hasqaren ins reginedefined. resarisanc toer idnfetious ... · toward sensitized red cells was only...

ANTIMICROBIAL FACTORS OF NORMAL TISSUES AND FLUIDS

ROBERT C. SKARNES' AND DENNIS W. WATSONDepartment of Bacteriology and Immunology, University of Minnesota, Minneapolis, Minnesota

INTRODUCTION it readily inactivated by the simple procedure of

Observations by many investigators during dialysis against distilled water (25). Buchner

the last fifteen years of the 19th century demon- named this serum bactericidal substance "alexin"strated the existence of antimicrobial substances (Greek, to ward off) in 1891 (26). Since thesein blood, leucocytes and lymphatic tissues. Since early reports many investigators have added to

the turn of the century many tissues have been the knowledge of this material.shown to contain factors inimical to microorgan- In 1905-6 Ehrlich proposed the name "comple-isms. No doubt many of the recent findings ment" in place of his earlier term "addiment" torepresent rediscoveries of agents which previously apply to the heat labile normal serum substancehad been inadequately defined. A great deal of active in hemolytic systems (41). He claimed thatconfusin hasqaren regine gteir identiie serum contained a multiplicity of normal hemoly-andtheiroleins naturalresarisanc toer idnfetious sins which consisted of thermostable interbodiesprocesses

role in natural resistancetoinfectious(not inactivated at 55 C) and the thermolabileThisrev r complement. He supported the view that Buch-This review represents an attempt to discuss

and classify many of the antimicrobial agents ner's originally described alexin was actually aobt ained from normal tissues and body fluids. The dual system of interbody and complement. Sincesubject matter is divided into two broad groups, this time alexin has generally been regarded as a

based upon the antibacterial selectivity of the thermolabile substance which required for itsvarious factors toward gram negative or gram bactericidal action the participation of another

positive bacteria. It should be noted that only serum factor, normal or natural antibody.certain of the factors have been established as On the Identity of Alexin, Opsonin and Complementshowing a definite predilection toward one groupof bacteria or the other, while others are active The early controversy regarding the similari-against representatives of both groups or have not ties or differences between alexin and complementbeen adequately differentiated. In spite of these has been resolved in more recent years. Amonglimitations, the general predisposition of these those who have reviewed and worked with theagents towards either gram negative or gram subject extensively (57, 116, 130, 187), thepositive forms is patent and affords a convenient concensus is that they are identical.division. Other aids used in the classification of The term "opsonin" (Greek, to cater for) wasthese antimicrobial substances are tissue or fluid introduced by Wright and Douglas in 1903 (182)source, heat stability and, if known, chemical to denote the thermolabile phagocytosis-stimulat-composition. ing substance in normal serum. We agree with

Raffel (145) that the term opsonin be restrictedANTIMICROBIAL SUBSTANCES SELECTIVE FOR to Wright's original description to differentiate it

GRAM NEGATIVE BACTERIA from those specific antibodies, the bacterio-tropins, which also enhance phagocytosis.

In 188S8 Nuttall observed that normal dog or 'rpnwihas nac hgctssIn1888 rNuttallblobserved losthatsnormal dgcor Osborne (130) discussed the investigations which

prabbitydfrigramnatedgblo lsterit bactericidal led to the provisional theory that opsonin repre-property for gram negative bacteria after being setdcmlenwihuteforh o-heated at 52 to 55 C for Hi to 1 hr (129). The pent.citingeth workoofMtaerf(105) whi

ponent. Citing the work of Maltaner (105) whichnext year Buchner (24) repeated this observation indicated that leucocytes contained C4', theon cell free normal serum heated at 55 C for I/-

theory contended that phagocytes ingest micro-hr or 52 C for 6 hr. He attempted to identify the organisms buttered with opsonin and that theprotein material by chemical methods but found fourth component is added intracellularly. An

1 Present address: Laboratory of Chemical excellent literature survey of the role of comple-Pharmacology, National Cancer Institute, Be- ment components in opsonization is found inthesda, Maryland. Chapter 52 of the Topley-Wilson textbook (180).

273

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274 SKARNES AND WATSON [VOL. 21

Through the development of unique methods, of serum, he concluded that alexin did notMaal0e (102) demonstrated that all four com- originate from leucocytes. Later Korschunponents of complement were necessary for the showed that rabbit leucocyte extracts weremanifestations of bactericidal and opsonic bactericidal for typhoid organisms and that theactivity in normal serum. Thus it appears that active material was only slightly inactivated incomplement, in conjunction with normal anti- Hi hr at 72 C (91). However, if the extracts werebody produces the bactericidal, hemolytic and heated in the presence of serum the activity wasphagocytosis-promoting effects of normal serum. lost in }A hr at 56 C. These early observationsMaaloe has suggested that a single new term be implied that leucocytes contained at least twointroduced to replace alexin, complement and thermostable antibacterial factors.opsonin. Recently, Tullis and Surgenor isolated Gengou (53) found that uncoagulated cell freeby paper electrophoresis two normal serum plasma, obtained without the use of anticoagu-proteins which proved stimulatory to phago- lants, was not bactericidal whereas the serumcytosis (169). Although both of the phagocytosis- from the same blood was active. In carefulpromoting factors were thought to be distinct experiments Fuchs (50) showed that the clottingfrom complement and properdin, further studies of blood must begin before hemolytic complementwill be required to establish this. activity is manifested. He also noted that in

blood to which anticoagulants had been addedSource of Alexin or Complement hemolytic complement action was immediately

A controversy developed concerning the source apparent. He postulated that, until the clottingof alexin and complement and although the process was initiated, complement was not free toproblem has been clarified somewhat, no act in circulating blood. Maltaner (105) showeddefinitive conclusions are yet possible. In 1894 that extracts of carefully washed leucocytes, whileBuchner (27) and Denys and Havet (32) reported devoid of full complement activity, reactivatedthat leucocyte extracts were much more active ammonia-treated serum, thus indicating that theagainst gram negative bacteria than the cor- leucocytes furnished the C4' component of com-responding serum from the same experimental plement. This finding may explain the resultsanimals. They found the substance in leucocytes obtained by Gengou and Fuchs regarding theto be heat labile and thus believed that serum lack of bactericidal action in normal uncoagulatedalexin was derived from injured leucocytes. It plasma. The leucocyte injury which occurs as ashould be noted that the testing of leucocyte consequence of coagulation, or after the additionextracts was performed in the presence of serum, of anticoagulants, could result in the release ofa fact which probably accounted for the observed C4', thus providing full complement activity.heat lability. Many subsequent workers also This suggests that complement exists in a pre-reached the conclusion that leucocytes repre- cursor state in circulating blood and is not acti-sented the source of alexin or complement, usually vated until leucocyte injury occurs.as a result of finding greater antibacterial activity Normal Antibodyin leucocyte extracts than in corresponding sera(1, 22, 53, 62, 108). In some of these early reports In 1899 Moxter observed that alexin requiredit was shown that leucocyte extracts were active an additional normal serum component toagainst gram positive as well as gram negative manifest bactericidal action (113). Wright andbacteria. These results indicated the presence of Windsor (181) adsorbed normal human andother antibacterial factors in leucocytes which rabbit sera with small quantities of killed cholerawere unrelated to alexin or complement. or typhoid cultures and found that either organ-

Several investigators reported the absence of ism removed the bactericidal property, whilealexin or complement activity in carefully washed bacteria not susceptible to the killing action ofleucocytes (91, 112, 133, 152, 153, 186). Schatten- serum did not do so. Steinhardt (161) found thatfroh (152) tested extracts of washed leucocytes in the bactericidal action for both typhoid andsaline rather than serum and found that they dysentery bacilli was removed from normalwere active only against gram positive bacteria. serum by dead cultures of either organism butFurthermore, since the extracts required tempera- was partially restored upon the addition oftures of 75 to 80 C for inactivation in the absence normal serum which had been heated to 55 C to


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19571 ANTIMICROBIAL TISSUE FACTORS 275

destroy complement. She ascribed the result to guinea pig complement as a result of the inter-the action of a common, naturally occurring action with natural (normal) antibody. He foundimmune body which was adsorbed by the killed that bacteria, exposed to normal serum at 0 Cbacteria, resulting in a concomitant nonspecific for 1 hr, became sensitized to subsequent com-reduction in complement. plement action at body temperature. The cold

Ehrlich described the complex nature of normal adsorption of normal antibody was nonspecificserum hemolysins (41). He observed that when a since many typhoid strains as well as charcoalnormal serum, which dissolved certain unsen- and powdered glass removed it from serum. Thesitized foreign erythrocytes at 37 C, was mixed natural antibody resembled immune antibody inwith these cells at 0 C and centrifuged, the its ability to fix complement but differed in itssupernatant lost its ability to lyse red cells at nonspecificity and greater thermolability. Dunlophigher temperatures. This property could be found a variation in the heat stability of naturalrestored to the centrifuged serum by the ad- antibody, depending upon the serum source anddition of a quantity of normal serum which had the strain of bacteria against which it wasbeen heated to 55 C for }A hr. He referred to the tested. In some sera it appeared to be moresubstance adsorbed at 0 C as interbody and the labile than complement.heat labile substance as complement. Mackie and Finkelstein (103, 104) studied theMuir (115) reported the removal of bactericidal heat labile antibacterial normal serum system

activity of normal guinea pig serum by adsorption and found it to be active only against gramwith killed homologous or heterologous bacteria negative bacteria of the typhoid-dysentery-vibriowithout appreciable impairment of hemolytic groups. Antibacterial action was dependent uponactivity against sensitized erythrocytes. When two serum components, complement and a morenormal serum was adsorbed with the same species heat stable constituent which they referred to asof bacterium against which it was tested, a sensitizing or intermediary antibody. Comple-greater decrease in bactericidal effect was ob- ment activity was lost in heated serum after Hiserved than if the adsorption step had been per- hr at 55 C while the sensitizing antibody (naturalformed with heterologous species. These experi- or intermediary antibody) required temperaturesments suggested that normal serum contained of 57 to 60 C to be inactivated in ½ hr (104).one or a few closely related molecular species of The intermediary antibodies could be adsorbeda nonspecific substance having a differing affinity out by viable or killed typhoid organisms at 0for various bacteria. Since this time, the sub- to 2 C, and they sensitized susceptible bacteriastance has come to be known most commonly as to the killing action of complement, the latternormal or natural antibody. The investigations of not being adsorbed out by the cells in the cold.many workers have since confirmed and extended These authors suggested that normal serumthese early reports. Pettersson (140) introduced contained specific naturally occurring antibodiesthe term, "alpha-lysin," to describe the two- which were increased upon immunization. Theycomponent alexin system consisting of the pointed out that the complexity of naturalthermolabile substance and normal antibody. antibody specificities was affected by the serumGordon and Wormall (57) showed that heat- source and the species or strain of bacteria used

killed dysentery bacilli removed the bactericidal in adsorption studies. Gordon and Carter (58)power of normal guinea pig serum when incubated adsorbed normal guinea pig serum with washedfor 1 hr at 37 C although the hemolytic property bacterial suspensions at 0 C, which removed non-toward sensitized red cells was only slightly specifically the bactericidal power for a numberdiminished. The bactericidal power could be of gram negative bacteria. Variations in thisreinstated by the addition of normal serum activity of normal serum against different or-heated to 56 C for 'i hr. If large amounts of ganisms depended upon the sensitivity of theseadsorbing bacteria were employed, the specific bacteria to the nonspecific serum componenthemolytic action was also lost. The authors re- (normal antibody). In 1946 Maal0e concludedferred to the bactericidal factor removed at 0 C that normal serum probably contained only oneas normal immune body. or a few fairly nonspecific normal antibodies

In 1928 Dunlop (40) found that many strains (102). In discussing the differences between trueof Salmonella typhosa caused the fixation of normal antibodies and classical antibodies, Land-


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steiner suggested (94a) that there may exist in ment occurs in the absence of specific antibodies,serum a great variety of globulin molecules which, serum normal antibody represents a second"by virtue of accidental affinity to certain sub- necessary component in these systems.strates are picked out as (specific) antibodies." The role of alexin or complement in immuneRecent experiments by Adler (2) offer a clari- systems has been elucidated by Bordet (22) and

fication of the issue of normal antibody specificity. others (41). In vitro studies by many investigatorsIt is well known that enteric bacteria share com- have shown that the addition of small amounts ofmon antigens and the widespread occurrence of heated specific serum to normal serum resultedthe typhoid antigens, XII and IX, among enterics in marked increases in the bactericidal activitysuggests that subdetectable levels of antibody against gram negative bacteria. Exceptions tocould be produced to these antigens, whatever this phenomenon have been observed wheretheir bacterial source. Thus, the adsorption of certain bacteria survived or grew profusely in"normal" serum by a particular enteric strain fresh immune serum while being inhibited ormay result in the removal of specific antibody killed in normal serum from the same animalwith consequent reduction in serum bactericidal (108, 125, 172). At least a partial explanation forpower toward that bacterium. Gaines and Landy these seemingly contradictory findings is ap-reported a highly specific antibody to Pseudo- parent from the early experiments of Neisser andmonas aeruginosa present in "normal" sera at Wechsberg (125) and more recently, of Maaloelevels undetectable by conventional methods (102). The former authors showed that the addi-(51). Undoubtedly, some of the confusion re- tion of very small amounts of heated immunegarding specificity of normal antibody can be serum to normal serum enhanced the bactericidallaid to the presence of ordinarily undemonstrable effect while the addition of larger amounts of thelevels of specific antibody in apparently normal specific serum caused a reversal of antibacterialsera. This seems particularly likely when we action. Observations such as these led Mechnikovconsider that these specific antibodies are di- to become more strongly imbued with the cellularrected towards members of the normal enteric theory of immunity. He reasoned that it was theflora of most animal species. agglutinating action of specific antibody, result-A simple way to differentiate between normal ing in greatly increased phagocytosis, which

antibody and specific antibody is to measure heightened the resistance of immunized animalstheir respective heat stabilities, but unfortunately (108). Maalpe found that subdetectable amountsthis was not done in the two papers just cited. of specific agglutinins enhanced the bactericidalAdler found that the bactericidal antibodies action of normal serum whereas a two- or three-were able to survive 56 C for Hi hr (2), but no fold increase in agglutinin concentration did notfurther heat stability studies were undertaken. further increase this effect; in fact, high levelsMost experimenters have shown that normal of the specific antibody completely reversedantibody is inactivated after Hi hr between the bactericidal action (102). Maal0e also noted thattemperatures of 55 and 60 C, while classical high concentrations of normal serum interferedantibodies require temperatures of 65 to 70 C with antibacterial action presumably because offor inactivation. Normal antibodies from various the presence of excess normal antibody. Justanimal sources reasonably can be expected to recently Muschel and Muto demonstrated thatvary somewhat in thermostability and as Jacox the bactericidal action of normal mouse serumnoted (80), stored serum may vary significantly was dependent upon full complement, normalin pH, a factor which also may alter the heat antibody, and Mg++ ions (117).stability of serum components. In the future amore critical study of the heat stability of normal Properdinantibody would aid in its proper classification and Pillemer and co-workers (142) isolated a pro-avoid confusion with the more stable specific tein from normal human serum which requiredantibodies which may be present in subdetectable the presence of complement and Mg+ ions toamounts in "normal" serum. exhibit bactericidal action against gram negative

It is generally accepted that in experiments bacteria. It also neutralized certain viruses andwhere hemolysis of nonsensitized erythrocytes, was active in an unsensitized hemolytic system inbactericidal reaction, or phagocytosis enhance- the presence of complement and Mg++ ions.


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1957] ANTIMICROBIAL TISSUE FACTORS 277

This factor was named properdin (Latin, to infection was not always exact, and in some casesdestroy) and it was found to be a euglobulin, properdin levels were merely maintained in testrepresenting no more than 0.03 per cent of total animals which exhibited increased resistance. Theserum proteins. It formed complexes with a authors concluded that other defense mechanisms,yeast zymosan, bacterial products, large branched in addition to properdin, were responsible forpolysaccharides and charcoal (143) between the increased host resistance following administra-temperatures of 10 to 20 C, enabling its removal tion of lipopolysaccharides. Recently, Rowleyfrom serum without destruction of specific (148) reported a correlation between increasedhemolytic complement. At temperatures above resistance of animals to infection and the bac-20 C inactivation of hemolytic complement tericidal power of their serum following injection(C3') occurred in most cases. The substance of 0 antigen of E. coli. He found that pathogenicproved heat labile in serum at 56 C for Hi hr but strains of this organism were more anticomple-was more stable in a purified state. It was not mentary than nonpathogenic strains because ofremoved by antigen-antibody complexes, not their greater content of 0 antigen, and he sup-involved in the serum clotting mechanism, and ported the view that a substance like properdinwas assayed by its ability to inactivate C3' in could be involved.the presence of zymosan at 37 C. Properdin was Dubos and Schaedler (39) observed an in-isolated from the serum of several animal species creased resistance to gram positive infections(172). following the administration of lipopolysac-Properdin proved to be active against certain charides. Since gram positive microorganisms are

strains of several gram negative bacteria but not affected by properdin in vitro it was con-inactive for Micrococcus pyogenes var. aureus. cluded that other defense mechanisms wereThe presence of specific agglutinins in serum stimulated by injection of the foreign material.interfered with its bactericidal action against Landy (95) found that increased resistanceShigella dysenteriae. The re-addition of physiologic following intraperitoneal injection of lipopoly-levels of properdin to properdin-deficient serum saccharide extended only to gram negative bac-restored almost completely the bactericidal effect, teria while no change in susceptibility wasbut excess amounts of properdin reversed serum demonstrated to three species of gram positiveantibacterial activity (172). bacteria. The fact that different mouse strainsLandy and Pillemer (96) injected small were used by these investigators may explain

amounts of purified lipopolysaccharide into mice the conflicting results, since other variables wereand observed a transient increase in properdin similar in both studies.levels within 12 hr. If mice were treated with In 1893 Klein (87) first observed that thesmall amounts of lipopolysaccharide 24 hr prior intraperitoneal or subcutaneous injection ofto challenge with certain gram negative bacteria various killed bacteria prior to the administrationthey were protected, whereas control mice ex- of lethal doses of cholera organisms protectedhibited decreased properdin levels and death. experimental animals. The next year Jssaeff (78)Rowley (147) had previously shown this protec- and Pfeiffer and Issaeff (141) protected guineative effect with prior injections of cell walls of pigs from lethal doses of cholera by a previousEscherichia coli. He noted an immediate highly intraperitoneal injection of broth, peptone orsusceptible state within 2 hr after injection of urine. They observed a marked leucocytosis inthe cell walls but an increased resistance if chal- the body cavity at the time of challenge andlenge doses were administered 24 hr later. Serum attributed the increased resistance of treatedcomplement levels did not increase after E. coli animals to the release of bactericidal materialextracts were injected. Landy and Pillemer (97) from the leucocytes. Ledingham and Bullochcorrelated the maintenance of, or increase in, (98) and Bedson (15) found that the intravenousproperdin levels with increased resistance of injection of autolyzed bacteria was followedmice to challenge doses of gram negative bac- within 2 hr by a marked increase in serum opsonicteria. Small amounts of lipopolysaccharide activity and leucocytosis. The increased opsonic(endotoxin) completely protected animals if activity was nonspecific, was destroyed at 56 Cchallenge doses were given 6 to 24 hr later. The and disappeared after 24 hr. Hiss (74) and Hisscorrelation of properdin levels and resistance to and Zinsser (74a) observed that intraperitoneal


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injection of leucocyte extracts protected animals as it functions in sensitized hemolytic reactions.against subsequent infections with both gram At a temperature of 37 C the nonspecific adsorp-positive and gram negative bacteria. The authors tion results in destruction of the hemolytic com-reported a marked leucocytic infiltration in plement power of the serum. Normal antibodyprotected animals and found that protection was has been shown to be adsorbed out in the tem-manifested in either intraperitoneal or septicemic perature range of 0 to 37 C with killed bacterialinfections. Wright (184) observed increased suspensions and other materials but not withbactericidal and phagocytic activities of the zymosan, per se. Pillemer and co-workers haveserum of animals injected previously with gram reported that properdin cannot be adsorbedpositive bacterial vaccines. The addition of these from serum below temperatures of 5 to 10 C.vaccines to normal serum in vitro also brought However, no data have been published to sup-about an increased killing power. Wright noted port this finding for adsorbing materials otherthat both in vivo and in vitro antistaphylococcal than zymosan.activities were nonspecific and almost immediate Properdin is nonspecific in its antimicrobialin occurrence. He attributed the changes to the action toward various gram negative bacteria andrelease of opsonin from damaged leucocytes. certain viruses. Sensitivity of different bacterial

It seems quite clear that the numerous observa- species to properdin is variable, depending upontions of heightened resistance following the in- the age of the culture and the particular strainjection of various nonspecific materials are the employed (96).result of a general stimulation of multiple host There seems to be some doubt as to the speci-defenses. The marked leucocytosis commonly ficity of normal antibodies. Muir showed thatnoted in many of these reports certainly impli- serum adsorbed with killed suspensions of thecates the leucocytes as important participants same organism against which it was then testedin the altered response of the host (Suter, 162). (homologous strain) proved less active than if it

were tested against a heterologous bacterialCommon Characteristics of Properdin and Normal culture. However, by using a longer adsorption

(Natural) Antibody time or a larger number of adsorbing heterologous

Because of the strong similarities which became bacteria, all bactericidal action could be removedapparent between normal antibody and proper- before greatly affecting hemolytic complementdin, this section is devoted to a summary of this activity. Mackie and Finkelstein believed thatsubject and is compiled from the two preceding normal serum contained a number of naturallysections. Both substances require full comple- occurring somewhat specific antibodies whichment and Mg++ ions for the manifestation of were increased upon immunization. This con-bactericidal and unsensitized hemolytic activities. fusion about normal antibody specificity may beBoth function in bactericidal and hemolytic explained, in part, on the basis of Adler's findingssystems in the absence of specific antibody. Their (2). One must differentiate between low levels ofantibacterial action is directed almost exclusively classical antibody in "normal" serum and thetoward gram negative bacteria. Both materials normal antibody ordinarily present in serum.are regarded as being heat labile, i.e., inactiva- Since both normal antibody and specific antibodytion usually takes place at temperatures between can be adsorbed out of serum by a particular55 and 60 C in 20 to 30 min. Variations in heat bacterium, the distinction between the two canstability are the result of factors such as the be made on the basis of their differing heatanimal serum source, species and strains of micro- stabilities.organisms against which the heated sera are Most investigators of this problem have con-tested, pH of sera at time of heating and the eluded that normal or natural antibodies arecomplexity of the medium in which heating takes nonspecific or only slightly specific. As noted onplace. Both substances have been found in the p. 275, Dunlop was able to remove the bacteri-normal serum of many mammalian species and cidal action of serum toward several bacterialboth can be adsorbed nonspecifically from serum strains by first adsorbing normal serum withby similar materials. charcoal or powdered glass at low temperature.

Adsorption and removal from the serum of Gordon and Carter (58) were able to remove fromeither substance at temperatures below 15 to 20 C serum the bactericidal action for several differentcauses little, if any, inactivation of complement bacteria by cold adsorption with a single killed


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bacterial suspension. It is most reasonableto---accept the view that serum contains one or at I

most only a few normal antibodies of rather I OPSONIN SYSTEM I

nonspecific nature and that variable results can ICOMPLEbMENT SYSTEM:be explained on the basis of differing affinities of L- (CMUO)-bacteria for this substance. As others have stated,it is difficult to imagine that normal serum couldcontain the myriads of naturally occurringspecific antibodies necessary to sensitize greatnumbers of susceptible bacterial species andstrains, each by its own specific antibody. How- PROPERDINever, it is reasonable to expect that heterophile (PILLEMER, ET AL)reactions in serum explain, in part, those reportedobservations in which specificity seemed to beestablished. M

Wardlaw and Pillemer (172) found the bac-tericidal action of properdin to be inhibited byspecific antibody with Shigella dysenteriae. BACTERICIDAL HEMOLYTIC PHAGOCYTOSISFurthermore, an excess of properdin itself caused ACTIVITY ACTIVITY PROMOTINGa reversal of the antibacterial effect. As has been

( NGTI) (on-IMMUNE) (NOW-IMMUNE)stated, both specific and normal antibody in Figure 1. The thermolabile antimicrobial andexcess quantities also have been shown to inter- hemolytic systems of normal serum.fere with the thermolabile bactericidal system ofnormal serum. normal serum, and that the term, properdin, be

It is concluded from this discussion of the used in place of normal or natural antibody.numerous similarities shared by normal antibody Buchner's term, alexin, to describe his originaland properdin that they are likely the same important observation, did not envisage a com-substance. The extensive and careful work of plex bactericidal system in serum. He developedPillemer and co-workers has added much specific the concept that normal serum possessed a bac-information to our knowledge of the thermolabile tericidal property which was thermolabile. Sincebactericidal system in normal serum and their his time investigators have demonstrated that theterm, properdin, is more satisfactory than the antibacterial action of alexin required the par-ambiguous label of normal or natural antibody. ticipation of other factors. We feel it is unfortu-It is suggested that the old terminology be dis- nate that today alexin, opsonin and complementcarded unless it can be proved that normal have become identified, through usage, as oneantibody and properdin are distinct substances. substance. Since the bactericidal, hemolytic andWe cannot subscribe to the naming of the phagocytosis-promoting properties of normal

complete antibacterial system as the "properdin serum have been found to depend upon the inter-system." Since properdin itself represents only action of full complement, properdin (normalone of at least six components known to play a antibody) and Mg++ ions, we prefer to regardrequisite role in bactericidal action, this term alexin, opsonin and complement as similar systemsseems rather overweighted. Furthermore, the expressing the manifestations of bactericidallarge amount of work which began with the reaction, phagocytosis-promotion and hemolysis,searches of Nuttall, Buchner and Moxter laid respectively. Figure 1 is a schematic presentationthe foundation for what has since come to be of the antimicrobial and hemolytic activities ofregarded by many as the alexin system. These normal serum and is offered as a convenient wayearly important observations, as well as subse- of expressing certain general information.quent contributions, reveal that the alexinsystem bears close and, as yet, unconflicting Antibacterial Substances from Leucocytessimilarities to the recently described "properdin Among the earliest reports of the presence insystem." For these reasons it is suggested that leucocytes of antibacterial materials activeBuchner's original name, alexin, be retained to against gram negative bacteria were those ofdescribe the thermolabile bactericidal system of Buchner (27) and Denys and Havet (32) in 1894.


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280 SKARNES ANI) WATSON [VOL. 21

They found the bactericidal substance to be heat The recent researches of Amano and his co-labile at 55 C but it was heated in the presence workers (5, 7, 9) and others (77) have brought toof serum which probably accounted for their light a new role for the participation of lysozymeidentification of this material with serum alexin. in certain antibacterial reactions involving gramKorschun (91) prepared rabbit leucocyte extracts negative bacteria. It was found that purified eggby the freeze-thaw technique and reported a white lysozyme and lysozyme from leucocytessubstance which killed typhoid and cholera greatly accelerated the lysis of several gramorganisms. The material proved heat labile if negative pathogenic bacteria in the presence ofheated in the presence of serum but was only specific antiserum and complement. An accelera-slightly inactivated at 72 C for 1 s hr in the tion of the action of the immune bacteriolyticabsence of serum. Zinsser (186) also found an system was also observed in the presence ofantityphoid substance in rabbit leucocytes to be plakin, acid extracts of leucocytes or a basicrelatively heat stable, requiring a temperature of polypeptide of pancreatic origin. In none of the75 C for H hr for inactivation. -Manwaring numerous experiments were normal serum con-(106) prepared leucocyte extracts in water and trols substituted in place of specific antiserum;demonstrated an antityphoid factor which was thus the participation of other factors in immuneheat stable and nondialyzable. Amano and co- serum has not been eliminated. However, thisworkers (8, 9, 11) have recently reported sub- criticism may be minimized in view of the highstances from leucocytes, one of which bears re- dilutions of immune serum used in the experi-semblance to these antibacterial factors. ments. The following scheme represents the

Hirsch (72, 73) has demonstrated bactericidal interaction of lysozyme or other materials in thematerial present in rabbit, human and guinea immune bacteriolytic system.pig neutrophils that proved active against several F lsozymespecies of gram negative bacteria. The factor, [ an me

..'Complement and ~~+Illiwhich was ineffective against gram positive Lspecific antibody basic peptidepathogens, was associated with a globulin fraction aleubcocyte extractin the cytoplasm of the leucocytes. It lackedproperdin activity and was relatively heat stable accelertedat acid pH, being only slightly inactivated after antib e2 hr at 65 C. It was not dependent upon divalentions for its action and exhibited a bactericidal pH ANTIMICROBIAL SUBSTANCES SELECTIVE FORoptimum in the acid range. The substance has GRAM POSITIVE BACTERIAbeen named phagocytin by Hirsch and, as hesuggested, it is probably similar or identical to Beta-i ysinthe poorly defined substances recorded in the Fodor (48) was among the first to direct atten-early literature. Whether the phagocytins from tion to the anthracidal action of defibrinatedwhite cells of different animals are a single sub- blood in 1887. The next year Behring (16) re-stance or a group of closely related substances is ported that the anthracidal power of rat serumnot known. withstood heating at 60 C for I hr. The heat

stability of this serum anthracidal factor wasL~ysoz~ym~le as an Antibacterial Agent for Gram also confirmed shortly after by Pane (132), Bail

Negative Bacteria (12) and Sawtchenko (151). Pirenne (144) noted

Fleming (46), in his original paper on the that the thermostable rat serum anthracidediscovery of lysozyme, reported that it was active proved bactericidal for various gram positiveagainst certain gram negative pathogenic bac- bacteria but was inactive against gram negativeteria as well as several saprophytic forms. In species.his classic review, Thompson (165) found little In a series of papers (135-137, 139, 140)evidence that lysozyme in a purified state was Pettersson reported studies of a thermostableactive against pathogens. He cited literature bactericidal substance from human, horse andwhich showed that the antibacterial activity of dog serum, which he named beta-lysin. He foundsaliva and tears was due to the presence of that beta-lysin, in contrast to alexin, actedother factors in these fluids; for example, a heat principally against gram positive bacteria, re-labile factor has been shown to participate. quired temperatures of 64 to 75 C for H hr to


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inactivate, withstood dialysis and extraction with serum during the acute phase of various illnessesfat solvents, and was somewhat active at low with subsequent diminution upon recovery (75,temperature. Like alexin it was reputed to be a 80, 118, 166, 167, 185). The active serum sub-dual system composed of a heat stable "acti- stance or substances were relatively heat stable,vating" factor and a less heat stable "activable" withstanding 56 C for periods of at least 1i hr,factor which did not adsorb onto susceptible cells and were most potent against gram positivein the absence of the former substance. Petters- bacteria. Jacox (80) and Myrvik and Weiserson claimed that the addition of small amounts of (118) have shown that citrate inactivated thefresh serum to heat-inactivated serum (64 C) bactericidin. It is probable that these reportscaused a reactivation of the bactericidal power. concerned substances related or identical to theThat beta-lysin is a two-component system has serum beta-lysin of Pettersson. In some instances,been supported by Ostenfeld (131), but most gram negative antibacterial activity was alsoinvestigators either have not observed this or observed to increase during the acute phase ofhave been unable to confirm the duality of the illness. It is possible that substances other thanserum beta-lysins (104, 120). beta-lysin are also elaborated under these con-

In 1932 Mackie and Finkelstein (104) reported ditions of stress to account for increased anti-a thermostable substance (it withstood Hi hr at bacterial action against these forms.56 C) in normal serum which was active only Whether these various poorly characterizedagainst gram positive bacteria. Braun described substances, described as being relatively heata relatively heat stable normal serum factor stable and usually more active against gramwhich was active against Brucella abortus and positive bacteria, are identical or closely relatedMicrococcus pyogenes var. aureus and was found to the beta-lysins is not ascertainable at present.to be associated with serum globulins (23). Until more information is made available theyFishman and Shechmeister (45) also reported a will be regarded here as a group of closely relatedheat stable substance in normal serum which was substances in the category of beta-lysins.active against staphylococci. The beta-lysins were originally believed to haveMyrvik and Weiser (118) investigated a serum been derived from leucocytes, but their source is

bactericidin active against Bacillus subtilis which still undetermined. The presence of other anti-was relatively heat stable. Ekstedt (42, 43) bacterial factors in leucocytes has contributedworked with a normal serum antistaphylococcal to the confusion about the source of beta-lysins.factor which appeared to be similar to beta-lysin. Pettersson believed that beta-lysins were differentIt was relatively heat stable and active against from the endolysins of leucocytic origin (lateravirulent and weakly coagulase-positive strains named leukins) in that the latter material wasof staphylococci but not active toward good coagu- more heat stable and exhibited a somewhat dif-lase producing strains, an observation reported ferent antibacterial selectivity against gramearlier by Spink and Vivino (159). It was thought positive organisms (138). Mackie and Finkel-that coagulase neutralized the serum bactericidin stein (104) also differentiated the serum beta-thus enabling coagulase positive strains to lysins from leukins on the basis of the greatersurvive the antibacterial action. Calcium ions heat stability of the latter. In Pettersson's lastwere thought to be required for antibacterial paper on this subject (140) he concluded that itaction. Myrvik (120) was unable to confirm the would be difficult to differentiate between beta-role of coagulase in the neutralization of this lysin and leukin because of their similar proper-serum bactericidin. Unlike Ekstedt, he found ties. To attempt to distinguish between thesethat the addition of crude coagulase to test two substances on the basis of heat stability maysera did not impair bactericidal action towardsusceptible bacterial strains. Furthermore, his reveal a difference more apparent than real. Theobservations indicated that calcium ions may heat stability of beta-lysin was of necessitynot be required in the antibacterial action but tested in serum where the pH might vary any-

rather that the addition of citrate to serum in- where from 7.5 to 8.5, depending upon serumcreased ionic strength, accounting for inactiva- storage time. An alkaline pH has been showntion of the bactericidin. to decrease the heat stability of beta-lysin (104).

Several investigators have reported a non- On the other hand, leucocyte extracts are nor-

specific increase in the bactericidal power of mally acidic and this could account for the


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apparently greater heat stability of leukins when who extracted it from nuclei of avian red bloodheated. cells (92). Its antimicrobial properties haveMost of the antibacterial substances to be recently been confirmed (110, 124, 176). In

discussed have been identified as basic proteins, 1869 MUieseher extracted a nuclein from thepolypeptides or polvamines which have been nuclei of human neutrophils and he later charac-derived principally from cellular elements. They terized and named the basic portion of the salt-are generally most active against gram positive like nuclein, protamine (109). Protamines frombacteria, exhibiting little action against most various sources have since been shown to exhibitgram negative forms, except in higher concen- antimicrobial action against a virus (123), atrations. trypanosome (146), a yeast (107) and several

species of bacteria (19, 110, 123, 124, 176).Lysozymlle Grain positive bacteria generally appeared to be

As mentioned above, lysozyme, per se, has not more sensitive to the protamines.been shown to be active against pathogenic The basic nature of the histones and protaminesmicroorganisms (165). Recently, however, Glad- is well established. The histones possess largestone and Johnston (56) found purified lysozyme amounts of the two basic amino acids, lysineto act against certain encapsulated anthrax and arginine (30), whereas the protamines con-strains, in the presence of high levels of HCO3- tain large concentrations of arginine and areand CO2. This finding represents the first well- usually low in lysine (94).defined example wherein lysozyme has beenshown to inhibit a pathogenic microorganism. Tissue Basic PolypeptidesAttempts by the authors to inhibit other gram Bloom et al. (17) succeeded in extracting apositive pathogens under conditions of high CO2 nondialyzable anthracidal material from theconcentration were not successful. thymus, pancreas and caecum of differentThe role of lysozyme in defense against bac- animal species. The factor from calf thymus was

terial invaders should not be minimized, par- found to be an acid and heat stable basic poly-ticularly in view of its synergistic action with peptide containing approximately 30 per centcomplement and immune antibody as reported lysine and 3.5 per cent arginine and having anabove. Furthermore, the suggestion by Dubos isoelectric point of pH 11.2. The active substance(35) that potentially pathogenic bacteria may be was thought to have been derived from theprevented from establishing themselves in a host nuclear thymus histone (176). Basic tissuebecause of their lysozyme sensitivity is credible. polypeptides have also been derived from spleenThe basic nature and properties of lysozyme have (20, 21) and thyroid tissue (18). A mycobacteri-been reviewed (150a, 165). Amino acid analysis cidal peptide from calf thymus was studied byhas shown it to contain the three basic amino Dubos and Hirsch (37, 70, 71). This substanceacids, being particularly rich in arginine (49). wvas basic, possessing large amounts of lysineOther basic proteins of enzymic nature which and arginine, and having an isoelectric point

have been reported to possess antimicrobial between pH 10 and 11.properties are ribonuclease (3, 17, 86), deoxyribo- We have further characterized the thyimusnuclease and hyaluronidase (47). It is possible peptide of Bloom and co-workers and showedthat the antibacterial property of these enzymes, that it was derived from a histone fraction of theactive only in high concentrations, was due to calf thymus (156) as originally thought. Anthe presence of active impurities, as suggested amino acid analysis proved it to be identical toby the work of Fletcher et al. (47). the histone Fraction A, analyzed by Crampton,

et al. (30). This histone fraction was shown to beNucleins,Histones andProtainesmost active at alkaline pH and proved to be

Antibacterial nucleins, which are complexes of active predominantly against gram positive bac-nucleic acids and simple proteins such as histones teria.and protamines, were reported to be active It has been postulated that the antibacterialagainst gram positive species by Vaughan et al. properties of the basic tissue peptides are due toin 1893 (171) and by Kossel in 1896 (93). The the presence of large amounts of the basic aminofirst histone was discovered and named by Kossel, acid, lysine (160, 173, 176). Watson and Bloom


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(173) pointed out the significance of the high with microorganisms, since their positivelylysine content (29 per cent) of the antibacterial charged groups had reacted preferentially withthymus peptide. In comparing a synthetic poly- the free acidic polymers (83). The role of mucinlysine and thymus peptide they found that poly- and other large acidic molecules in the loweringamino lysine was about four times more active of natural resistance may be partly the result ofthan thymus peptide on a weight basis. They neutralization of the basic antibacterial agents inconcluded that antimicrobial activity resided tissues, as previously suggested (122, 154).within the lysine portion of the peptide molecule. The cause of death or inhibition of microor-The work with synthetic monoamino polypep- ganisms, once an antimicrobial factor becomestides of lysine (28, 83, 160, 173) and arginine and fixed upon the cell surface, is not clear. Few andornithine (83) lends strong support to this con- Schulman (44) showed that the basic peptide,cept. The basic polyamino acids proved very polymyxin E, was adsorbed in large amountsactive against both viruses and bacteria while upon the cell walls of susceptible bacteria. Theneutral and acidic polyamino acids exhibited no result of this adsorption was a disorganizationantimicrobial action at high concentrations (83). of the cell components taking part in the main-Monomers of the basic amino acids were inactive, tenance of osmotic equilibrium. Amano et al. (4)indicating that molecular weight was also im- reported that the bactericidal peptide, plakin,portant for activity of these peptides, a conclu- adsorbed onto bacterial surfaces causing damagesion previously offered by Massart in his study of to membrane permeability. Since molecularan antimicrobial protamine (107). weight, and perhaps linearity, was shown to be

Several investigators have suggested that a requisite for antibacterial action of certainantimicrobial basic proteins and polypeptides basic polypeptides, it is possible that surfacecombine with cell nucleoproteins or other nega- steric stresses result after combination withtively charged surface constituents of bacteria or susceptible bacteria, thus altering membraneviruses, thus disrupting important cell functions integrity. This subject has been reviewed recently(19, 83, 85, 160, 176). The union of the basic by Newton with respect to the polymyxin pep-substances with negatively charged cell surfaces tides (126).is believed to occur through electrostatic bonding.Bloom and Blake (18) observed that bacteria Leukins

clumped by the tissue polypeptide could be re- In 1891 Hankin (63) obtained an anthracidaldispersed by agitation, and after several washings material from lymph nodes of the dog and catthese cells were again clumped upon the addition which he characterized as a beta-globulin. Heof ribonucleic acid. It was concluded that the postulated that the active principle was derivedfirst clumping resulted from the attraction of the from the damaged leucocytes in the lymphaticbasic linear peptide to oppositely charged cell tissue. Since this initial observation many investi-surfaces while the second clumping indicated that gators have succeeded in extracting antibacterialthe peptide, which was in firm union with cell substances from leucocytes, particularly neutro-surfaces, combined with the ribonucleic acid by phils. In the majority of early reports leucocytemutual discharge of electrostatic bonds. extracts proved to be most active against gramThe fact that nucleic acids and long chain positive bacteria and relatively heat stable (88,

polysaccharides neutralize the antimicrobial 134, 152, 153, 175), withstood heating at 56 Cproperties of basic proteins or peptides (19, 83, but was destroyed in Hi hr at temperatures of84, 154, 156, 173, 176) offers indirect evidence 60 to 80 C. In 1909 Schneider named this groupsupporting the view that the basic materials of antibacterial substances "leukins" and dis-exert their deleterious effects by union with cell tinguished them from alexins on the bases of theirnucleoproteins. It is thought that the positively greater heat stability, antibacterial selectivitycharged ammonium groups of the linear basic for gram positive bacteria and their source (153).substances unite with negatively charged acidic Later confirmations of the presence of these leu-groups on the surfaces of susceptible microor- kins in leucocyte extracts have been publishedganisms. The reversal of antibacterial action by (17, 52, 64, 138).large acidic macromolecules likely results from Conflicting results were occasionally reportedthe inability of the basic materials to combine concerning the heat stability and antibacterial


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284 SKARNES AND) WATSON [VOL. 21

selectivity of the leukins. No doubt this confusion probably a reflection of its further purification asarose from the fact that there are at least two compared faith the early crude fractions.different bactericidal factors in leucocyte extracts: Leukins or leukin-like products have been ob-the leukins and the phagocvtins. For example, tained from leutcocytes of the human, rabbit, dog,Gengou (54) reported an acid-extractable leuco- guinea pig and rat, but whether these variouscyte material which inhibited many gram nega- fractions share similar properties is uncertain. Ittive species in high concentrations and was heat is reasonable to assume that they are protamineresistant at 80 C for H hr at acid pH but not at or histone fractions of nucleoprotein origin.alkaline pH. Since Gengou did not test his ma- Few attempts have been made to obtain anti-terial against gram positive species it is not pos- bacterial substances from leucocytes other thansible to know whether he was working with the neutrophils. However, Bloom et al. reportedleukin, phagocytin or a combination of both. an inhibitory action of rat mononuclear extractGay and Clark (52) demonstrated the presence of upon tubercle bacilli (21). While Gengou re-two different antibacterial substances in single ported little or no antibacterial activity in macro-leucocyte extract preparations. Unheated extract phage cell extracts (53), Gay and Clark demon-killed both gram negative and gram positive strated antibacterial action against both grambacteria while heated extract proved active only positive and gram negative bacteria with macro-against the latter. phage extracts (52). It would not be surprisingAmano and co-workers (6, 9) and others (77) to find that the nuclei of all types of leucocytes

described acid extracts of leucocytes which proved contain leukin-like material obtainable by suit-active in accelerating immune bacteriolytic able extraction methods.systems against gram negative bacteria. The Plakinauthors named two factors from the acid ex-tracts, "leucozyme A" and "leucozyme B" (10). Blood platelets represent another source ofThe method of preparation and heat stability of antibacterial material, as first demonstrated bythese two substances suggests that they belong Gruber and Futaki in 1907 (61). They obtainedto the category of leukins. Furthermore, it is an anthracidal substance from blood platelets ofprobable that the two names describe but one the horse. Barreau found in the platelets of severalsubstance which manifests itself in two different animals a relatively heat stable substance whichways, depending upon the presence or absence proved active against anthrax bacilli (14). MXoreof calcium or magnesium ions. However, these recently Amano and co-workers extracted fromfindings may elucidate a new relationship of horse platelets a plakin which inhibited cellimmunity whereby a substance active only respiration of two gram positive saprophytes (3).against gram positive bacteria can also function The product was relatively heat stable in acidagainst gram negative forms in the presence of medium, requiring heating for Hi hr at 80 C forspecific antibody. inactivation, but was less stable at neutral pH.We have recently characterized a leukin from Amano and his colleagues also found plakin to be

rabbit polymorphonuclear cells (155). Presum- operative in immune bacteriolytic systems (7).ably this leukin was derived from nucleoprotein Although plakin did not attack gram negativeof the white cell and is a protamine split product bacteria directly, it caused an acceleration of thewith a definite predilection for gram positive immune bacteriolysis. The relationship of plakinpathogenic bacteria. The product proved to be to leukin is unclear but the possibility exists thatactive in low concentrations (2 to 5 gg/ml) in the former substance was derived from nucleo-vitro and was very heat stable at acid or neutral protein of the platelets and consequently ispH, surviving 100 C for 2 hr. It was shown to similar to leukin.contain a large amount of the basic amino acid,arginine, which likely was responsible for the eantibacterial action. Large acidic polymers In 1914, Kammerer observed that meso-blocked its activity in the manner described for hematin was inhibitory to many gram positivethe basic polypeptides cited on p. 283. The ap- saprophytes and pathogens, and that hematinparently greater heat stability of this leukin as was less active (81). Whitney et al. (178) extractedcompared with leukins previously reported is an antibacterial material from trypsin-treated


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bovine red blood cells. The factor was stable to cus strain. This apparent conflict of results withautoclaving for 15 min at alkaline pH only and those of Dubos and Hirsch may be explained onthe authors suggested that it was a peptide. The the basis of the different bacterial species used.source and preparation of this factor made it moreprobable that antibacterial action was due to Lacteninthe presence of hematins, as suggested by van Hesse was the first to report the antibacterialHeyningen (66). This investigator obtained from effect of cow's milk against several bacterialhorse red blood cells hematin and mesohematin species (65). Wilson and Rosenblum studied thewhich, in very low concentrations inhibited a large bactericidal protein, lactenin, found in the wheynumber of gram positive saprophytic bacilli, of human, cow and goat milk. The substance inIvanovics and Koczka (79) found mesohematin low concentrations was selectively active againstbactericidal for many gram positive bacteria but Group A streptococci and was inactivated at 80not for gram negative forms, with the exception C (179).of two Hemophilus strains. The susceptible bac- There are many other reports in the literatureteria adsorbed large amounts of mesohematin not included in this review which deal with anti-whereas resistant species did not, unless pre- microbial substances of tissue origin. No doubttreated with organic acids or ethylene glycol. some of these agents (89, 100, 119, 128, 158, 168)The authors postulated that resistant gram are related or identical to the basic factors alreadynegative bacteria possessed a polysaccharide discussed but a current lack of information pre-layer at their cell surface which prevented pene- vents their inclusion under any of the headingstration of mesohematin. The mechanism of listed. The various antimicrobial substancesaction of the antibacterial heme compounds may discussed in this review are briefly characterizedbe due to a competitive action of these porphyrins in table 1.with those essential to metabolism of the cell, assuggested by Dubos (38).

In 1930 Douglas and Smith noted the viricidalSpermine,Spermidine action of a heat labile normal serum component

In 1951 Dubos (36) extracted a tuberculostatic (33). The next year Mueller attributed this anti-factor from various animal tissues. Its action viral activity to the alexin system of serum (114).was later found to be dependent upon the pres- Since this time many investigators have con-ence of a bovine albumin fraction and it was firmed the virus neutralizing effect of normalidentified as spermine (67). Hirsch further ex- serum (13, 55, 157), an effect which not only re-panded this work and also found spermidine to moved viral infectivity but hemagglutinatingbe equally active against certain strains of activity as well. Much evidence has accumulatedMycobacterium tuberculosis whereas other related pointing to complement as the heat labile viralpolyamino compounds proved inactive (68, 69). inhibitor in serum (34, 76, 101, 111, 150, 177),The dependency of tuberculostatic action of the and it was observed that complement power wastwo basic polyamines upon the presence of an heightened in the presence of specific viral anti-alpha-globulin fraction was explained on the body. McCarty and Germer (121) found whatbasis of an enzymatic alteration which released appeared to be two heat labile serum componentsthe active principle. Rozansky et al. (149) demon- necessary for virus neutralization. Recently thisstrated that spermine inhibited growth of several observation was enlarged by Wedgwood et al.gram positive bacteria and two strains of Neis- (174) who defined the complete serum systemseria, although it was inactive against enteric operating in viral neutralization. They foundmicroorganisms. The antibacterial power of that complement, properdin and Mg++ ions werespermine was seen to increase greatly as pH was required and that properdin probably combinedchanged from acid to alkaline, a finding in keeping directly with virus, the complement componentswith the results obtained with other basic anti- and divalent ion acting as cofactors. Koprowskibacterial substances. Grossowicz et al. (60) observed that a relatively heat stable serum com-reported that spermine and spermidine were not ponent was responsible for the neutralization ofdependent upon the presence of the bovine al- yellow fever virus and certain other viruses ofbumin fraction for activity against a staphylococ- the encephalitogenic group (90). Casals and


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TABLE 1Tissue antimicrobial substances

Name Common Source Heat Stability Chemical Class

I. Antibacterial selectivity: gram negative

Complement* Serum Labilet Euglobulin-carbohydrate-albumin

Properdin (normal) an- Serum Labile to rela- Euglobulintibody tively stable

Phagocytin Neutrophilic leucocytes Relatively stable Globulin fractionLysozyme (special case) Ubiquitous distribution Stable Small basic protein

II. Antibacterial selectivity: gram positive

beta-Lysin Serum Relatively stable Protein (?)Lysozyme Ubiquitous distribution Stable Small basic proteinHistone Lymphatics (nucleopro- Stable Small basic protein

tein)Protamine Sperm cells (nucleopro- Stable Small basic protein

tein)Tissue polypeptides Lymphatics (nucleopro- Stable Linear basic peptides

tein)Leukin Neutrophilic leucocytes Stable Basic peptides (protamine)Plakin Blood platelets Relatively stable Peptide (?)Hematin, mesohematin Red blood cells Stable Iron porphyrinsSpermine, spermidine Pancreas, prostate Stable Basic polyaminesLactenin Milk Relatively stable Protein (?)

* We do not apply the names alexin or opsonin to single substances; rather we consider them to denotesimilar systems which require full complement in addition to properdin (normal antibody).

t Labile, inactivated 56 C, 12 hr; relatively stable, resist 56 C, Y2 hr but destroyed below 80 C, 12 hr;stable, resist 80 to 100 C, YM hr or more. (pH at heating ca. neutral or acid.)

Olitsky reported a heat stable lipid fraction jected by a different route where they would bewhich inactivated neurotropic viruses and which rapidly neutralized by embryonic tissues beforewas obtainable from normal sera of several getting to the site of virus inoculation.animals (29). Utz prepared a lecithin-like fractionfrom normal sera which neutralized the infectivity RELATIONSHIP OF ANTIMICROBIAL SUBSTANCES

of influenza and Newcastle disease viruses but TO NATURAL RESISTANCEdid not alter hemagglutinating activity (170). In 1922 Ledingham cautioned against ascribing

Antiviral action has also been reported for too much significance in immunity to the variousprotamine (59, 123), thymus polypeptide (173) bactericidal factors described in the literature,and synthetic polylysine (59, 160, 173). It was particularly since most such factors have beenpostulated that the basic ammonium groups on tested in vitro only (99). This caution is eventhe polypeptide combine by ionic linkage to more warranted today and has been reiteratedvirus nucleoprotein in a manner analogous to and expanded recently by Wilson and Milesthat previously suggested for bacteria. Green et al. (180). In addition to the limitations inherent in(59) reported in vivo activity of synthetic poly- the application of in vitro experimental results tolysine and protamine. Protection of chicken the in vivo environment, another importantembryos resulted when both the antiviral agent qualification is apparent. Often, the preparativeand the virus were injected at the same site, methods used for obtaining antimicrobial agentsindicating that the protective action was only will give rise to artifacts which bear little resem-local. It is doubtful whether protection would blance to naturally occurring tissue products.have resulted if the basic substances were in- However, this latter criticism is lessened in those


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experimental procedures where harsh chemical When authors report measurements of heatextractions were not necessary to evidence anti- stability, the experimental procedures should bemicrobial action. For example, serum factors and well described. That is, heat stability can becrude preparations of leukins and phagocytins examined under conditions of varying pH, vary-can be demonstrated quite simply. ing concentrations of the active material, and inDubos has offered some tenable views regarding the presence of carefully defined media. The

the significance of many of the antimicrobial degree of purity of the particular factor beingagents described above (38). He has also dis- tested will also influence the observed heat sta-cussed the influence on host resistance of anti- bility. The determination of antibacterial selec-bacterial fatty acids, high CO2 tension, and the tivity is useful in classification; however, it isaccumulation of organic acids such as lactic acid important to take cognizance of the variationin inflammatory sites. It is likely that many anti- in individual species and strain sensitivities. Suchmicrobial agents are released locally in damaged properties as coagulase production amongtissues and that their activities are augmented staphylococcal strains (42, 159, 163) and theor enhanced by ensuing changes within the im- phase of shigella strains, whether rough or smoothmediate environment of inflammatory loci (31, (164), affect individual susceptibilities to the72, 155, 173). antagonizing agent and make classification moreThe role of antimicrobial tissue factors in re- difficult. Measurements of potency, optimal pH

sistance may represent one facet of the whole of action, effects of inhibitors and ionic influencesdefense system. We do not suppose that these upon particular antimicrobial factors also maysubstances exist, as such, in normal tissues but aid in their identification.that they arise in response to physiologic changes REFERENCESwhich accompany stress. They may act in thebody to kill or to slow the growth of invading 1. ADAMI, J. G. 1909 Inflammation. Macmil-microorganisms, enabling other host defenses to lan and Company, London.operate more efficiently in the removal of inimical 2. ADLER, F. L. 1953 Bactericidal action ofagents from the tissues. Allied mechanisms of normal sera against a strain of Salmonellanatural resistance to infectious disease, with typhosa. J. Immunol., 70, 69-78.whichatural eantimicrobial factors maybea inte-

3. AMANO, T., KATO, K., AND SHIMIZU, R. 1952which the antimicrobial factors may be inter Studies on the role of plakin. Med. J.grated, have been well reviewed by Nungester Osaka Univ., 3, 293-311.(127), Kass and Finland (82) and Suter (162). 4. AMANO, T., SHIMA, M., AND KATO, K. 1953

The damage of semipermeability of bac-CONCLUSIONS teria caused by plakin. Med. J. Osaka

This review, though incomplete, should aid in Univ., 4, 277-284.eliminating some of the confusion which has 5. AMANO, T., INAI, S., SEKI, Y., KASHIBA, S.,

prevailedregarding the identis of my of te1FUJIKAWA, J., AND NISHIMURA, S. 1954

prevantmildregardine.fuidenti any oforthe Accelerating effect on the immune bacterio-

antheimicroialtsure. and flutaid sstances reported lysis by lysozyme-like substance of leuco-i the literature. In certain instances we have cytes and egg-white lysozyme. Med. J.generalized beyond present-day knowledge con- Osaka Univ., 4, 401-418.cerning the classification of these host factors but 6. AMANO, T., SEKI, Y., KASHIBA, S., Fuji-this oversimplification is used to facilitate a better KAWA, K., AND ORIHARA, M. 1954 Bac-general understanding of the subject. teriolytic substance in the acidic extract of

Ideally it would be most desirable to determine leucocytes. Med. J. Osaka Univ., 5, 639-exact chemical compositions of the various host 643.factors to arrive at a more accurate classification. 7. AMANO, T., NISHIMOTO, M., KINJO, K.,Unfortunately, as this is not yet possible, we INOUE, K., INAI, S., SEKI, Y., AND KASH-mustrelyupon gross observations to define IBA, S. 1954 Influence of immune bacte-must relyeup ngsbservationscto defin riolysis upon bacterial respiration. Med.

certain of these antibacterial products. Until J. Osaka Univ., 5, 145-165.such time as definitive characterizations become 8. AMANO, T., NISHIMOTO, M., INAI, S., SEKI,available, the following suggestions may prove Y., KASHIBA, S., FUJIKAWA, S., AND ORI-fruitful in categorizing antimicrobial agents in HARA, M. 1954 Studies on the immunethe future. bacteriolysis. V. Fractionation of the


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active substance from the acidic extract of stances extracted from normal rat spleen.bovine spleen. Med. J. Osaka Univ., 5, J. Infectious Diseases, 92, 70-73.631-638. 22. BORDET, J. 1909 Studies in immunity.

9. AMANO, T., SEKI, Y., KASHIBA, S., FUJIKAWA, (translated by F. P. Gay). Wiley & Sons,K., ORIHARA, M., AND MORIOKA, T. 1955 Inc., New York.The effect of leucocytes extracts on Hae- 23. BRAUN, W. 1949 The nature of the selec-mophilus pertussis and the immune bacte- tive serum factor affecting the variationriolysis of Haemophilus pertussis. Med. J. of Brucella abortus. J. Bacteriol., 58,Osaka Univ., 6, 67-79. 291-297.

10. AMANO, T., SEKI, Y., KASHIBA, K., FUJIKAWA, 24. BUCHNER, H. 1889 Tber die bakterientod-K., MORIOKA, T., AND ICHIKAWA, S. 1956 tende Wirkung des zellfreien Blutserums.The effect of leucocyte extract on Staphylo- Centr. Bakteriol. Parasitenk., 6, 1-11.coccus pyogenes. Med. J. Osaka Univ., 7, 25. BUCHNER, H. 1889 Ueber die nahere Na-233-243. tur der bakterientodtenden Substanz im

11. AMANO, T., SEKI, S., KASHIBA, S., FUJIKAWA, Blutserum. Centr. Bakteriol. Parasitenk.,K., MORIOKA, T., AND ICHIKAWA, S. 1955 6, 561-565.Separation of the accelerating substance 26. BUCHNER, H. 1891 Zur Nomenklatur derof immune bacteriolysis from bovine spleen schutzenden Eiweisskorper. Centr. Bak-by column chromatography. Med. J. teriol. Parasitenk., 10, 699-701.Osaka Univ., 6, 717-723. 27. BUCHNER, H. 1894 Neuere Fortschritte

12. BAIL, 0. 1903 Untersuchungen uber natuir- in der Immunitatsfrage. Munch. med.liche und kuinstliche Milzbrandimmunitat. Wochschr., 41, 497-500.Centr. Bakteriol. Parasitenk., Abt. I, 33, 28. BURGER, W. C., AND STAHMANN, M. A.343-353. 1952 The agglutination and growth in-

13. BANG, F. B., FOARD, M., AND KARZON, D. T. hibition of bacteria by lysine polypep-1951 Mode of action of heat-labile serum tides. Arch. Biochem. Biophys., 39, 27-inactivating substance on Newcastle dis- 36.ease virus. Bull. Johns Hopkins Hosp., 29. CASALS, J., AND OLITSKY, P. K. 1947 In-88, 83-100. activation of certain neurotropic viruses

14. BARREAU, E. 1909 tber die Wirkung von in vitro by serum lipids. Science, 106,Blutplattchenstoffen gegen Milzbrander- 267-268.reger. Arch. Hyg., 70, 331-354. 30. CRAMPTON, C. F., MOORE, S., AND STEIN,

15. BEDSON, S. P. 1915 Analysis of the effect W. H. 1955 Chromatographic fractiona-of nuclein and nucleic acid on the normal tion of calf thymus histone. J. Biol.antibody. J. Pathol. Bacteriol., 19, 191- Chem., 215, 787-801.209. 31. CROMARTIE, W. J., BLOOM, W. L., AND

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DOUGLAS, S.Rmunol., 27, 451-468.An experimental investigation of the role 186. ZINSSER, H. 1910 On bactericidal sub-of the blood fluids in connection with stances extracted from normal leucocytes.phagocytosis. Proc. Roy. Soc. (London), J. Med. Research, 22, 397-433.72, 357-370. 187. ZINSSER, H., ENDERS, J. F., AND FOTHER-

183. WRIGHT, A. C., AND DOUGLAS, S. R. 1904 GILL, L. D. 1946 Immunity principlesAn experimental investigation of the role and application in medicine and publicof the blood fluids in connection with health. Macmillan Co., New York.


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