bacteria: 0.005 ml of S.typhosa strain 0901; 0.004 ml of S. dysenteriae type 1; and 0.0001 ml of E. coli strain 0127.

TABLE 3. Alterations in level of antibodies for Salmonella typhosa, Shigella dysenteriae, andEscherichia coli in CAF, mice receiving S. dysenteriae endotoxin


Test strain Hours after 50 1g of S. dysenteriae endotoxinBefore

endotoxin*1 3 6 12 24 48 96

S. typhosa strain 0901 1.0 1.0 1.2 1.9 1.9 1.7 1.7 1.0S. dysenteriae type 1 1.0 0.4 0.4 0.6 0.6 0. 6 1.5 3.8E. coli strain 0127 1.0 1.2 2.6 2.3 2.5 2.5 1.9 1.3

* Quantity of normal serum effecting a 50% reduction in an inoculum of 106 bacteria: 0.007 ml ofS. typhosa strain 0901; 0.006 ml of S. dystenteriae type 1; and 0.0001 ml of E. coli strain 0127.

TABLE 4. Alterations in level of antibodies for Salmonella typhosa, Shigella dysenteriae, andEscherichia coli in CAF, mice receiving E. coli strain 08 endotoxin


Test strain Hours after 10lg of E. coli strain 08 endotoxinBefore

endotoxin*½ 1 2 4 6 12 24

S. typhosa strain 0901 1.0 1.1 1.5 1.8 1.7 1.6 1.6 1.6S. dysenteriae type 1 1.0 1.2 2.5 - 2.3 1.8 1.8E. coli strain 0127 1.0 1.1 1.9 2.5 - 3.4 2.6 3.4

* Quantity of normal serum effecting a 50% reduction in an inoculum of 106 bacteria: 0.007 ml of S.typhosa strain 0901; 0.006 ml of S. dysenteriae type 1; and 0.0001 ml of E. coli strain 0127.

tered, antibody levels for the heterologous or-ganisms tested rise sharply within 1 hr and remainelevated for 48 hr but return to normal limits by96 hr. By contrast, the antibody level for anantigenically homologous strain of S. typhosa ismarkedly reduced in the first 12 hr and does notreach normal levels until 24 hr or elevated levelsuntil 96 hr. By 8 days, antibody to this organismis elevated to a level much higher than that foundto heterologous strains at any time. Similar find-

ings are seen in Table 3 where 50,g of S. dysen-teriae endotoxin were injected, except that therise in antibody to heterologous strains occurredsomewhat later, appearing 3 to 6 hr after endo-toxin had been given. Table 4 illustrates thechanges found after injection of 10lg of E. colistrain 08 endotoxin, which is antigenically un-related to any of the test organisms. Here eleva-tion of antibody for all the test strains had takenplace after 1 hr and then persisted throughout the

442 [VOL . 25


http://mm

br.asm.org/

Dow

nloaded from



TABLE 5. Alterations in level of antibody forEscherichia coli in N.I.H. mice receiving

Salmonella enteritidis endotoxin

Relative serum antibody levels for E. colistrain 0127

S.

enteritidis Before Hours after endotoxinendoto-

xsnnt*° 1 3 6 12 24 48 72

10 1.0 4.2 2.9 2.3 1.8 1.8 1.0 0.91 1.0 0.9 1.9 1.8 2.4 1.5 0.9 1.20.1 1.0 0.9 1.8 2.2 2.5 1.0 1.1 1.20.01 1.0 1.0 0.9 - 0.8 1.1 -

* Quantity of normal serum effecting a 50%reduction in an inoculum of 106 E. coli strain0127: 0.0002 to 0.00015 ml. The values in the tableare related to the normal serum value on the dayof test.

TABLE 6. Alterations in level of antibody forShigella dysenteriae in N.I.H. mice

receiving Salmonella enteritidis endotoxin

Relative serum antibody levels forS. dysenteriae type 1

S.enterinids Hours after endotoxined

Before

endotoxin*6 24 48

Ag

10 1.0 3.3 2.7 1.61 1.0 3.4 2.0 1.00.1 1.0 2.3 1.2 1.0

* Quantity of normal serum effecting a 50%reduction in an inoculum of 106 S. dysenteriae was0.004 to 0.0035 ml.

test period (24 hr). Illustrations of the effect ofdifferent doses of S. enteritidis endotoxin on anti-body levels are found in Tables 5 and 6, whichshow that a dose of 0.01 j.g fails to produce anychange in antibody levels, whereas doses of 0.1Mug or more are followed by elevation of antibodylevels. There is evidence that the duration of theeffect is related to the dose of endotoxin, lastingonly 12 hr after 0.1 Mig, 24 hr after 1 Mug, and 24 to48 hr after 10 Mg.

These results illustrate that, after endotoxinadministration, increased levels of antibody ac-tive in a bactericidal system were found. Thequestion whether this antibody showed specific orcross-reactive characterization was next con-

TABLE 7. Specificity of bactericidal antibody ofendotoxin-treated mice

Relative bactericidal activityof serum

After absorp-Unabsorbed tion. with

Treatment of mice Unabsorbed Salmonellatyphosa

S. ly- Escker-phSas ichia coli ty- E. coli

None............... 1.0 1.0 0 1.0Shigella dysenteriaeendotoxin (50 yg,iv) 24 hr pre-viously............ 1.7 2.5 0 2.3

sidered. The finding (Tables 2 and 3) that; after alarge dose of endotoxin, bactericidal activity forantigenically heterologous strains rises, whereasthat for antigenically homologous strains falls,argues for a specific basis for the bactericidalresponse. The most probable explanation of thedifference is that complexing of antigen (endo-toxin) with antibody occurs in the host. A non-specific substance should be equally active againsthomologous strains; however, evidence of speci-ficity was also provided by other means.The specific nature of this response has been

established by absorption studies (16) and by in-hibition tests in the bactericidal reaction withendotoxins of differing antigenic specificity.When sera were absorbed with a bacterial suspen-sion, it was found that antibody activity againstthe absorbing strain could be removed, whereasactivity against other strains was retained. Table7 shows an example of this finding: 24 hr after 50Mg of S. dysenteriae endotoxin had been adminis-tered, serum obtained from mice possessed 1.7times the bactericidal activity against S. typhosaand 2.5 times as much activity against E. colistrain 0127 as normal mouse serum. When theactivity against S. typlosa was removed by ab-sorption with heat-killed bacteria, the bactericidalactivity against E. coli was not significantly af-fected. Other absorption studies have been car-ried out with similar results.

Further evidence for specificity was obtainedby adding antigenically homologous and heterol-ogous endotoxins to reaction mixtures in thebactericidal reaction (17). An example of such anexperiment is illustrated in Table 8 where it can

19611 443


http://mm

br.asm.org/

Dow

nloaded from



Relative bactericidal activity forEscherichia coli strain 0127

Un-treatedserum

2

Somatic antigen added totest serum

Salmo-nella

typhosa,100 Ag

1

2

Shigelladysente-

riae,1002g

1

2

be seen that, although 10 jg of homologous endo-toxin will abolish bactericidal activity, even 100tig of antigenically unrelated endotoxins are with-out effect. Thus it can be concluded that there isa specific basis for the increase in bactericidalantibody which follows administration of endo-toxin to animals and no evidence for cross-react-ing antibodies was obtained.

Finally, the possibility was investigated thatadministration of endotoxin leads to the releaseof some nonspecific substance which will increasethe efficiency of antibody in the bactericidal re-

action. The findings so far reported would be ex-

plicable on such a basis because absorption or

blocking studies would behave in a specific man-

ner in such a situation. An example of experimentsdone to test this hypothesis is now given. Anti-body for S. typhosa was absorbed from serum ob-tained from mice which had received 50 jig ofS. dysenteriae endotoxin. After absorption, thisserum was found still to possess increased bac-tericidal activity for E. coli; therefore, if a non-

specific cofactor existed, it still remained in theserum. Furthermore, a portion of this absorbedserum added as a supplement to a bactericidaltitration of normal mouse serum against S.typhosa did not increase the titer or activity of thenormal mouse serum. Such results make it un-

likely that a nonspecific cofactor is involved andlead to the conclusion that the increase in bac-tericidal titer after endotoxin administration tomice was due to a low level, general increase ofspecific antibodies to gram-negatijve species.

SUMMARY AND CONCLUSIONSEndotoxin administered to mice leads to

changes in the metabolic activity of the mono-nuclear phagocytes. With small doses, only stimu-lation occurs, but with higher doses there is aninitial period of depression followed by a periodof stimulation, which with large doses may lastfor at least 8 days. The metabolic changes mayreflect the improved phagocytic performance ofthese cells but opsonic factors are still necessaryfor adequate ingestion of enterobacteriaceae.Nonetheless, the metabolic changes alone mayexplain the longer duration of nonspecific re-sistance in staphylococcal and mycobacterial in-fections, where opsonic factors are not an absoluterequirement for phagocytosis to occur.

Following endotoxin administration there is ageneral release of substances active against entero-bacteriaceae which show the specific activitycharacteristic of antibodies. These alterations inthe level of antibody were produced with endo-toxins in amounts known to modify resistance toexperimental infection. Moreover, the serum ef-fect was dose dependent in a manner analogousto changes in resistance. A major activity ofspecific antibody is the ability to opsonize or-ganisms for phagocytosis. As the antibodies de-scribed here prepare bacteria for the lethal ac-tion of complement, it is reasonable to assumethat they can also function as opsonins. Althoughno direct correlation has been established, thechanges reported here should be intimately in-volved in determining the outcome of infection,since they affect both the very cells which areconcerned in host defense against an intraperi-toneal challenge and a humoral factor which isknown to increase the efficiency of these cells asphagocytes.Emphasis has long been laid on the lack of

specificity of the phenomenon known as nonspe-cific resistance. This designation was based onthe criterion that administration of any endotoxinled to increased resistance and activity againstantigenically unrelated strains. This basis for non-specificity cannot be questioned, but we havefound that, as far as serum changes are concerned,the result of endotoxin administration is to in-crease the general level of specific antibody in thehost. It may be appropriate, therefore, to considerwhether "nonspecific resistance" is a suitable des-ignation for this effect or whether a title more

TABLE 8. Specificity of increased bactericidalactivity of serum from endotoxin-treated

mice

Mouse serum

Normal ..........At 24 hr after ad-

ministration of10,ug of Salmo-nella enteritidisendotoxin .....

444 [VOL. 25


http://mm

br.asm.org/

Dow

nloaded from



truly descriptive of the immunological changes in-duced by endotoxin should be sought.

LITERATURE CITED

1. BENACERRAF, B., M. M. SEBESTYEN, AND S.SCHLOSSMAN. 1959. A quantitative study ofthe kinetics of blood clearance of P32-labelledEscherichia coli and staphylococci by thereticulo-endothelial system. J. Exptl. Med.110:27-48.

2. BENACERRAF, B., AND P. MIESCHER. 1960.Bacterial phagocytosis by the reticulo-endothelial system in vivo under differentimmune conditions. Ann. N. Y. Acad. Sc.88:184-195.

3. Biozzi, G., B. BENACERRAF, AND B. N. HAL-PERN. 1955. The effect of Salmonella typhiand its endotoxin on the phagocytic ac-tivity of the reticulo-endothelial system inmice. Brit. J. Exptl. Pathol. 36:226-235.

4. BOYD, W. C. 1956. Fundamentals of immunol-ogy. 3rd ed. Interscience Publishers, Inc.,New York, 776 p.

5. COHN, Z. A., AND S. I. MORSE. 1960. Functionaland metabolic properties of polymorpho-nuclear leucocytes. I. Observations on therequirements and consequences of particleingestion. J. Exptl. Med. 111:667-687.

6. COHN, Z. A., AND S. I. MORSE. 1960. Functionaland metabolic properties of polymorpho-nuclear leucocytes. II. The influence of alipopolysaccharide endotoxin. J. Exptl.Med. 111:689-704.

7. DUBOS, R. J., AND R. W. SCHAEDLER. 1956. Re-versible changes in the susceptibility of miceto bacterial infections. I. Changes broughtabout by injection of pertussis vaccine or ofbacterial endotoxins. J. Exptl. Med. 104:53-65.

8. FIELD, T. E., J. G. HOWARD, AND J. L.WHITBY. 1955. Studies on the rapid produc-tion of a non-specific type of immunity toSalmonella typhi infection in mice. J. Roy.Army Med. Corps 101:324-334.

9. FISCHER, J. 1959. Zur Beeinflussung der Serum-bakterizidie. Schweiz. Z. allgem. Pathol. u.Bakteriol. 22:827-843.

10. HOWARD, J. G., D. ROWLEY, AND A. C. WARD-LAW. 1958. Investigations on the mechanismof stimulation of non-specific immunity bybacterial lipopolysaccharides. Immunology1:181-203.

11. JENKIN, C., AND D. L. PALMER. 1960. Changesin the titre of serum opsonins and phagocytic

properties of mouse peritoneal macrophagesfollowing injection of endotoxin. J. Exptl.Med. 112:419-429.

12. LANDY, M., AND L. PILLEMER. 1956. Increasedresistance to infection and accompanyingalteration in properdin levels following ad-ministration of bacterial lipopolysacchar-ides. J. Exptl. Med. 104:383-409.

13. LANDY, M., J. G. MICHAEL, AND J. L. WHITBY.1962. A bactericidal method for the measure-ment in normal serum of antibody to gram-negative bacteria. J. Bacteriol. 83 (in press).

14. LEE, L., AND C. A. STETSON. 1960. Studies onthe mechanism of the Shwartzman phe-nomenon. Accelerated cutaneous reactivityto bacterial endotoxins. J. Exptl. Med. 111:761-770.

15. MACKANESS, G. B. 1960. The phagocytosis andinactivation of staphylococci by macro-phages of normal rabbits. J. Exptl. Med.112:35-53.

16. MICHAEL, J. G., J. L. WHITBY, AND M. LANDY.1961. Increase in specific bactericidal anti-bodies after administration of endotoxin.Nature 191: 296-297.

17. MICHAEL, J. G., J. L. WHITBY, AND M. LANDY.1961. Studies on natural antibodies to gram-negative bacteria. J. Exptl. Med. (in press).

18. PFEIFFER, R., AND ISSAEFF. 1894. Ueber diespezifische Bedeutung der Choleraimmuni-tat. Z. Hyg. Infektionskrankh. 17:355-400.

19. PHILIPSON, J. 1937. Experimental studies onenhanced resistance to infection followingsome non-specific measures. Acta Pathol.Microbiol. Scand. Suppl. No. 32:1-148.

20. ROWLEY, D. 1955. Stimulation of natural im-munity to Escherichia coli infections. Ob-servations on mice. Lancet 1:232-234.

21. ROWLEY, D. 1956. Rapidly induced changes inthe level of non-specific immunity in lab-oratory animals. Brit. J. Exptl. Pathol. 37:223-34.

22. ROWLEY, D., AND J. L. WHITBY. 1959. The bac-tericidal activity of mouse macrophages invitro. Brit. J. Exptl. Pathol. 40:507-515.

23. ROWLEY, D. 1960. The role of opsonins in non-specific immunity. J. Exptl. Med. 111:137-144.

24. SBARRA, A. J., AND M. L. KARNOVSKY. 1959.The biochemical basis of phagocytosis. I.Metabolic changes during the ingestion ofparticles by polymorphonuclear leucocytes.J. Biol. Chem. 234:1355-1362.

25. SHILO, M. 1959. Nonspecific resistance to in-fections. Ann. Rev. Microbiol. 13:255-278.

4451961]


http://mm

br.asm.org/

Dow

nloaded from



26. TEAGUE, O., AND H. I. MCWILLIAMS. 1917. Ex-periments with a possible bearing upon

treatment of typhoid fever with typhoidvaccine administered intravenously. J.Immunol. 2:185-191.

27. WHITBY, J. L., AND D. ROWLEY. 1959. The roleof macrophages in the elimination of bac-

teria from the mouse peritoneum. Brit. J.Exptl. Pathol. 39:358-370.

28. WOODS, M. W., M. LANDY, J. L. WHITBY, AND

D. BURK. 1961. Symposium on bacterial en-

dotoxins. III. Metabolic effects of endotoxinson mammalian cells. Bacteriol. Rev. 25:447-456.

446 [VOL. 25


http://mm

br.asm.org/

Dow

nloaded from


Documents

SYMPOSIUM ONBACTERIAL ENDOTOXINS1 II. POSSIBLE …bactericidal power of serum for Escherichia coli wasalso elevated after bacterial cell wall (21) or endotoxin administration (9)