Role of Macrophages NaturalResistance to Infectionslishment ofinfection and, if infection has...

Vol. 43, No. 1MICROBIOLOGICAL REVIEWS, March 1979, p. 1-260146-0749/79/01-0001/26$02.00/0

Role of Macrophages in Natural Resistance to Virus InfectionsS0REN C. MOGENSEN

Institute ofMedical Microbiology, University ofAarhus, Aarhus, Denmark

INTRODUCTION .............................................................THE MONONUCLEAR PHAGOCYTE SYSTEM ................................

ASSESSMENT OF VIRUS-MACROPHAGE INTERACTIONS IN VITRO ......Sources of Macrophages ....................................................In Vitro Studies ofVirus-Macrophage Interactions ..........................

Adsorption ...............................................................Direct assay of virus growth .......... ..................................

Indirect assays of virus growth ...........................................(i) Infectious center assays ...........................................

(ii) Cytopathology ...................................................(iii) Immunofluorescence ..............................................(iv) Electron microscopy...............................................(v) Tracer techniques .................................................

ASSESSMENT OF VIRUS-MACROPHAGE INTERACTIONS IN VIVO .........General Markers of Pathogenicity ..........................................

Morbidity and mortality ..................................................Virus clearance and virus growth ........................................

Histological lesions .......................................................Modulations of the Function of the Mononuclear Phagocyte System .........Macrophage blockade ....................................................Macrophage stimulation ..................................................Macrophage transfer .....................................................

ASPECTS OF MACROPHAGE-DEPENDENT RESISTANCE TO VIRUS INFEC-TIONS ................................................................

Virus Strain- and Virus Type-Related Resistance ...........................Geneticaly Determined Resistance .........................................Age-Related Resistance .....................................................Role ofNonspecifically Stimulated and Activated Macrophages in Resistance

CONCLUDING REMARKS ....................................................SUMMARY ...................................................................LITERATURE CITED .........................................................

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INTRODUCTIONIn analyzing host defense mechanisms against

virus infections, two aspects have to be consid-ered. The first aspect concems the mechanismsby which the host displays resistance to andrecovers from viral infections encountered forthe first time in life; the second concerns thedefense mechanisms mounted by an immunehost to reinfection. In both instances, the prin-cipal tasks of host defenses are to prevent estab-lishment of infection and, if infection has hap-pened, to interfere with virus multiplication,limit the spread of infection, and finally elimi-nate the virus. To achieve this, a complex inter-action of both specific and nonspecific host de-fense mechanisms has to be at work.

In primary infections, nonspecific host resist-ance factors represent the main line of defenseduring the first few days of infection. Theseconsist of preexisting defense mechanisms, suchas barriers to virus penetration (cutaneous epi-thelium, ciliated respiratory epithelium, vascu-

lar endothelium, lack of membrane surface re-ceptors, etc. [94]), nonspecific viricidins in bodyfluids (138), and phagocytosis leading to virusdestruction (98). A further series of nonspecificantiviral principles are induced fairly rapidly.These are factors like interferon (15, 67), ele-vated body temperature (86), and acid pH andhypoxia in inflammatory exudates (14). By in-terfering with the early stages of virus invasion,multiplication, and spread to susceptible organs,the nonspecific host defenses may be importantin determining the outcome of a virus infection.Final recovery from a fully established infection,however, is probably determined by the specificimmune response, appearing some days after theinitiation of infection.

In addition to being of importance in recoveryfrom fully established primary infections, spe-cific host responses are the main defense mech-anisms against reinfection with the same or an-tigenically closely related organisms. They aremounted by cells of the lymphoid system and

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involve the production by the B-lymphocytesystem of antibodies with the capacity to neu-tralize extracellular virus and cellular immunitymediated by sensitized T lymphocytes. Beingimmunological in nature, they are specificallyinduced and act specifically against the virusconcerned. An intense interaction between B-and T-cell-mediated immune responses to virusinfections occurs, and other factors, such as com-plement and cells from the mononuclear phago-cyte system (macrophages) are involved ashelpers or mediators in the immune response(3).The objective of this review was to analyze

the role played by macrophages in natural re-sistance to primary virus infections. Special em-phasis is placed on the key role of macrophagesas a first line of defense at the portal of entry orin target organs of crucial importance for theoutcome of an infection. The review does notemphasize immunological aspects of virus-mac-rophage interactions; information on this topiccan be found in other reviews (3, 23).

THE MONONUCLEAR PHAGOCYTESYSTEM

The term macrophage ("large eater") wasintroduced by Metchnikoff in 1892 (95). He clas-sified phagocytic cells as "macrophages" and"microphages" (polymorphonuclear leukocytes)on morphological and functional grounds. Hedemonstrated that these cells play an importantrole in resistance to bacterial infections by en-gulfment and digestion of the invading micro-organisms. Mononuclear phagocytes resident invarious organs and tissues were, according toMetchnikoff, closely related and belonged to a"macrophage system."This system was further elaborated by Aschoff

(8), who grouped reticular cells of the spleen andlymph nodes and reticuloendothelial cells of thelymph and blood sinuses as well as histiocytes,splenocytes, and blood monocytes in a unifyingcell system called the "reticuloendothelial sys-tem." Despite much criticism (92) and sugges-tions of other terms, such as the "reticulohistio-cyte system" (144, 156), the concept of Aschoff'sreticuloendothelial system has been widely ac-cepted and used until today. However, our cur-rent knowledge of phagocytic mononuclear cellsrecently demanded a reevaluation of the classi-fication of these cells. On the basis of morphol-ogy, function, and kinetics of development, allhighly phagocytic mononuclear cells and theirprecursors can fit into one cell lineage called the"mononuclear phagocyte system" (154).The morphology of cells of the mononuclear

phagocyte system depends to certain degrees onthe species of animal, the anatomical site, and

the degree of development or stimulation (60).Certain common characteristics can, however,be outlined. They are fairly large cells withvarying amounts of cytoplasm and a reniform oroval nucleus. Cytoplasmic organelles for secre-tory activities are abundant in the more matureforms present in the tissues (154). Ruffling ofthe surface membrane, easily recognized inphase-contrast or electron microscopy, is a spe-cific morphological characteristic of the mono-nuclear phagocytes as compared with lympho-cytes (60).The functional properties shared by cells of

the mononuclear phagocyte system are avidphagocytosis, pinocytosis, and the ability to ad-here firmly to a glass or plastic surface (154).Glass adherence is a major means of isolatingthe cells. Other cell types (for example, fibro-blasts, reticular cells, and endothelial cells) canalso phagocytize foreign particles, but to a muchlower degree (116). Furthermore, the ingestionof particles by mononuclear phagocytes is en-hanced by the presence of specific immunoglob-ulins with or without complement ("immunephagocytosis"), because these cells have Fc (18)and complement (78) receptor sites at the cellmembrane. This is probably not the case withthe other cell types.

Cytokinetic studies have shown that the cellsof the mononuclear phagocyte system and theirprecursors represent a single cell lineage (153).Mononuclear phagocytes originate from precur-sor cells of the bone marrow; here, these cellsmature into promonocytes and monocytes.Monocytes are transported via the blood to or-gans and tissues, where they finally mature intomacrophages.These are histiocytes ofconnectivetissue; Kupffer cells of the liver; alveolar mac-rophages of the lungs; free and fixed macro-phages of the spleen, lymph nodes, and bonemarrow; and macrophages monitoring serousspaces, such as the pleural and peritoneal cavi-ties. Osteoclasts of bone tissue and microglialcells of the nervous system have more tenta-tively been included in the system (154). Bloodmonocytes continuously "feed" these compart-ments with new cells. In sites of inflammation,the recruitment of monocytes from the blood isgreatly accelerated, although local multiplica-tion ofmature macrophages apparently may alsooccur (111).

ASSESSMENT OF VIRUS-MACROPHAGE INTERACTIONS IN

VTROSources of Macrophages

Macrophage cultures are easily started andcan be maintained in vitro for weeks (68); in fact,

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MACROPHAGE RESISTANCE TO VIRUS INFECTIONS

macrophages were one of the first cell types tobe established in culture (27). In mammals andbirds, cells can be obtained from serous cavitiesor various organs. The choice of a macrophagedonor depends on the particular virus-host in-teraction under study and the cell number re-quired for the investigations. For many pur-poses, the mouse is a convenient experimentalanimal, because different inbred and congenicstrains are available. From the peritoneal cavi-ties of mice as well as rats, rabbits, and guineapigs, resident macrophages can be harvested bysimple lavage of the cavities with buffered salineor culture medium (31, 36). To increase the yieldof cells, various inflammatory agents, such asmineral oil, glycogen, proteose-peptone, etc., areoften used to induce an exudate in the peritonealcavity. However, in addition to increasing theyield, individual cells harvested will be in a stateof activation and may not reflect the propertiesof native, uninduced cells (36). Other sources ofmacrophages have been used. Large numbers ofalveolar macrophages can be obtained by lavageof the lower respiratory tracts of experimentalanimals (108). Bovine macrophages in enormousquantities can be harvested from the mammarygland after infusion of lipopolysaccharide in thegland (157). By explanting fragments of liversfrom newbom mice onto a reconstituted collagensubstrate (37) in a medium supplemented withhorse serum and chicken embryo extract, Bangand Warwick (13) obtained migration of livermacrophages away from the explants. Recently,a technique has been described by which livermacrophages can be isolated by perfusion anddigestion with Pronase (38).

In humans, the only readily accessible sourceof cells of the mononuclear phagocyte system ismonocytes from peripheral blood. Several meth-ods for the isolation of these cells have beendevised, including gradient centrifugation ondense albumin (17) or Ficoll-Hypaque (24) so-lutions combined with a gla adherence step.After 2 or 3 days in culture, the cells showmacrophage characteristics.

In Vitro Studies on Virus-MacrophageInteractions

The ability of virus particles to grow in mac-rophages can be a major pathogenicity or viru-lence factor in virus diseases, because macro-phages monitor the main body compartmentsand thereby may determine the access of virusparticles to susceptible tissues or organs (98).Several in vitro methods have been used in thestudy of virus particle uptake into and replica-tion in macrophages. Although the outcomes ofvirus-macrophage interactions as regards these

parameters have been elucidated for many vi-ruses, little is still known about the intracellularevents which enable a virus to replicate in themacrophage instead of being digested or whatfactors determine the failure of macrophages todigest the virus particle, making a replicationpossible (99). Because the yields of infectiousvirus released from infected macrophages arevery low for many viruses as compared with theyields from other cell types, indirect assays areoften used to estimate the ability of a virus toreplicate in these cells.Adsorption. The first step in virus-macro-

phage interaction is adsorption of the virus par-ticle to the cell. Adsorption experiments can beconducted with both monolayer and suspensioncultures of macrophages. Two different ap-proaches to the assessment of adsorption havebeen applied. The first uses measurement of thefractions of residual, unadsorbed virus in theculture medium after different times of adsorp-tion (101, 135, 141). Appropriate controls with-out macrophages have to be included to takeaccount of heat inactivation and adherence ofvirus to the glass or plastic surface. By thesecond method, the amount of intracellular virusis determined after various times of adsorptionand thorough washing of the cultures (62, 146).This method misses the eclipsed virus and thusdoes not accurately reflect the total amount ofadsorbed virus. The information gained of dif-ferences in virus particles replication in variousvirus-macrophage systems is only valid if theadsorption rates are equal.Direct assay of virus growth. In some vi-

ruses, it has been possible to measure directlyvirus growth in cultured macrophages by simpletitration of extracellular virus released into theculture medium or of intracellular virus releasedfrom the cells by freezing and thawing or ultra-sonic vibration. In one of the most productivesystems studied, Shif and Bang (135) were ableto grow mouse hepatitis virus type 2 (MHV-2)in cultures of peritoneal macrophages from sus-cegtible Princeton (PRI) mice to titers close to10 macrophage tissue culture mean infectivedoses per ml. The replication ofMHV-3 in mac-rophages has also been demonstrated by a sim-ple titration approach (155). Lactic dehydrogen-ase virus replicates mainly, or perhaps exclu-sively, in mouse macrophages (35) and grows tohigh titers in cultures of these cells (34). Linden-mann et al. (82) adapted a strain of avian influ-enza A virus to grow to high titers in mousemacrophage cultures as assessed by virus titra-tion and hemagglutinin measurement. Less effi-cient is the replication of flaviviruses in mousemacrophages, but direct titrations of West Nilevirus and yellow fever virus yields from mouse

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macrophage cultures prepared from resistantand susceptible strains of mice have been sensi-tive enough to reveal the importance of macro-phages in determining resistance to these viruses(48,49). Growth of vaccinia virus in macrophagecultures from mice (118) and rabbits (131) hasbeen used to evaluate the role of activated andimmune macrophages in host defenses. Forherpes simplex virus type 1 (HSV-1) (69, 141)and murine cytomegalovirus (132), the yieldshave generally been less than 10% of the initialinocula.Indirect assays of virus growth. Indirect

assay methods have been used to measure virusgrowth in virus-macrophage systems in whichthe yields are too small for direct titration. Fur-thermore, these techniques (i.e., immunofluores-cence, electron microscopy, and autoradiogra-phy) have been used in attempts to disclose theintracellular events occurring in virus-infectedmacrophages.

(i) Infectious center assays. In infectiouscenter assays, the infected macrophages are co-cultivated with a permissive cell monolayer.Two major modifications of the technique havebeen applied. Either macrophages are grown ona glass or plastic surface, infected with the virus,and finally overlaid with a monolayer of suscep-tible target cells (62, 69, 101, 146), or a suspen-sion of infected macrophages is added to a pre-formed permissive monolayer (117, 132, 150).The release of infectious virus from the macro-phages can then be estimated by counting thenumber of infectious centers appearing in theindicator cell monolayer either as plaques (62)or as fluorescent foci (69). It is important thatall nonadsorbed virus be eliminated before co-cultivation; this is usually achieved by washingthe macrophages with antiserum. Another im-perative demand in these assays is to work withpure macrophage populations without contami-nation with permissive cells, such as fibroblasts.The infectious center assays indicate the numberof macrophages in the culture that support virusreplication, but they do not give any informationof how well the individual cells do so.

(ii) Cytopathology. Direct observation ofcytopathic changes of virus-infected macro-phages has not been widely used to evaluatevirus-macrophage interactions because cellularchanges are not very prominent with many vi-ruses studied. However, MHV-2 causes suchmarked degenerative changes in macrophagesfrom susceptible mice that this effect has beenused as the main marker for susceptibility orresistance of whole animals to the virus (13, 71),and it has been possible to develop a plaqueassay for this virus in macrophage monolayers

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(134). Similarly, MHV-3 induces another easilydetectable cytopathic effect in macrophagesfrom susceptible mice with the formation ofmultinucleated giant cells (90, 155). A macro-phage-adapted strain of avian influenza virusinduces rounding and clumping of macrophagesfrom susceptible mice with a characteristic blur-ring of the outlines of the cells (82). Occasionalproduction of polykaryocytes by HSV-1 inmouse macrophage cultures was described byStevens and Cook (141).

(iii) Immunofluorescence. Immunofluo-rescent visualization of virus antigen in infectedmacrophage cultures has proven useful in study-ing both quantitative and qualitative aspects ofvirus-macrophage interactions. Both direct andindirect techniques have been used. By thesemethods, information has been gained of theactual numbers of macrophages being infectedwith West Nile virus (49, 150), ectromelia virus(122), HSV (69, 141), lymphocytic choriomen-ingitis virus (145), andMHV (155). Furthermore,the time courses of intracellular development ofvirus antigen have been followed in macrophagecultures infected with HSV (69) and ectromeliavirus (122), and the transmissions of newly syn-thesized HSV (69) and MHV (152) to surround-ing macrophages have been monitored. Specificresults obtained by using the immunofluores-cence technique in various virus-macrophagesystems are given in more detail in later sectionsof the review.

(iv) Electron microscopy. Although elec-tron microscopy should offer a good means ofstudying the morphogenesis of virus particles inmacrophages, it has only been applied in a fewstudies. Johnson (69) found that suckling mousemacrophages infected by HSV-1 contained largenumbers of apparently normal virus particles inthe cytoplasm. Stevens and Cook (141), on theother hand, found in their study of adult mousemacrophages that only a few morphologicallycomplete HSV particles were seen in the cyto-plasm of infected macrophages, whereas mostcells contained empty capsids lacking a densecentral core. Other models with similar age-de-pendent restrictions of virus growth or modelswith genetically determined or virus strain-re-lated restrictions could probably take advantageof the technique in a search for the mechanismsby which virus replication is restricted in mac-rophages.

(v) Tracer techniques. Macrophages do notdivide in vitro (16), and deoxyribonucleic acid(DNA) synthesis in macrophages infected withDNA viruses can generally be regarded as virusspecific. Macrophages labeled with [3H]thymi-dine during infection incorporate the radioactive


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material into nascent virus-specified nucleicacid, which can then be assayed by countingdisintegrations in extracted DNA (40, 141). Theviral origin of the DNA can be verified by buoy-ant density determination after equilibrium sed-imentation in CsCl gradients (141) or by com-

parisons of reassociation kinetics (40). The pro-

portion of infected macrophages producing viralDNA may be determined by autoradiography(69, 141). Virus-induced protein synthesis in in-fected macrophages can be detected by labelingthe infected cells with [3H]thymidine and 14C-labeled reconstituted protein hydrolysates andidentified by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (141).

ASSESSMENT OF VIRUS-MACROPHAGE INTERACTIONS IN

VIVO

In vivo studies are as essential for the under-standing of the participation of macrophages inthe pathogenic events during virus infections as

are in vitro investigations. First, a complexity ofvirus-host interactions is involved in in vivoinfection as compared with the much simplerand more readily controllable virus-cell interac-tion in tissue culture. Second, during collectionand cultivation, macrophages might lose theirnative properties, or new ones might be gainedin vitro. The latter point is of special importancewhen the cells are induced by inoculation of aninflammatory agent before collection to increasethe yield. These stimulated macrophages are

qualitatively different from the cells normallyresident in the animal. Furthermore, cells col-lected after injection of different compoundsmay differ in a number of characteristics (36),for example, in their ability to support virusreplication (152).

General Markers of PathogenicityIn most studies on the role of macrophages in

host defenses against virus infections, generalmarkers of pathogenicity are used to evaluatethe outcomes of virus-macrophage interactionsand the effects of various modulations of themononuclear phagocyte system.Morbidity and mortality. Data on disease

and death are perhaps the parameters mostcommonly used in the study of virus-host inter-actions. As integrated parts of other in vivo andin vitro studies, they represent valuable markersfor pathogenicity or virulence of the virus or forsusceptibility or resistance of the host. Specificsigns of virus-induced disease, for example,pneumonia, hepatitis, and encephalitis, may, fur-thermore, reveal the target organ for the infec-tion. However, they provide little information

on the site of initiation of infection, the spreadof infection through the body, the cells support-ing virus growth, and the antiviral host defensemechanisms that are counteracting the infec-tion.Virus clearance and virus growth. Assays

of organs and tissue suspensions for virus atdifferent times after infection reveal, like mor-bidity and mortality, only gross features of thepathogenesis of a virus infection. Clearancecurves for the disappearance of inoculated virusand growth curves for virus in the main targetorgans and tissues may be constructed, but,taken alone, no information is gained on thetypes of cells that are infected and the factorsthat are acting against the infectious process.But, again, infectivity titrations analyzed in thelight of information obtained by other means areoften valuable or even necessary for interpreta-tion of the accumulated data on the pathogenicevents.

Histological lesions. Examination of sec-tioned organs and tissues taken from animalsduring infection has been extensively used in thestudy of the pathogenesis of virus diseases. Byroutine histological examinations, the develop-ment of pathological lesions in the main targetorgans can be followed, and the cells undergoingdegeneration or developing inclusion bodies canbe studied. Most often, the affected areas areinfiltrated with inflammatory cells, such aspolymorphonuclear leukocytes, lymphocytes,and mononuclear phagocytes, and an estimationof the time course of their appearance and theirrelative number in relation to, for example,changes in the amount of virus recovered fromthe organ may give information on their partic-ipation in host defenses against the infectiousprocess.By the fluorescent antibody technique, in-

fected cells can be readily identified. The pri-mary uptake of the virus by macrophages intissues and organs can be studied, and the intra-cellular fate ofthe virus particles can be followed(fading or increasing brightness of fluorescence).Eventual spreading of the infection to surround-ing parenchymal celLs can be monitored. Exten-sive application of this technique has been of theutmost importance for the elucidation of the roleof macrophages in the pathogenesis of ectrome-lia virus infection in mice, which thereby hasbecome one of the best studied models of gen-eralized viral infection (22, 98, 121).

Modulations of the Function of theMononuclear Phagocyte System

Macrophage blockade. One way of deline-ating the role ofmononuclear phagocytes in host

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resistance to virus infections in vivo is to selec-tively inhibit the macrophage population of theanimal. This can be done by treating the animalby either specific antimacrophage serum or cer-tain chemical agents. Although crude antimacro-phage serum was prepared as early as 1899 byMetchnikoff (96), antiserum reacting specificallywith macrophages was first produced by Cayeuxet al. (28) and Unanue (147), by immunizingrabbits with cultured mouse peritoneal macro-phages. The first attempts to modify the courseof virus infection by antimacrophage serum weremade in mice by Panijel and Cayeux (115), withequivocal results: increasing susceptibility to yel-low fever virus infection contrasting with anapparent protection against encephalomyocar-ditis virus infection. Hirsch et al. (61) achievedsome increase in the susceptibility of baby miceto vesicular stomatitis virus infection by inocula-tion of antimacrophage serum intraperitoneallyat daily intervals for 3 days. The resistance ofadult mice to infection with HSV (167) andrabies virus (146) was also significantly reducedby intraperitoneal inoculation of antimacro-phage serum for 3 days.

Chemically induced macrophage blockade hasbeen more widely applied. Several compoundshave been used, but among these silica (silicondioxide) seems second to none as regards po-tency and specificity (4). It has been shown thatsilica is selectively toxic to macrophages (5, 73,114). In mice, intraperitoneal doses of 50 mg andintravenous doses of 3 mg are well tolerated, andsuch doses have been used to examine the roleof macrophages in infections with HSVs (34,104,167), yellow fever virus (168), Coxsackie B-3virus (117), murine cytomegalovirus (132),Friend leukemia virus (75, 163), rabies virus(146), lactic dehydrogenase virus (34), influenzavirus (55), and Semliki Forest virus and enceph-alomyocarditis virus (4). In most studies, a Ger-man sample of silica, Dorentrup no. 12, <5 ,umin diameter, distributed by A. C. Allison, ClinicalResearch Centre, Harrow, England, has beenused, which makes possible a comparison of theresults obtained with different viruses.Macrophage stimulation. Another way of

assessing the potentials of macrophages to limitvirus spread in vivo uses the administration be-fore or during infection of various immuno-modulators which are known to elicit augmentedrecruitment to tissues and organs of hyperactivemononuclear phagocytes from the bone marrowand blood. If such treatments result in increasedresistance to the virus under study, the mono-nuclear phagocyte system may also be assigneda role in the natural defenses against the virus.Both live microorganisms, such as attenuatedMycobacterium bovis (Bacille Calmette-Guerin

[BCG]) (9, 76, 140) and Staphylococcus aureus(133), and nonviable microbial preparations,such as staphylococcal phage lysate (133, 140)and typhoid and Brucella vaccines (140), havebeen used, as well as synthetic compounds, suchas levamisole, an anthelmintic drug (140).Macrophage transfer. Since a successful

transfer of cells can be achieved only in synge-neic animals, most macrophage transfer studieshave been conducted in mice, where macro-phages have been transferred from inbred adultmice to usually more susceptible young or suck-ling mice of the same inbred strain. Protectionof the recipient mice against subsequent infec-tion can then be ascribed to the transferred cells.This approach has been used to analyze age-related resistances of mice to infections withHSVs (62, 69, 103), murine cytomegalovirus(132), Coxsackie B-3 virus (117), and rabies virus(146). Usually, a total of 106 to 107 cells are givenby the intraperitoneal route immediately beforeinfection, but transfer of macrophages fromadult mice to 3-week-old mice by intravenousinoculation has also been applied (103).

ASPECTS OF MACROPHAGE-DEPENDENT RESISTANCE TO VIRUS

INFECTIONSBy their position at sites of initial infection

(e.g., the respiratory tract) and their wide distri-bution in major organs of the body (e.g., theliver), macrophages may be decisive in deter-mining the susceptibility or resistance of an an-imal to virus infections (98). The uptake orclearance of a virus particle from the circulationor extracellular fluids has been shown first of allto depend on the particle size (98), as is the casewith inert particles (165), larger particles beingcleared more rapidly than smaller ones. How-ever, other factors, such as the presence of virus-specific receptor sites on macrophages and thepresence of opsonizing antibody (25), are alsoimportant. By uptake and digestion of virusparticles, macrophages may delay or even pre-vent the spread of the infection to susceptiblecells. However, if the virus can replicate in mac-rophages, the infection may go further, andwidespread destruction of organs and tissuesmay occur. Furthermore, infected monocytes inthe circulation can, provided they support virusreplication, be a source of dissemination of theinfection by their migration through the body(45, 51, 98). It thus seems clear that virus-mac-rophage interactions may be of crucial impor-tance for the outcome of the infection.Much information on the importance of mac-

rophages in resistance to virus infections hasbeen gained by studying experimental infectionsin which differences exist between the interac-

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tions of macrophages with different strains ortypes of the same virus. Likewise, models inwhich differences in handling a certain virus arefound among macrophages from animals of dif-ferent ages or different strains of the same spe-cies have been extensively studied. Aspects ofthese studies and the antiviral capacities of ac-tivated and stimulated macrophages are consid-ered in the next sections.

Virus Strain- and Virus Type-RelatedResistance

In studies ofthe pathogenesis ofvirus diseases,much valuable information can be obtained bycomparing the pathogenic events in vivo andvirus-cell interactions in vitro of closely relatedvirus strains or virus types (10). In this way, ithas been possible to recognize a number of vir-ulence or pathogenicity markers of a particularvirus and resistance determinants of the host(138). In some cases, the ability of a virus strainor virus type to replicate in macrophages froman animal has been shown to be correlated withvirulence of that particular virus strain or typefor the intact animal, whereas the ability ofmacrophages to restrict virus replication hasbeen correlated with resistance of the animal.Both naturally occurring strain or type variantsand virus strains obtained by adaptation to aparticular host have been used. Of these, thenaturally occurring strains or types seem to bemost reliable in disclosing real resistance factorsof the animal, since it cannot be ruled out thatthe various adaptation or attenuation proce-dures to which the laboratory strains have beensubjected have resulted in mere artifacts withlittle bearing on the natural situation.The earliest comparative study of the inter-

action of virulent and avirulent strains of a viruswith macrophages in vitro was performed byBang and Warwick (11). They examined thegrowth of a virulent strain and an avirulentvaccine strain of Newcastle disease virus inchicken cells. The virulent strain of the virus,which kills embryos and chickens rapidly, de-stroyed chicken fibroblasts and macrophages inculture, whereas the avirulent strain, which onlycauses mild respiratory infection in hatchedchicks, grew poorly in chicken macrophages. Al-though the avirulent strain also grew less abun-dantly than the virulent strain in fibroblasts, themacrophage cultures showed a more consistentdifference in susceptibility to the two strains ofvirus.Roberts (121) used the differences in virulence

for mice of two strains of ectromelia virus toanalyze host defenses against liver infection bythis virus. The Hampstead mouse strain, whichhad only been passed in mice, was found much

more virulent than the Hampstead egg strain,which had gone through more than 60 egg pas-sages. After intravenous inoculation, virusgrowth in livers was followed by infectivity titra-tions, and the number of infected Kupffer cellsand the spread of the infection to parenchymalhepatic cells were estimated by fluorescence mi-croscopy. It was found that the avirulent strainwas much less efficient in infecting Kupffer cellsand that Kupffer cells which were actually in-fected yielded less virus than did cells infectedwith the virulent strain. No difference was foundin the ability of the two strains to grow andspread in liver parenchymal cells. These studieswere followed up by quantitative in vitro studiesof the susceptibility of mouse peritoneal macro-phages to infection with the two virus strains.By the fluorescent antibody technique, it wasfound that peritoneal macrophages were about10 times as susceptible to infection by the viru-lent strain of virus as they were to infection bythe avirulent strain (122). No in vitro studies ofthe infectivity and growth of virulent and avir-ulent strains of ectromelia virus in other celltypes were conducted to support the in vivoobservation that the difference in infectivity wasonly expressed in macrophages.A similar differential action of mouse Kupffer

cells against two vaccinia virus strains was men-tioned by Mims (97). The CL-R strain grew inKupffer cells and thereby gained access to he-patic parenchymal cells. The CL strain, on theother hand, did not grow in Kupffer cells, whichthereby protected hepatic cells from the virus.The intrinsic ability of the CL strain to grow inhepatic cells was, however, disclosed by inocu-lating the virus up the bile duct to infect paren-chymal cells directly.

Tosolini and Mims (145) analyzed the behav-ior of two strains of lymphocytic choriomenin-gitis virus in mice. No antigenic differences havebeen demonstrated in different strains of thisvirus, and after intracerebral and footpad inoc-ulation both the WE-3 and the Armstrongstrains infected local tissues. However, after in-travenous inoculation, only the WE-3 straincaused marked infection as detected by viraltitrations and immunofluorescent and histologi-cal examinations of liver and spleen. Clearancestudies revealed that the Armstrong strain wasnot detectably cleared from the blood after in-travenous inoculation, whereas about 90% of theWE-3 strain was cleared within 5 min. Immu-nofluorescent examination of peritoneal macro-phages harvested 2 days after intraperitonealinfection with the two strains of virus showedinfection of 70% of macrophages obtained frommice injected with the WE-3 strain, whereasonly 4% of the cells from mice inoculated with

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the Armstrong strain were infected. After infec-tion of macrophage cultures, the figures were 40and 3%, respectively. It was concluded that therelatively inefficient uptake of the Armstrongstrain by cells of the reticuloendothelial systemwas responsible for the inability of this strain toestablish significant infection of the viscera, es-pecially the liver.MHV exists in several closely related sero-

types with various degrees of pathogenicity inmice (88). In various studies, it has been foundthat a correlation exists between the pathoge-nicity of a certain type of the virus and its abilityto grow in macrophages. Thus, the nonpatho-genic type MHV-1 does not multiply in mousemacrophages (3), whereas the pathogenic typesMHV-2 and MHV-3, which after intraperitonealinoculation in mice produce fatal hepatitis, read-ily infect mouse peritoneal macrophages, inwhich they multiply to high titers with distinctcytopathic effects (13, 90). Likewise, a close cor-relation between the ability of two variants ofMHV-2 to replicate in C3H macrophages andtheir virulence for C3H mice was described byShif and Bang (136).A specific pathogenic difference has been

shown to exist between infections with HSV-1and HSV-2 in mice. On intraperitoneal inocula-tion with HSV-2, progressive focal necrotizinghepatitis develops in most strains of mice,whereas HSV- 1 only occasionally produces a fewtiny, self-limiting foci of necrosis in the liver(Fig. 1) (106). This marker of pathogenicity,which has later been confirmed by others (120,

A

149), was shown to be very stable, as it wasclosely correlated with the antigenic type of thevirus, irrespective of the anatomical site of pri-mary virus isolation (genital or nongenital) orpassage history of the strain. An in vitro corre-late of the difference in pathogenicity betweenthe two virus types was found in their abilitiesto replicate in peritoneal macrophages frommice (101). By an infectious center assay, it wasfound that although macrophages were ratherrestrictive in the replication of both virus types,the restriction of HSV-1 was far more pro-nounced than that of HSV-2 as judged by thenumber of plaques and progression of the infec-tion in the target cell monolayer (Fig. 2). Thedifference in the yields of infectious virus fromperitoneal macrophages infected with HSV-1and HSV-2 was not caused by a relative inabilityof HSV-1 to adsorb to mouse macrophages, asthe adsorption rates of the two virus types inmacrophage cultures were equal. Moreover, itwas shown that the more pronounced restrictionof HSV-1 replication was specific for macro-phages, as HSV-1 grew to higher titers than didHSV-2 in embryonic fibroblast cultures pre-pared from the same mouse strain. Further evi-dence in support of the participation of macro-phages in the pathogenic distinction betweenliver infections with these two closely relatedvirus types was obtained by selectively blockingthe macrophage function of the animals by silica(104). Administration of 3 mg of silica intrave-nously 2 h before virus infection seemed to in-duce an effective blockade of liver macrophages

B

FIG. 1. Livers from 3-week-oldAH mice 4 days after intraperitoneal inoculation of2 x 104 plaque-formingunits of HSV-1 (A) or HSV-2 (B). A few tiny lesions are seen on the margin of the liver from the HSV-1-infected mouse (arrow). Magnification, x2.5. From Mogensen (100).

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A B

FIG. 2. Infectious centers in macrophage culturesseeded with 5 x 105 peritoneal macrophages from 4-week-old BALB/c mice. The cultures were infectedwith 5 x 105plaque-forming units ofeither HSV-1 (A)or HSV-2 (B), thoroughly washed, overlaid with sus-ceptible mouse embryonic fibroblasts, and stainedafter 2 days of incubation. The arrow indicates asmall HSV-1 plaque. Magnification, x1.9. From Mo-gensen (101).

for at least 2 days and a partial blockade foranother 2 days, as HSV-1- and HSV-2-inducedlesions progressed equally well for the first 2days of infection followed by a relative retarda-tion of the growth of HSV-1-induced lesions.Intraperitoneal inoculation of 50 mg of silicaseemed even more effective in inducing macro-

phage blockade, as the difference in size andnumber of HSV-1 and HSV-2 lesions on day 4of infection was almost abolished by this treat-ment schedule (Fig. 3).

Genetically Determined Resistance

It is a well-known fact that most viruses or

groups of viruses differ in host range, some spe-cies of animals being susceptible to infectionwith a certain virus, whereas others are resistant.More interesting, however, is the variation innatural or innate resistance to infectious diseasesdisplayed by members of the same animal spe-cies, indicating that factors in the genetic con-

stitutions of the animals play an important partin the phenomenon (F. B. Bang, Adv. Virus Res.,in press). Genetically determined resistanceagainst infections is well documented as regardsbacteriophages and plant viruses (1). The earli-est reports on a genetic basis for resistance to

FIG. 3. Livers from 4-week-old BALB/c mice in-jected intraperitoneally with 50mg ofsilica 2 h beforereceiving 105 plaque-forming units of HSV-1 (A) orHSV-2 (B) intraperitoneally. The mice were sacrificedafter 4 days. Magnification, X2.5. From Mogensenand Andersen (104).

animal virus infections were published morethan 40 years ago (50, 87, 158). Since then, theinheritability of resistance to virus infections hasbeen demonstrated in several animal model sys-tems, and the genetics behind the variation hasbeen explored. Instances in which resistance (48,49, 82, 102, 127, 130) or susceptibility (13, 29,112) is determined by a single dominant genehave generally offered the best prospects of elu-cidating the virus-host interactions involved,whereas polygenic traits (80, 85, 164) have beenmore difficult to evaluate.Webster (158) was the first to work out the

Mendelian ratios for the inheritance of resist-ance to a virus infection by crossing resistantand susceptible strains of mice followed by ap-propriate intercross and backcross matings. InFig. 4 and 5, the expected ratios of susceptibleand resistant animals are outlined in examplesof virus infections where resistance or suscepti-bility is inherited as a monogenic autosomaldominant trait with genetic segregation of re-sistance and susceptibility occurring in the sec-ond filial (F2) generation and in backcrosses(BC1) to the recessive parental strain. When twoor more loci are involved, genetic evaluation ismore difficult because of the possibilities of in-teraction or linkage between the loci or varyingcontributions to the phenotype of the loci in-volved.

In some virus-animal systems, macrophageshave been found to express at the cellular levelthe genetic resistance seen in vivo (Table 1).The best clarified examples of this are the re-

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sistance of mice to flaviviruses (arbovirus groupB) and the susceptibility of mice to MHV-2,both of which are inherited as monogenic auto-somal dominant characters, thus representingthe two cases illustrated in Fig. 4 and 5.During the thirties, Webster developed by

selective breeding strains of mice which differedgreatly in resistance to two flaviviruses, St. Louisand louping ill viruses. By crossing susceptiblewith resistant lines of mice and testing hybridand backcross progeny, he achieved segregationof the two characters in ratios close to thoseexpected on the basis that resistance be inher-ited as a single, dominant factor (Fig. 4) (158).Sabin (127) extended the work of Webster by

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showing that nonselected PRI and C3H strainsof mice differed in resistance to yellow fevervirus and a number of other flaviviruses tested(PRI, resistant; C3H, susceptible). He intro-duced the terms "multiplication-depressing fac-tor" and "cellular vulnerability," both of whichwere of importance for relative resistance. Fur-thermore, evidence was presented that inheritedresistance operated at the cellular level, sincevirus multiplied freely in foreign tumor cellsimplanted in resistant PRI mice (128).

In extensive studies of the genetics of resist-ance to West Nile virus, another flavivirus,Goodman and Koprowski (48,49) confirmed Sa-bin's findings and further investigated the mech-anism of inherited resistance. After intraperito-

rr Rr Rr RR

FIG. 4. Autosomal dominant inheritance in miceof resistance to virus infection. The symbols indicateboth female and male mice with the resistant (*) orsusceptible (O) phenotype. RR, Rr, and rr indicatethe genotypes. BC1 is the first backcross generationin a test cross between mice of the F1 generation andthe recessive parental strain. In F, all mice are re-

sistant, in F2 75% are resistant, and in BC, 50%o areresistant.

SS Ss Ss ss Ss ss

FIG. 5. Autosomal dominant inheritance in miceof susceptibility to virus infection. The symbols indi-cate both female and male mice with the susceptible(O) or resistant (*) phenotype. SS, Ss, and ss indicatethe genotypes. BC, is explained in the legend to Fig.4. In F1 all mice are susceptible, in F2 75% are suscep-tible, and in BC, 50%o are susceptible.

TABLE 1. Inheritance of macrophage-dependent resistance to virus infection in mice

Mouse strains Property testeda (mode of inherit-Virus Reference

Resistant Susceptible ance)

Flaviviruses PRI, BRVR C3H/He, BSVS R (autosomal dominant) 48, 49

MHV-2 C3H/He PRI S (autosomal dominant) 13

MHV-3 A/J C57BL/10, DBA/2, 155(C3H/He, A2G)b

HSV-2 GR/A BALB/c R (X-linked dominant) 102

Murine cyto- CBA/J C57BL/6 R (autosomal dominant) 132megalovirus

Influenza A A2G A/J R (autosomal dominant) 82

a R, Resistance; S, susceptibility.b Intermediate susceptibility.

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neal inoculation, the virus was cleared at com-

parable rates within 10 to 12 h from the blood ofboth susceptible C3H and resistant PRI andBRVR mice. In susceptible mice, however, a

second period of viremia soon occurred, whereasvirus did not reappear in the blood of resistantmice. Specific antibody production or putativenonspecific serum inhibitors could not accountfor this difference nor was any febrile reactionobserved in the resistant mice. Attempts to mod-ify resistance by various treatments (X rays,

cortisone, Thorotrast, thioguanine, endotoxin)did not reveal the nature of the resistance mech-anism. However, a cellular expression of inher-ited resistance was found in the capacity ofcultures of splenic and peritoneal macrophagesfrom resistant and susceptible mice to supportvirus multiplication. Whereas virtually no virus

production was seen in cultures of these cells

from resistant PRI and BRVR mice, titers of 102to 104 plaque-forming units per ml were re-

covered from supernatants of cultures preparedfrom the susceptible C3H strain. Furthermore,resistance and susceptibility in vitro were found,as expected, to segregate close to 1:1 in macro-

phage cultures prepared from individual BC8mice (48). Groschel and Koprowski (52) devel-oped a homozygous inbred mouse line (C3HRV)which was congenic with the virus-susceptibleC3H/He strain, only differing from the latter atthe gene for flavivirus resistance. The new

mouse line was found to be just as resistant as

the PRI mouse from which the resistance genehad been transferred, and macrophage culturesreflected this resistance completely.The basic nature of the depressed flavivirus

multiplication in macrophages from resistantmice is not fully understood. Conflicting resultsexist as regards the specificity of macrophagesas the only cell type expressing the genetics ofresistance. In the original reports (48, 49), no

difference was found in the capacities of kidneyand lung cells from susceptible and resistantmouse strains to support virus multiplication,whereas later investigators (56, 150) claim thatsuch a difference exists, although to a lesser andmore variable degree than that displayed bymacrophages. Differences in interferon produc-tion have been shown not to play any role invirus resistance: interferon levels were demon-strated to be lower in resistant than in suscep-tible mice (151), and interferon production levelsin macrophage and fibroblast cultures from re-

sistant and susceptible strains ofmice were equal(56). However, cells from the virus-resistantmouse strain were more readily protected byinterferon from virus infection than were cellsoriginating from the susceptible strain (56). It

thus appears that Sabin's inheritable multipli-cation-depressing factor may be the interferonsensitivity of the host cells, principally macro-phages (constituting the primary barrier to suc-cessful virus invasion) but also the target cellsfor the infection.MHV-2 infection in mice provides an example

of susceptibility inherited as a dominant unifac-torial trait (Fig. 5). In 1959, Bang and Warwickshowed that this virus caused selective destruc-tion ofthe macrophage population in outgrowthsof liver explants from susceptible PRI mice,leaving both the parenchymal liver cells and theepithelial cells and fibroblasts intact (12). Inter-est in this phenomenon was greatly intensifiedwhen it turned out that C3H mice, which wereresistant to the infection, yielded macrophageswhich were themselves resistant when infectedin vitro (13). Tests of other strains of mice re-sistant or susceptible to MHV-2 in vivo showedcomplete agreement between mouse and mac-rophage susceptibilities. Genetic crosses be-tween susceptible PRI and resistant C3H miceyielded F1 offspring all of which were suscepti-ble, indicating that susceptibility is the domi-nant feature. Segregation of the characters wasachieved in the F2 (3:1) and in the BC1 (1:1)generations in ratios in accordance with a singlegene for susceptibility. A similar segregation ofsusceptibility and resistance occurred in macro-phage cultures prepared from individual mice ofthe F2 and BC1 generations (13). Further back-crosses showed that the PRI gene for macro-phage susceptibility was constant and com-pletely manifest even when present in a C3H(resistant) background: the plaquing efficiencyof the virus in macrophage cultures from suscep-tible mice of the BC7 generation was as high asthat in PRI cultures (134); macrophage culturescontinued to segregate in ratios close to 1:1during 20 backcross matings; and macrophagesobtained from the virus-susceptible inbred lineof C3HSS mice, which are congenic with resist-ant C3H mice and only differ from these at thelocus for MHV susceptibility, were just as sus-ceptible as macrophages from the PRI mice,from which the gene for susceptibility was trans-ferred (160). The expression of the genotype forsusceptibility or resistance was not confined toliver macrophages, since the same pictureemerged when macrophages were cultured fromother tissues (13) or from peritoneal exudates(70, 71).A further investigation of the differential abil-

ities of macrophages from C3H and PRI strainsof mice to support replication of MHV-2 wasundertaken by Shif and Bang (135). Theyshowed that the virus was adsorbed equally well

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to resistant and susceptible cells. After adsorp-tion, the virus disappeared into the eclipse phasein the susceptible macrophages and subse-quently replicated to high titers within 20 h,whereas in the resistant cells the input viruspersisted in an infective state for several dayswithout multiplication and without causing anyapparent damage to the cells. It thus appearsthat the resistance of C3H macrophages is re-lated to the ability of these cells to maintain thisparticular virus in a nonreplicative state. In thisconnection it is of interest to note that the sametwo strains of mice have been used in studies ofthe genetics of macrophage resistance to bothMHV-2 and flaviviruses but that the strain sus-ceptible to MHV-2 is resistant to flavivirusesand vice versa. Furthermore, when C3H macro-phage cultures are inoculated with high multi-plicities of MHV-2, a variant virus emergeswhich is capable of killing C3H mice and ofreplicating in and destroying C3H macrophages(136). It is thus clear that the capacity of C3Hmacrophages to restrict the growth of the strainof MHV-2 used in these studies does not stemfrom a generalized capacity ofC3H macrophagesto restrict virus replication and that the impor-tance of macrophage-virus interactions requiresindividual assessment with each virus-host sys-tem involved.

Several attempts have been made to elucidatethe factor or factors which determine resistanceand susceptibility of macrophage cultures toMHV-2. Evidence has been presented that it isa property of the individual cells: macrophagecultures from resistant C3H mice remained re-sistant after several changes of media, and infec-tion ofmixed cultures prepared from equal num-bers of PRI and C3H macrophages resulted inthe destruction of about 50% ofthe macrophages(13). Likewise, no sign of interferon activity wasfound in resistant C3H cultures after infection(136). However, a factor related to susceptibilitywas recognized in extracts from uninfected sus-ceptible PRI macrophages. Resistant C3H mac-rophages exposed to this extract before infectionwith the virus became susceptible, whereas ex-tracts from C3H macrophages did not changethe susceptibility of PRI macrophages (70). Al-though the factor has been partly characterized(a relatively large, heat-stable molecule onlyslightly sensitive to deoxyribonuclease and in-sensitive to ribonuclease treatment), the natureof the conversion from resistance to susceptibil-ity has not been revealed (71). Further attemptsto modify the resistance of C3H mice have notthrown much light on the mechanisms by whichmacrophages display this character. Cortisonetreatment rendered C3H mice partly susceptible

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to the infection, and macrophages treated invitro with cortisone also became partly suscep-tible to the destructive effect of the virus (46),but a causal relationship between these findingswas not established. Cyclophosphamide treat-ment likewise increased in vivo susceptibility,although the drug had no effect on in vitroreplication of the virus in macrophages (162). Itwas shown that the conversion of resistance tosusceptibility in vivo by the drug was not due toinhibition of specific antibody production, andthe effect ofcyclophosphamide remains obscure.

Recently, brief reports have pointed to a pos-sible effect of T lymphocytes and lymphokineson the expression of the genetic resistance orsusceptibility of macrophages to MHV-2: thus,supernatant fluid from allogeneic mixed lympho-cyte cultures converted macrophages from re-sistant C3H mice to susceptibility to the virus(159), whereas supernatants of concanavalin A-stimulated lymphocyte cultures had the oppo-site effect, rendering macrophages from suscep-tible PRI mice resistant (161). Furthermore,concanavalin A-treated PRI mice showed in-creased resistance to the infection, and macro-phages from concanavalin A-pretreated micewere resistant in vitro. The significance of thesefindings is at present unknown, but they clearlyshow that lymphocytes and products of stimu-lated lymphocytes can modify the inborn de-fense mechanisms.

Susceptibility of mouse macrophages toMHV-3 was also found to parallel the suscepti-bility of mice of different strains to infectionwith this virus (155). Most strains of mice werefully susceptible and developed fuihninant hep-atitis upon infection. When infected in vitro,macrophages from these mice showed extensivegiant cell formation, and the virus replicated tohigh titers. Mice of the A strain, on the otherhand, were completely resistant, and no growthof virus was detected in their macrophages, evenby the sensitive immunofluorescence technique.C3H and A2G mice showed intermediate suscep-tibility both in vitro and in vivo with develop-ment of a persistent infection and late neurolog-ical involvement. No data on the mode of inher-itance were reported, but it is interesting to notethat the C3H mice, which are resistant to MHV-2, were found to be intermediately susceptible toMHV-3, again stressing the risk of drawing toohasty conclusions as to resistance mechanismsagainst even closely related viruses.

Resistance ofmice to the development of focalnecrotizing hepatitis by HSV-2 was found byMogensen (100) to be under genetic control. Afurther analysis of the patterns of inheritancehas revealed that resistance is determined by


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one dominant gene (or a complex of closelylinked genes) located on the X chromosome(102). This unique feature of sex linkage of re-sistance to a virus infection emerged from mat-ings of resistant male GR mice to susceptiblefemale BALB/c mice and backcross matings ofresistant F1 females to susceptible BALB/cmales (Fig. 6). The characters for susceptibilityand resistance segregated between male and fe-male offspring of the F1 generation (all malessusceptible, all females resistant) and in segre-gated ratios of about 1:1 in mice of both sexes inthe BC1 generation (Fig. 7). A cellular expressionof virus resistance was found in the ability ofperitoneal macrophages from resistant mice torestrict the replication of the virus in vitro. Therestriction was most pronounced in macro-phages from GR mice, and this character wasretrieved in macrophage cultures from female F1progeny. Moreover, a segregation of high andlow restriction close to the expected 1:1 ratiowas found in the BC1 generation (Fig. 8). Nodifference was detected in the replication of thevirus in embryonic fibroblasts from resistant andsusceptible strains of mice.As regards the genetics of resistance to other

herpesviruses, Lopez (85) presented resultswhich indicated that resistance to HSV-1 infec-tion in mice is dominant but that it is governedby at least two, and probably more, pairs ofgenes. The marker for susceptibility/resistance

|Irr Rr r RFIG. 6. X-linked dominant inheritance in mice of

resistance to virus infection in females (circles) andmales (squares) with resistant (closed symbols) andsusceptible (open symbols) phenotypes. For explana-tion ofBCi, see legend to Fig. 4. In F1, all females are

resistant and all males are susceptible. In BC1, 50%are resistant irrespective of sex.

1,

-

M._

a.3_

1--2 -

Balb c GR F1 BC1

FIG. 7. Titers of virus in livers of 8-week-old fe-male BALBIc, GR, (GR6 x BALB/cl)F1, and(BALB/c& x [GR x BALB/c]9)BC, mice 4 days afterintraperitoneal inoculation of 106 plaque-formingunits (PFU) of HSV-2. Also shown are virus titers ofF1 male mice. Symbols: 0, mice with lesions; 0, micewithout lesions; O), mice with tiny lesions on livermargins. From Mogensen (102).

was, however, the death of the animal, which isusually caused by central nervous system infec-tion. This is a much more complex pathogenicevent than infection of the liver and might implyhematogenic and neurogenic spread of virus tothe brain. Indeed, several reports are availableof polygenic inheritance of resistance to diseasehaving a single-locus component at a certainpoint in the structure of resistance (63). Geneticcontrol of resistance of mice to another herpes-virus, murine cytomegalovirus, was described bySelgrade and Osborn (132). They also found thatresistance was the dominant feature, but it wasindependent of resistance to HSV-1 infection.Although transfer and blockade experimentssuggested some importance of macrophages inhost defenses against the virus, infection in vitroof macrophages from resistant CBA mice was noless productive than was infection of cells fromsusceptible C57BL mice. These findings do notsupport the concept of a simple barrier functionof macrophages in genetic resistance to this in-fection, and it was proposed that the importanceofmacrophages in resistance was related to theirrole in the inductive phase of cellular immunity.

Resistance of A2G mice to various myxovi-ruses has been attributed to a single dominant

4. :

L -

0

*~~~00 i0oo 00

o voo 000000 000 (000

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200 -

180 -

1i n -

: 140-

0 120

zV100-A.

/-

Z 0

20 -

n-Ball c GR F1 BC1

FIG. 8. Number of infectious centers in macro-

phage cultures prepared from individual 8-week-oldfemale BALBIc, GR, (GRd x BALB/c9)F,, and(BALB/c6 x [GR x BALB/c]9)BC, mice and infectedwith 5 x 105 plaque-forming units of HSV-2. FromMogensen (102).

gene designated Mx for myxovirus resistance(81, 83). The resistance is operative against neu-rotropic influenza viruses inoculated intracere-brally, pneumotropic strains injected intrana-sally, and a hepatotropic strain injected intra-peritoneally (55). Attempts to define the phe-notypic expression of the Mx gene have untilrecently been unsuccessful. No virus inhibitorswere detected in body fluids or tissue extracts ofresistant mice (83), and no difference has beenfound in the abilities of fibroblasts, kidney cells,and nerve cells from resistant and susceptiblemice to support virus multiplication in vitro (82).Various immunosuppressive treatments failed toabolish resistance (55). However, recently, Lin-denmann et al. (82) presented evidence thatmacrophages represent the key cell in the resist-ance of A2G mice to myxoviruses. An avianstrain of influenza virus grew to high titers andcaused a rapid and distinct cytopathic effect inperitoneal macrophage cultures from a numberof mouse strains susceptible to influenza virus,whereas no signs of virus growth were detectedin macrophage cultures prepared from resistantA2G mice or from F1 hybrids between these andsusceptible strains of mice. Furthermore, resist-ance and susceptibility of individual mice andtheir macrophages cosegregated in a ratio closeto 1:1 in the BC1 generation of resistant F, mice

to a susceptible mouse strain. The fact thatresistance is also manifest upon intracerebralchallenge of A2G mice is not a common featureof infections in which macrophages have beenfound to be of importance in resistance; whetherthis phenomenon in the case of myxovirus re-sistance is due to such macrophage analogs asmicroglial cells in nervous tissue or to othermechanisms is at present unknown.Even in cases where macrophages are of doc-

umented importance for resistance against virusinfections, they do not represent the only de-fense mechanism. This was illustrated in thegenetics of resistance ofmice to ectromelia virus.Although virus growth in macrophages wasfound to be correlated with the difference invirulence for mice of two strains of this virus,the virulent virus strain grew equally well inmacrophages from strains of mice with high,intermediate, and low resistance to infection(122). As pointed out by Schell (130), the differ-ences in susceptibility of these strains of miceare probably attributable to differences in im-mune response, as differences in virus growth inthe two strains of mice first appeared at a timewhen neutralizing antibodies and cell-mediatedimmunity were demonstrable. Furthermore, nodifference was demonstrated in clearance of in-travenously inoculated virus or in subsequentgrowth of virus in the liver.

Age-Related ResistanceA general characteristic of most virus infec-

tions in both humans (26) and animals (137) isthe increased resistance to disease which devel-ops in the course of growth and maturation ofthe host. The changes are particularly dramaticin the neonatal period. If the effects of antibodyacquired passively from the mother are ex-cluded, viral infections are often very severe inthe neonatal period, becoming steadily milderduring infancy and early childhood. In old age,infections again tend to become severe, presum-ably owing to progressing inadequacies of hostdefense mechanisms (89). Several explanationsof the age-dependent increase in resistance havebeen advanced, including maturation of immu-nological reactivity, augmented interferon pro-duction, changes in viral receptors, and higherand more stable body temperature (44).

In many experimental viral infections, age-related resistance mechanisms are only opera-tive after peripheral inoculation, both adult andneonatal animals being susceptible to intracere-bral inoculation (113). This difference in suscep-tibility depending on the route of inoculationhas led to the concept of development of "bar-riers" to the spread of virus from the periphery

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to highly susceptible target organs (129). In anincreasing number of experimental systems, ev-

idence has accumulated which suggests that amaturation of macrophages occurs during thefirst few weeks of life. The mature macrophagesof older animals seem to constitute a more effec-tive barrier to the spread of virus than do im-mature macrophages from newbom and wean-ling animals. Examples of such age-related mac-rophage-dependent resistance to virus infectionsare given below.

In HSV-1 infections in mice, the developmentof age-related resistance to encephalitis afterperipheral inoculation ofthe virus was first dem-onstrated by Andervont, in 1929 (7), and laterconfirmed by others (74, 79, 125). The existenceof a cellular basis for the age-dependent resist-ance to this infection was claimed by Johnson inhis comprehensive study of 1964 (69). By the useof the immunofluorescence technique, heshowed that macrophages represent the cellularlevel at which the spread ofextraneural infectionis arrested in adult mice. Infection of peritonealmacrophages both in vivo and in vitro revealedthat macrophages from adult and suckling micewere infected with equal ease. However, adultmouse macrophages infected in vitro did notspread the infection to other cells, whereasspreading of infection always occurred whensuckling mouse macrophages were infected so

that giant clumps offluorescent cells had formed72 h after inoculation. The in vitro capacity ofmacrophages to disseminate virus was shown togradually decrease with the age of the macro-phage donor in parallel with the development ofresistance in vivo.The importance of macrophage maturation in

age-related resistance to HSV-1 infection was

later confirmed by Allison's group. Selectiveblockade of macrophage function by silica andantimacrophage serum rendered weanling mice,which are relatively resistant to 104 plaque-form-ing units of HSV-1 much more susceptible tothis virus dose, with a resulting wide dissemi-nation of the virus and early death (167). Usingan infectious center assay, Hirsch et al. (62)found that infected macrophages from sucklingmice consistently released more virus than didadult mouse macrophages. Furthermore, intra-peritoneal transfer of peritoneal macrophagesfrom adult mice protected suckling mice fromsubsequent infection with a low dose of virus (10plaque-forming units), but only when more than6 x 106 stimulated macrophages were trans-ferred. Smaller numbers of stimulated macro-phages or large numbers of unstimulated mac-

rophages did not alter the total mortality signif-icantly, although a delay in the occurrence ofdeath was seen. These findings might explain

why Johnson (69) in his original investigationswas unable to transfer augmented resistance byintraperitoneal transfer of macrophages. Heused smaller numbers of unstimulated macro-phages from younger donors and measured totalmortality, which seems a rather insensitive pa-rameter for macrophage protection (103).

Focal necrotizing hepatitis in mice caused byHSV-2 represents another model system inwhich the role of macrophages in age-relatedresistance has been investigated. On intraperi-toneal inoculation with this virus, numerous fo-cal areas of necrosis develop in the livers ofyoung mice up to the age of 4 weeks (106). Whenolder mice are infected, fewer animals show le-sions, and the number and size of the foci dimin-ish (100). Whereas the studies of age-relatedresistance to HSV-1 encephalitis have focusedon the increase in resistance from the neonatalperiod to the age of 3 to 4 weeks, when the miceare weaned, the hepatitis model has revealedthat mice do not reach maximum resistance tothis infection until they reach the fertile age, i.e.,about 8 weeks of age. In vitro infection of peri-toneal macrophages from 3-week-old and 8-week-old mice showed that age-related resist-ance was concomitant with an increased restric-tion of replication of the virus in peritonealmacrophages, as judged by the number ofplaques appearing in an infectious center assay.Furthermore, age-dependent resistance to HSV-2-induced hepatitis could be overcome by pre-treatment with silica. Transfer of 2 x 106 unstim-ulated macrophages from 8-week-old mice to 3-week-old mice by the intravenous route con-ferred to the latter a defense capacity againstHSV-2 hepatitis almost as effective as that seenin adult mice (103). One reason for this goodrate of protection as compared with the resultsof Johnson (69) and Hirsch et al. (62) probablyis that death from encephalitis, as used in theirstudies, is less sensitive than the degree of hep-atitis as a marker for macrophage protection.When the virus enters the central nervous sys-tem, it seems to be beyond the reach of the hostdefense mechanisms which terminate the infec-tion in peripheral sites, probably because mac-rophages are less effective in the central nervoussystem than in the organs of the mononuclearphagocyte system (2). Furthermore, Roser (123,124) has shown that approximately 60% of peri-toneal macrophages injected intravenously inmice are localized in the livers of the recipientswithin a few hours of injection, so that theintravenous route of transferring cells might beparticularly suited to protect the liver from sub-sequent infection.The human correlate to these animal model

infections is neonatal infection with HSV (109).

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The infection is usually acquired during thepassage through the birth canal and hence ismost often caused by HSV-2, which is the genitaltype of the virus. The infection is characterizedby widespread dissemination of the virus withlesions in many organs, including the centralnervous system and the liver, and is most com-monly fatal. An unproportionate number ofcases are seen in premature infants (1:1.4 againstthe expected 1:8 to 1:10), suggesting that imma-ture host defenses (for example, macrophagefunction) are responsible for the ease ofcontract-ing a disseminated infection and developingovert disease. However, once established, theherpetic infection in full-term infants seems tobe as severe as that observed in prematures(109).Murine cytomegalovirus, another member of

the Herpesvirus family, is similar to HSV in thatsuckling mice are much more susceptible to in-fection than are adults (91). The acute stages ofinfection are characterized by focal necrosis bothin the liver and in other viscera. Henson et al.(58) showed that accumulation of Kupffer cellsaround lesions in the liver coincided with a de-crease in virus titer and preceded the appearanceof neutralizing antibodies by several days, sug-gesting that macrophages limit the spread of thevirus in the livers of adult mice. This idea wassupported by the finding of Selgrade and Osborn(132) that macrophage blockade with silica in-creased the susceptibility of adult mice to theinfection. Furthermore, pretreatment ofsucklingrice with 106 stimulated adult macrophages sig-nificantly increased their resistance to subse-quent murine cytomegalovirus infection. Pre-treatment with 107 to 108 nonadherent spleno-cytes (lymphocytes) also conferred some protec-tion to suckling mice. However, from their re-sults it cannot be ruled out that this effect mightbe due to an even slight (1%) contamination ofthe lymphocyte suspension with macrophages.As regards the ability of the virus to replicate inmurine macrophages, conflicting results havebeen reported: the data of Selgrade and Osbornshowed only low levels of virus replication inmurine macrophages, irrespective of the age ofthe macrophage donor, in contrast, Tegtmeyerand Craighead (142) achieved extracellular virustiters in macrophage cultures from adult micejust as high as those seen in cultures of mouseembryonic fibroblasts. However, the delayed ap-pearance of virus in the macrophage cultures (6to 12 days) as compared with fibroblast cultures(s 3 days) renders it possible that outgrowth offibroblasts in the macrophage cultures might beresponsible for the high virus titers obtained inthese experiments.

MICROBIOL. REV.

Resistance of mice to MHV-2 is geneticallydetermined and closely correlated with resist-ance of macrophages in vitro (13). However, inthe first few days of life, even genetically resist-ant C3H mice are susceptible. The ontogeny ofmacrophage resistance to this infection wasstudied by Galily et al. (47). Although newbornC3H mice were susceptible to the infection, theirsurvival time (6.5 to 8.0 days) exceeded that of"genuinely" susceptible PRI mice (2 to 4 days),and hence they were described as delayed sus-ceptible. Resistance rapidly increased with ageso that 14-day-old mice were fully resistant. Thedelayed susceptibility of infant C3H mice wasreflected in the in vitro susceptibilities of liverand peritoneal macrophages explanted frommice of different ages. The maturation processoccurred only in intact animals, as continuedgrowth in vitro of liver macrophages from 1-day-old C3H mice did not increase the resistance ofmacrophages to the destructive effect of thevirus, although the in vitro conditions of culti-vation were able to modify the degree of resist-ance (47, 77). In susceptible C3HSS mice, whichare congenic to resistant C3H mice, a temporaryincrease in resistance as the mice passed 7 weeksof age was noticed by Weiser et al. (160). Theincreased resistance in vivo was, however, notreflected in increased resistance of peritonealmacrophage cultures from mice of this age andwas ascribed to incomplete penetrance of thedominant gene for susceptibility.Most strains of mice are highly susceptible to

infection with yellow fever virus by the intrace-rebral route, but not by the intraperitonealroute. Suckling mice, on the other hand, arehighly susceptible by either route (143). Impair-ment ofmacrophage function by silica was foundto increase the susceptibility of adult mice toperipherally inoculated virus t168). Similar re-sults have been obtained by administration ofantimacrophage serum (115). Passive transfer ofspecific antibodies to silica-pretreated mice 1 to2 days after virus inoculation was fully able tosubstitute this function ofmacrophages, whereasadministration of antilymphocytic serum to nor-mal mice had no potentiating effect on the in-fection (168). These data suggest that macro-phages are important in preventing the access ofvirus to the central nervous systems of adultmice, with antiviral antibody (normally first ap-pearing on day 3 of infection) as a second line ofdefense. Cell-mediated immune reponses, on theother hand, seem not to be important.The nature of the maturation process that

renders macrophages from adult mice able toconfine virus infections is not understood atpresent. The question has been most fully inves-


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tigated in the case of HSV. A general cellularmaturation cannot explain the limitation ofvirusspread, since cells of ectodermal, mesenchymal,and endodermal origin remain highly susceptibleto productive infection (69, 141). No evidence ofinterferon production by virus-infected adultmouse macrophages was found by Johnson, andHSV-1-infected macrophage cultures were sus-ceptible to rechallenge with the same virus orectromelia virus (69). These findings were con-firmed by Hirsch et al. (62); however, their dataindicate that adult mouse macrophages, in con-trast to suckling mouse macrophages, do pro-duce low levels of interferon when infected withHSV-1, but that the interferon produced is in-effective in protecting other adult macrophagesfrom being infected. The significance of thesefindings seems unclear, but probably interferonproduction is not the factor responsible for age-dependent maturation. Similarly, attempts tomodify the capacity of macrophages to spreador contain infection by extracts of adult or suck-ling mouse macrophages analogous to the trans-fer of susceptibility to MHV among macrophagecultures from inbred strains of mice as describedby Kantoch and Bang (70) were not successful(69).The molecular and morphogenetic events in

the restricted replication ofHSV-1 in adult mac-rophages were systematically analyzed by Ste-vens and Cook (141). They concluded that thevirus undergoes an abortive infection in thesecells. Both viral DNA and the major virus-spec-ified proteins were synthesized, yet electron mi-crographs of infected macrophages showed apaucity of virus particles with central densecores and very few enveloped capsids. This sug-gests that virus-specified macromolecules areeither structurally defective or produced in in-sufficient amounts or that errors exist in theassembly of the virus particle. Furthermore, itwas shown that infected macrophages releasevirus-destructive products, probably lysosomalenzymes, which are abundant in these cells.Recent studies have revealed differences in

the ultrastructures of peritoneal macrophages ofnewborn and adult mice (57). The most conspic-uous difference in the intracellular organizationsof adult and newborn mouse macrophages isconcerned with the rough endoplasmic reticu-lum, which is abundantly represented in adultmouse macrophages but is practically nonexist-ent in macrophages from newbom mice. Fur-thermore, differences were revealed in the or-ganizations and charge densities of the cell mem-branes. Whether the differences reported rep-resent significant elements of the different anti-microbial potencies of macrophages from new-

born and adult mice remains, however, to beelucidated.

Role of Nonspecifically Stimulated andActivated Macrophages in ResistanceBesides constituting an immediate, "ready to

function" barrier to virus penetration and dis-semination in the body, macrophages participatein the defenses mounted by an infected host anddirected against the invading pathogen. Duringthe courses of many infections, macrophagesbecome hyperactive and activated with in-creased microbicidal capacity. This process isimmunologically mediated by specifically sensi-tized T lymphocytes, presumably via the secre-tion of soluble lymphokines, and macrophagesare now recognized as important ultimate effec-tors of both antiviral and antibacterial cell-me-diated immunity (23). The increased antimicro-bial activity is at least partly nonspecific in thatactivated macrophages arising during an infec-tion with one microorganism show increasedactivity against other, unrelated agents as well(20). In addition to increased microbicidal ca-pacity, activated macrophages have been shownto exhibit a number of other distinctive features:increased phagocytic ability, accelerated"spreading" on glass surfaces (20), increased ly-sosomal enzyme content (31, 119), augmentedmetabolic functions (72), and antitumor activity(59). Many of these properties are shared bynonspecifically stimulated macrophages elicitedin vivo or in vitro by various irritants, such asmineral oil, glycogen, proteose-peptone, thiogly-colate, etc. (36), and it has been speculated thatactivated and stimulated macrophages are fun-damentally alike (20).

It is well documented that specific macro-phage recruitment and activation occurring inthe course of a virus infection are of crucialimportance for recovery (23). Much more openquestions are the significance of nonspecificmacrophage activation and stimulation in thenatural resistance of the host to virus infectionsand the prospects of nonspecific macrophage-activating immunomodulation in the prophy-laxis and treatment of virus infections.

In vitro, both stimulated and immunologicallyactivated macrophages have shown great anti-viral potentials. Thus, Hirsch et al. (62) demon-strated by an infectious center assay that pro-teose-peptone-stimulated peritoneal macro-phages from adult mice were considerably morerestrictive in the replication of HSV-1 than wereunstimulated cells. Suckling mice, on the otherhand, did not respond to proteose-peptone stim-ulation with the production of a macrophagepopulation with these characteristics, a fact that

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might partly explain the inefficiency of sucklingmouse macrophages to restrict replication ofviruses in vivo and in vitro. In another assaysystem, Lodmell et al. (84) demonstrated signif-icant inhibition of plaque formation and virusyield in HSV-infected rabbit kidney cells incu-bated with rabbit peritoneal leukocytes (mainlymacrophages) elicited with sodium caseinate.The kinetics of the inhibition suggested that theantiviral effect of the stimulated macrophageswas exerted through nonspecific damage of bothinfected and noninfected cells, thus preventingcell-to-cell spreading of the virus. A similar an-tiviral effect toward HSV-2, vaccinia virus, andencephalomyocarditis virus was demonstratedby Morahan et al. (107) with both activated andstimulated mouse macrophages, whereas normalunstimulated macrophages were relatively inef-ficient in preventing virus growth. As a lastexample, mouse peritoneal macrophages immu-nologically activated by previous inoculations ofS. aureus showed a considerably increased ca-pacity to restrict replication of an influenza Avirus, as revealed by the development of hem-adsorption and the formation of S and V anti-gens (133).

In spite of the marked antiviral potency ex-hibited by stimulated and activated macro-phages in vitro, most attempts to increase theresistance of experimental animals to virus in-fections by administration of macrophage-acti-vating immunostimulants have yielded resultsof only marginal significance. Thus, the highlyreduced capacity of influenza virus to replicatein vitro in macrophages from S. aureus-treatedmice was only reflected in a moderate, althoughsignificant, increased survival time after influ-enza virus infection of similarly treated mice,whereas no significant reduction of total mortal-ity was achieved (133). Adult rabbits inoculatedwith live M. bovis (BCG) were only partiallyprotected from the development of encephalitisafter subsequent corneal infection with HSV-2(76). Likewise, BCG conveyed no substantialprotection after vaginal HSV-2 infection of rab-bits (76) or adult mice (9), although a combina-tion of macrophage activation and passive ad-ministration of HSV-2 antiserum showed amarked effect in the latter study, probably be-cause of the opsonizing effect of the antibodies.Among several putative macrophage stimulatorsexamined by Starr et al. (140), only live BCGadministered 6 days before viral challenge wasable to reduce the mortality among sucklingmice inoculated intraperitoneally with HSV-2,whereas levamisole, staphylococcal phage ly-sate, and inactivated typhoid and Brucella vac-cines were without effect. It is noteworthy that

MICROBIOL. REV.

BCG was the only agent containing live orga-nisms and that at least 6 days were required toachieve the state of increased resistance, sug-gesting that immunological mechanisms are in-volved in the activation process. As a conse-quence of the 6-day period elapsing before pro-tection was noted, it is questionable whether analready disseminated human neonatal HSV in-fection, which usually rns a rapid fatal course(109), might be influenced by BCG administra-tion. Since, however, the incubation period ofthe infection is about 6 days, BCG might stillprove effective if administered prophylacticallyto newborns delivered vaginally from motherswith active genital infection at the time of deliv-ery or to babies with localized infections. It hasbeen claimed that BCG administration is capa-ble of controlling recurrent genital herpes infec-tion in a clinical trial (6).

Further evidence of the potentil importanceof activated macrophages in host defensesagainst viral infections has emerged from twoartifcial experimental animal models, namely,mice with the graft-versus-host (GVH) reactionand congenitally athymic nude mice. A commonfeature shared by these experimental animals isa combination of severe immunosuppressionwith concurrent nonspecific hyperactivity of themononuclear phagocyte system, making themsuitable to expose the potentials of the macro-phage-mediated resistance against invading vi-ruses.The development of a GVH reaction in adult

F1 hybrid mice by inoculation of immunologi-cally competent lymphoid celis from one of theparent strains has been shown to induce a stateof immunological incompetence against bothbacterial and viral antigens affecting both thehumoral (19, 22, 66) and the cell-mediated (21,22) immune responses. On the other hand, thephagocytic activity of the mononuclear phago-cyte system is profoundly increased as measuredby the clearance of intravenously injected col-loidal particles (64). The antibacterial potencyof these active macrophages has also been foundto be enhanced in that mice with the GVHreaction were more resistant to infection withListeria monocytogenes than were normal mice(21). As regards resistance to viral infections,Blanden (22) found that clearance of intrave-nously inoculated virus was significantly en-hanced in mice with the GVH reaction and thatvirus growth in the livers and spleens of thesemice was consistently lower during the first 4days of infection as compared with controls. Theoverall mortality was, however, higher in thegroup of mice with the GVH reaction. Thesefindings strongly suggest that the hyperactivity


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of macrophages caused by the GVH reactionimproved the primary protection of these organsby being more efficient in the uptake and de-struction of invading virus. The ultimate supe-riority of normal mice in controlling infectionwas not manifested until 6 to 8 days after infec-tion, when the necrotic foci in the livers ofnormal mice became smaller and more denselypopulated with mononuclear cells. This featurewas not seen in the liver lesions of mice with theGVH reaction, probably because of inefficiencyof cell-mediated recruitment of mononuclearphagocytes.

Recently, several authors have revealed evi-dence suggesting that the nude mouse with con-genital thymic aplasia represents another animalmodel in which nonspecific macrophage activa-tion can be studied. It is widely agreed that nudemice, because of their immunodeficiency, arehighly susceptible to several naturally occurringbacterial and viral infections (53). However, inrecent reports, the mice have been shown topossess unexpected enhanced primary resistanceto experimental infections with a number ofpathogens, including L. monocytogenes (30, 39,166), Brucella abortus (30), Salmonella typhi-murium (43), and Candida albicans (33). Anal-yses of the courses of infection have pointed toan increased microbicidal capacity of mononu-clear phagocytes as the cause of the augmentedresistance of nude mice to these pathogens, aview that has been corroborated by in vitrostudies of the interaction of the microorganismsand peritoneal macrophages in vitro (30, 166).Moreover, peritoneal macrophages from nudemice have been shown to exhibit nonspecificcytotoxicity to tumor cells in vitro (93), a findingthat is consistent with the observed low inci-dence of spontaneous tumors in nude mice (32,126).In 1976, Mogensen briefly reported an unex-

pected high resistance of nude mice to inductionof focal necrotic hepatitis by HSV-2 (100). Alater study (105) confirmed that nude mice be-yond the age of 4 to 5 weeks were considerablymore resistant than congenic mice of the back-ground strain and phenotypically normal heter-ozygous littermates, differing in genetic consti-tution from the nudes by the nu gene only,which codes for thymic aplasia and nakedness.Studies of the courses of infection showed thatnude mice were able to restrain virus multipli-cation in the liver far better than were normalmice during the first 5 days of infection (Fig. 9).In vitro investigation of peritoneal macrophagesrevealed that macrophages from 6-week-oldnude mice exhibited accelerated spreading after1 h in culture, a well-accepted characteristic ofactivation. Assessment of the replication of

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1 2 3 4 5 6 7DAYS AFTER INFECTION

FIG. 9. Courses of infection in the livers of6-week-old homozygous nude (nu/nu) mice (, ),heterozygous (nu/+) littermates (*,-*-*-), andBALBIc mice (0, -- -) inoculated intraperitone-ally with 106 plaque-forming units of HSV-2. Eachpoint represents one mouse. Lines are drawn betweenthe means of each group. From Mogensen and An-dersen (105).

HSV-2 in macrophage cultures by an infectiouscenter assay revealed that macrophages fromnude mice were much more restrictive in thisrespect than were macrophages from normalmice, since the number of macrophages fromnude mice that supported virus replication wasonly one-third the number from normal mice.As further evidence of the role of activated mac-rophages in the resistance phenomenon, it wasfound that blockade of the mononuclear phago-cyte system by silica treatment abolished theincreased resistance of nude mice. The causalrelationship between the athymic state and re-sistance was established by showing that resto-ration of the T-cell function of the nude mice bythymus cell grafting resulted in a normal, non-elevated defense capacity of the mice. However,in spite ofthe increased native resistance ofnudemice, they seemed inferior to normal mice in the

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final elimination of the infectious process (Fig.9), probably because of the absence of a cell-mediated immune response. Likewise, Starr andAllison (139) found that nude mice were more

susceptible than normal mice to infection withmurine cytomegalovirus as measured by overallmortality; however, during the acute stages ofinfection a relative sparing of the liver of nudemice was noted as compared with the severehepatitis seen in immunologically intact animals.The causal relationship between the athymic

state of nude mice and the restriction of virusreplication in their macrophages was also dem-onstrated by Rao et al. (118). In their assaysystem, vaccinia virus showed a 30-fold increasein titer in macrophage cultures from normalmice, whereas macrophages from nude micefailed to support virus replication. However,macrophages harvested from nude mice havingreceived a thymus transplant from heterozygouslittermates 5 to 6 weeks earlier replicated thevirus to control levels. Macrophages from nudemice raised under germfree conditions did thesame. Similarly, Meltzer (93) demonstrated thatmacrophages from germfree nude mice were nottumoricidal, as were macrophages from conven-tional nudes. This shows that environmentalstimuli are involved in the phenomenon. It hasbeen shown that nude mice have poorly devel-oped Peyer's patches and a low level of gutimmunoglobulin A plasma cells (54). Probably,the intestinal bacterial flora of nudes is respon-sible for a sustained nonspecific macrophageactivation through the absorption of bacteriallipopolysaccharide and phospholipid, which areknown to elicit macrophage activation in mice(42, 43, 65). In favor of this hypothesis is alsothe finding of Nickol and Bonventre (110) thatantibiotic elimination of the intestinal bacterialflora reduced the augmented resistance of nudemice to a challenge with L. monocytogenes.

CONCLUDING REMARKSIn this survey, aspects of the importance of

the mononuclear phagocyte system as a barrierto the establishment and dissemination of virusinfection in the body have been presented. Froman anatomical point of view, cells of the mono-nuclear phagocyte system are well suited for thisfunction. They are strategically placed at theportals of entry of many viruses and are widelydistributed in close contact with the circulatingblood in most organs of the body, thus encoun-

tering the virus early in the infection. From a

functional point of view, the ability of macro-phages to take up and destroy invading virusparticles may be of the utmost importance inthe race between the destructive growth of the

MICROBIOL. REV.

virus and the induction of a specific immuneresponse. If, on the other hand, the virus is ableto multiply more or less freely in macrophages,the "viral burden" and the destruction may haveproceeded beyond the limit of the capacities ofthe specific immune responses, when they even-tually turn up.No attempt has been made in this review to

assess the role of other phagocytic cells, such aspolymorphonuclear leukocytes, in natural resist-ance to virus infections. Polymorphonuclear leu-kocytes do not constitute a fixed barrier systemin the body, and they are generally not a veryconspicuous feature in the pathological lesionsproduced by viruses. Although they are capableof taking up virus particles, there is no evidencethat they play a very prominent role, if any, inthe antiviral defense mechanisms of the body(98, 104).The collaboration of macrophages with other

host defense mechanisms against virus infectionsis a subject of current interest. For example,macrophages have been shown to be of impor-tance in the production by specifically sensitizedlymphocytes ofimmune (type II) interferon (41).However, the role of macrophages seems moreimportant in the immune response. Thus, mac-rophages are recognized as important ultimateeffectors of antiviral cell-mediated immunity(23). Probably, macrophages also play a role inthe inductive phase of the immune response bycapturing the virus and by processing the viralantigens and presenting them to lymphocytesfor immune recognition (148).As previously stated, the basic nature of the

capacity of macrophages to restrict the replica-tion of viruses is at present unknown. A betterunderstanding of this question, together withmore insight into the collaboration of macro-phages with other host defense mechanisms,might open new fields of specific immunomod-ulation. This could be of importance in the pro-phylaxis and treatment of virus infections inpersons with increased susceptibility, for exam-ple, premature infants and persons with geneti-cally determined or induced immune deficien-cies.

SUMMARYThe scope of this survey was to analyze the

role played by macrophages in natural, innateresistance to virus infections.The term "macrophage" was introduced in

1892 by Metchnikoff to designate mononuclearphagocytes resident in various organs and tis-sues. On the basis of morphology, function, andkinetics of development, all highly phagocyticcells and their precursors are today grouped in


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one cell lineage called the mononuclear phago-cyte system. By their wide distribution in mostorgans of the body and by their ability to takeup and destroy virus particles, macrophages arewell suited to function as a barrier to the estab-lishment and dissemination of virus infection.Most of the information on the importance of

macrophages in natural resistance to virus infec-tions has been gained by studying experimentalinfections in laboratory animals, especially mice.Aspects of these studies have been dealt with infour sections: (i) studies in which macrophagerestriction of virus growth has been shown to beof importance for the difference in the resistanceof animals to closely related virus strains or virustypes; (ii) studies in which variation in innateresistance to a virus infection displayed by mem-bers of the same animal species (genetically de-termined resistance) has been correlated withthe ability of the virus to replicate in macro-phages from the animals; (iii) studies in whichan age-related increase in resistance to a virusinfection has been found to be caused by amaturation ofmacrophages occurring during thefirst few weeks of life (the mature macrophagesconstitute a more effective barrier to the spreadof virus than do immature macrophages fromnewborn and weanling animals); and (iv) studiesin which restricted virus replication in nonspe-cifically activated macrophages accounts foraugmented resistance to virus infections.Although the basic nature of the capacity of

macrophages to restrict the replication ofvirusesis at present unknown, an increasing amount ofdata from studies of the categories above listedpoints to a key role ofmacrophages as a first lineof defense at the portals of entry ofmany virusesor in target organs of crucial importance for theoutcome of infections.

ACKNOWLEDGMENTISI thank H. K. Andersen, S. Haahr, and E. A.

Freundt for their valuable criticism and suggestions atvarious stages of the preparation of this review.Thanks are due to Aase Hvergel for excellent technicalassistance and proofreading and to Grethe H. J0rgen-sen for expert secretarial assistance in the typing ofthe manuscript.

Part of my work described in papers cited in thisreview was supported by the Danish Medical ResearchCouncil grant no. 512-5648.

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against virus infections. Arch. Gesamte Virus-forsch. 17:280-294.

2. Allison, A. C. 1970. On the role of macrophagesin some pathological processes, p. 422-444. InR. van Furth (ed.), Mononuclear phagocytes.Blackwell Scientific Publications, Oxford.

3. Allison, A. C. 1974. Interactions of antibodies,complement components and various cell typesin immunity against viruse and pyogenic bac-teria. Transplant. Rev. 19:3-55.

4. Allison, A. C. 1976. Fluorescence microscopy oflymphocytes and mononuclear phagocytes andthe use of silica to eliminate the latter, p. 395-404. In B. R. Bloom and J. R. David (ed.), Invitro methods in cell-mediated and tumor im-munity. Academic Press Inc., New York.

5. Allison, A. C., J. S. Harington, and M. Bir-beck. 1966. An examination of the cytotoxiceffects of silica on macrophages. J. Exp. Med.124:141-153.

6. Anderson, F. D., R. N. Ushijima, and C. L.Larson. 1974. Recurrent herpes genitalis.Treatment with Mycobacterium bovis (BCG).Obstet. Gynecol. 43:797-805.

7. Andervont, H. B. 1929. Activity of herpeticvirus in mice. J. Infect. Dis. 44:383-393.

8. Aschoff, L 1924. Das reticulo-endotheliale Sys-tem. Ergeb. Inn. Med. Kinderheilkd. 26:1-118.

9. Baker, M. B., C. L. Larson, R. N. Ushijima,and F. D. Anderson. 1974. Resistance of fe-male mice to vaginal infection induced by Her-pe8Virus hominis type 2: effects of immuniza-tion with Mycobacterium bovis, intravenousinjection of specific Herpesvirus hominis type2 antiserum, and a combination of these pro-cedures. Infect. Immun. 10:1230-1234.

10. Bang, F. B., and C. N. Luttrell. 1961. Factorsin the pathogenesis of virus diseases. Adv. Vi-rus Res. 8:199-244.

11. Bang, F. B., and A. Warwick. 1957. The effectof an avirulent and a virulent strain of New-castle virus (Myxovirus multiforme) on cells intissue culture. J. Pathol. Bacteriol. 73:321-330.

12. Bang, F. B., and A. Warwick. 1959. Macro-phages and mouse hepatitis. Virology 9:715-717.

13. Bang, F. B., and A. Warwick. 1960. Mousemacrophages as host cells for the mouse hep-atitis virus and the genetic basis of their sus-ceptibility. Proc. Natl. Acad. Sci. U.S.A. 46:1065-1075.

14. Baron, S. 1963. Mechanisms of recovery fromviral infection. Adv. Virus Res. 10:39-64.

15. Baron, S. 1973. The defensive and biologicalroles of the interferon system, p. 267-293. InN. B. Finter (ed.), Interferons and interferoninducers. American Elsevier Publishing Co.,Inc., New York.

16. Bennett, B. 1966. Isolation and cultivation invitro of macrophages from various sources inthe mouse. Am. J. Pathol. 48:165-181.

17. Bennett, W. E., and Z. A. Cohn. 1966. Theisolation and selected properties of bloodmonocytes. J. Exp. Med. 123:145-159.

18. Berken, A., and B. Benacerraf. 1966. Proper-ties of antibodies cytophilic for macrophages.J. Exp. Med. 123:119-144.

19. Blaese, R. M., C. Martinez, and R A. Good.1964. Immunologic incompetence of immuno-logically runted animals. J. Exp. Med. 119:211-224.

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21. Blanden, R. V. 1969. Increased antibacterial re-

sistance and immunodepression during graft-versus-host reactions in mice. Transplantation7:484-497.

22. Blanden, R. V. 1971. Mechanisms of recoveryfrom a generalized viral infection: mousepox.III. Regression of infectious foci. J. Exp. Med.133:1090-1104.

23. Blanden, R. V. 1974. T Cell response to viraland bacterial infection. Transplant. Rev. 19:56-88.

24. Boyum, A. 1968. Isolation of mononuclear cellsand granulocytes from human blood. Scand. J.Clin. Lab. Invest. Suppl. 97:77-89.

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28. Cayeux, P., J. Panijel, R. Cluzan, and R.Levillain. 1966. Streptococcal arthritis andcardiopathy experimentally induced in whitemice. Nature (London) 212:688-691.

29. Chang, S. -S., and W. H. Hildemann. 1964.Inheritance of susceptibility to polyoma virusin mice. J. NatL Cancer Inst. 33:303-313.

30. Cheers, C., and R. Waller. 1975. Activatedmacrophages in congenitally athymic "nude"mice and in lethally irradiated mice. J. Immu-nol. 115:844-847.

31. Cohn, Z. A., and B. Benson. 1965. The differ-entiation of mononuclear phagocytes. Mor-phology, cytochemistry, and biochemistry. J.Exp. Med. 121:153-169.

32. Custer, R. P., H. C. Outzen, G. J. Eaton, andR. T. Prehn. 1973. Does the absence of im-munologic surveillance affect the tumor inci-dence in "nude" mice? First recorded sponta-neous lymphoma in a "nude" mouse. J. Natl.Cancer Inst. 51:707-711.

33. Cutler, J. E. 1976. Acute systemic candidiasis innormal and congenitally thymic-deficient(nude) mice. RES J. Reticuloendothel. Soc. 19:121-124.

34. duBuy, H. 1975. Effect of silica on virus infec-tions in mice and mouse tissue culture. Infect.Immun. 11:996-1002.

35. duBuy, H. G., and M. L. Johnson. 1966. Studieson the in vivo and in vitro multiplication of theLDH virus of mice. J. Exp. Med. 123:985-998.

36. Edelson, P. J., and Z. A. Cohn. 1976. Purifica-tion and cultivation of monocytes and macro-phages, p. 333-340. In B. R. Bloom and J. R.David (ed.), In vitro methods in cell-mediatedand tumor immunity. Academic Press Inc.,New York.

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37. Ehrmann, R. L., and G. 0. Gey. 1956. Thegrowth of cells on a transparent gel of recon-stituted rat-tail collagen. J. Natl. Cancer Inst.16:1375-1404.

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