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The macrophage and mycobacterial infections
TRANSACTIONS
OF THE ROYAL
SOCIETY
OF TROPICAL
The macrophage
MEDICINE,
Unit for Laboratory
77, No.
5, 646-655
(1983)
and mycobacterial D. B.
M.R.C.
VOL.
Studies of Tuberculosis,
LOWRIE
Royal Postgraduate W12 OHS
This paperdiscusses almostexclusively the interaction betweentubercle bacilli and macrophages,primarily because this has been studied far more extensivelythan interaction with Mycobacterium leprae or other pathogenicmycobacteria.But first I wish to indicatehow increasingunderstandingof that interaction might leadto far-reachingbenefitsvia improved tuberculosistreatment. It surprisesmost British people to learn that tuberculosiskills at least three million peopleevery year worldwide, a numberthat is increasing(STYBLO, 1980;WHO, 1981). They are also surprisedto find that tuberculosisis still the causeof many hundredsof deathsper annum in England andWales.,or about as many asall of the other notifiable infectiousdiseases added together (ENGLAND& WALES, 1982). The situationis still sobad, despitethe existenceof highly effective chemotherapy, becauseof two fundamentalpractical problems:(1)finding the cases,(ii) applying treatment. The first problem will not be consideredhere. The main componentof the second is that somuch chemotherapyis still requiredreliably to achievesterilization of an infection (a minimum of six months treatment even with the most potent rifampicin- and pyrazinamide-containingregimens, MITCHISON, 1980; Fox, 1981) that it is beyond practical reach on the scaleneededin the countries most affected. In the UK the difficulty increasingly arisesfrom reactivation of infection after many years of dormancy in the tissuesand late diagnosis(FOX, 1981). Treatment that also eliminated dormancy would therefore be useful. Whilst we can continue to hopefor new and better antimicrobialchemotherapeuticdrugs from the drug companiesand can aim further to improve our useof thosecurrently available,a moment’sreflectionon the intensity of interest, academicand commercial,in the development of immunopharmacological agents (see:- Abstracts, 1982)indicatesthat we would do well to consider using such agents as adjuncts to antimicrobialchemotherapy.If their useresultedin a substantial reduction in the length of treatment needed their impact on the tuberculosis problem would be enormous. Fortunately, from the kind of laboratory studiesof the macrophagewhich I will describe, and from parallel studiesof the cellsthat regulatemacrophage function, we are increasinglyin a position to define the kinds of immunopharmacoIogica1 agents that might be of value in tuberculosis and to devise appropriatein vitro teststo screenfor suchagentsor monitor their use. The foundation of thesedevelopmentsis the availability in the laboratory of systemsin which macrophagescan be activated so that they
infections
Medical
School, Ducane Road, London
acquire activity against pathogenic mycobacteria. Several such systemshave been described. Acquisition of bacteriostaticeffect againstphagocytosedmycobacteriahasbeena frequent finding when macrophageshave beentreated with lymphokinesin vitro. Lymphokineshavebeenpreparedin a variety of ways and used to activate various kinds of macrophages.Bacteriostasishas been conferred against Mycobacterium tuberculosis in resident and exudate mouseperitonealmacrophages(PATTERSON & YouMANS,1970;KLUN & YOUMANS,1973a,b; CAHALL & YOUMANS, 1975a, b; TURCO-REet al., 1976; MUROAKA et al., 1976a, b) and in macrophages differentiated from human peripheral blood monocytes (CROWLE& MAY, 1981). Lymphokine treatment also inhibited growth of M. lepraemurium and M. microti (vole acid-fast bacillus) in rabbit and human peripheral blood monocytes(GODAL et al., 1971) and of M. lepraemurium in mouseperitoneal macrophages (ALEXANDER& SMITH, 1978).In none of thesesystemswere macrophagesactivated to the point of showinga substantialor unequivocalcapacity to kill phagocytosedpathogenicmycobacteriain zlirro, increasingdoubts as to whether macrophagesever really killed these organismsat all, even in viva. The killing of pathogenic mycobacteria is now known with certainty to be within the capabilitiesof
1-j 0
,
1
3.5
24 Time
Fig. 1. Killing of intracellular mouse peritoneal macrophages. control macrophages.
(h /
M. microti by lymphokine-activated 0 = activated macrophages;
0
=
D.
B
macrophages, at least in one particular instance (WALKER & LOWRIE, 1981a). When resident mouse peritoneal macrophages were maintained as monolayers for three days in a serum-rich medium which was replenished daily they had little effect on phagocytosed M. microti. In contrast, if the maintenancemediumhad beensupplementedwith lymphokines from immunologicallyactivated mousespleen cells they killed >90% of the phagocytosedbacteria within 24 hours (Fig. 1). This finding wasobtained consistentlyin the absenceof antibiotics. This was important becausethe assumptionthat antibiotics do not interfere with testsof intracellularkilling because they do not penetratephagocytesadequatelyhasbeen shown to be erroneous(COLE & BROSTOFF, 1975; TULKENS
& TROUET,
1978;
LOWRIE
et al.,
5. 5
1979a,
b-b
5
4
2 1
0
15
3
?O
60 Tlme I minutes
90
.OWRIE
647
Usingthis systemit hasbecomepossibleto examine the mechanisms by which macrophages kill pathogenicmycobacteria.As anticipatedfrom studies by NATHAN et al. (1979), the mousemacrophages treated with lymphokinesm this way had acquireda substantialcapacity to releasehydrogen peroxide in respqnseto stimulation with phorbol myristate acetate, which is a convenient membraneperturbing agent, and in responseto phagocytosisof M. microti (Fig. 2). Direct evidencethat macrophageperoxide wasinvolved in killing M. microti wasobtainedwhen it was shown that catalaseadded to the system abolishedkilling (Fig. 3). This systemtherefore not only provides the basisfor directly relevant tests of lymphocyte and macrophagefunction, that will make it much easierto study the control of tuberculocidal immunity, but also has indicated that the control systemsfor induction and expressionof the hydrogen peroxide responsiveness of macrophagesmight be goodtargetsfor immunopotentiatingdrugsin tuberculosis. A rational approachto the possibilityof therapeutic enhancementof peroxide releasefrom macrophages that are either infected with, or encountering,tubercle bacilli dependson understandingthe underlying biochemistryof the macrophageand its responsivenessto environmentalsignals.Progressin this area seemslikely to follow from the discovery (L. Walker & D. B. Lowrie, unpublished)that normal mouse peritoneal macrophagesthat were maintained in a serum-less mediumwere thereby activated to kill M. microti (Fig. 4). This finding arosefrom an experiment to test the hypothesis that some degree of nutritional deprivation of the macrophagemight be necessaryfor tuberculocidalactivation in viva and in
120
I
Fig. 2. Release of hydrogen peroxide from lymphokine-activated macrophages. 0 = without stimulation; A = in response to phorbol myristate acetate; n = during phagocytosis of M. microfi.
100
20
IO 1 0
, 22.5
2.5 Time Ihl
Fig. 3. The protective effect of added catalse against killing of M. micrott by activated macrophages. 0 = catalase present; n = heat-denatured catalase present A = no added catalase.
Fig. 4. Killing of M. mirrori inside macrophages that have been activated by maintenance in a serum-less medium. Resident peritoneal macrophages were obtained from normal BALBc mice and maintained as monolayers for 3 days with daily medium changes in the serum-less medium of NEUMAN & TYTELL (1960) supplemented with human transferrin (5 pg ml-‘). After phagocytosis the macrophages contained an average of about 3 bacilli per cell. ExtraceUular bacilli were removed by frequent rinsing. No antibiotics were present at any time. 0 = Medium without iron supplement; 0 = medium supplemented with 2 pg Fe3+ ml-‘.
THE
648
MACROPHAGE
AND
MYCOBACTERIAL
INFECTIONS
own 0
30
60
90
120
MlllUkS
Fig. 5. Release of hydrogen peroxide from macrophages that had been maintained in serum-less medium with transferrin then stimulated with phorbol myristate acetate. 0 = Medium without iron supplement; 0 = medium supplemented with 2 pg Fe’+ ml-‘.
vitro. Indeed, consistentwith this hypothesis,increasing the iron content of the mediumfrom 0.2 pg per ml to a “phvsiological” level of 2 to 3 ug per ml reduced the subsequentkilling of phagocyiosedM. microti. When the abilitv of the cells to release hydrogen peroxide in responseto stimulation was tested they were found to be as activated as those activated -with lymphokine in serum-rich medium (Fin. 5). The enhancement of neroxideresnonsiveness by &intenance in serum-fr&medium waslessif the medium had been supplementedwith iron. The implication of thesefindings is that iron-deprivation might play a part in priming macrophageperoxidemediatedkilling in viva. However, the role of iron in infection is complex (KOCHAN, 1977; WEINBERG, 1978) and M. microti grown with iron asthe growthlimiting nutrient was found to have an increased resistanceto killing by lymphokine-treated macrophages(WALKER & LOWRIE, 1981b). Indirect evidencethat macrophagesusehydrogen peroxide to kill M. tuberculosis aswell asM. microti hasbeenaccumulatingfor almost30 years. After the tuberculocidaldrug isoniazidhad been in usefor a few years,isoniazid-resistant mutantsof M. tuberculosis were isolated that had a low virulence in the guinea-pig,were catalase-negative and were susceptible to killing by hydrogen peroxide (BARNETT et al., 1953; COHN et al., 1954; MITCHISON, 1954; MORSE et al., 1954;PEIZER 81 WIDELOCK, 1955).At that time hydrogenperoxide production by mammaliantissues had not even beendemonstratedbut the implication that peroxide might reach tuberculocidalconcentrations-insideleucocyteswasappreciatedby COLEMAN & MIDDLEBROOK (1956).This ideawasconsiderablv strengthenedin the early 1960swith reports that a substantialproportion of isolatesof M. tuberculosis from patientsin the Indian subcontinentwere of low virulence in the guinea-pigand peroxide-susceptible even though they had a normal catalasecontent and isoniazid susceptibility (SUBBAIAH et al., 1960; MITCHISON et al.. 1963). JACKETT; et al.. (198la, b) investigated whether oeroxide-mediatedkilling of isoniazid-resistanttuber&e bacilli in the guinia-pig could be attributed predominantly to immunologically activated mac-
0
3
6
Clays
Fig. 6. Effect of peroxide susceptibility of M. ~berculoris on the course of intravenous infection in the lungs of normal and BCGvaccinated guinea-pigs. l ,O = Parent M. ruberculosis strain H37Rv; A, a = peroxide-susceptible (iwniazid-resistant) mutant of H37Rv; solid symbols = cells from normal animals; open symbols = cells from vaccinated animals.
rophages. They compared the fates of isoniazidresistant (peroxide-susceptible)mutants and their parent strains in the organs of normal and BCGvaccinatedguinea-pigsand in parallel examinedthe capacity of macrophages obtained from the infected lungs to releaseperoxide. The mutants consistently survived lesswell than the parent bacilli (Fig. 6). In the normalanimalthe effect of the bacterialmutation to peroxide susceptibilitywasthe samebetweendays 1 to 3 after infection as between days 4 to 6, suggestingthat peroxideavailability wasthe samein the two periods.In contrast, in the vaccinatedanimal the effect of peroxidesusceptibilitvwasgreaterin the secondperiod suggestingan increasingavailability of oeroxide. Durine the first neriod the effect of peroxide suscepcbility was expressedequally in normal and vaccinated animals suggestingequal initial availability of peroxide in normal and vaccinated animals. These implicationsabout peroxide availability depend on the assumptionthat the only significant consequenceof the mutation to isoniazid resistance .wasacquisitionof peroxide susceptibilityand this was not necessarilythe case. Nevertheless,the implications were borne out by measurements of peroxide releasefrom macrophages obtainedfrom the infected lungs. Vaccination did not affect the releaseof peroxide from macrophagesthat were removed and tested immediately after infection of the animal. Phagocytosisof opsonizedtubercle bacilli enhanced peroxidereleaseand the incrementper bacillustaken up by the cells was at first modestand equal with normal and vaccinated donors. The increment then increasedwith time elapsedsince infection of the animal (Fig. 7). The increase in responsiveness occurredfaster in the vaccinatedthan in the normal animal,presumablyasa consequence of lymphokine generation.Surprisingly, this increasein responsivenesswas selectivein that the macrophagesdid not differ with time or between normal and vaccinated
D.
I
,
1
0
3
6
B.
LOWRIE
Days
Fig. 7. Increasein alveolartnacrophagehydrogenperoxide response to phagocytosisof tuber& bacilli during the 6 daysafterintravenous infection. 0 = cells from normal animals; 0 = cellsfrom vaccinated animals.
animalsin respectof peroxide releasedin responseto phorbol myristate acetate (JACKETT et al., 1981b). Thus a selectiveincreasein the efficiencyof linkageof phagocyticstimulationto peroxideresponsemay have contributed to an enhancedperoxide-mediatedkilling in macrophageswith the development of acquired immunity. Releaseof superoxidebroadly paralleled the release of peroxide but tubercle bacilli are resistantto this potentially toxic product (JACKETT et al., 1978a).The nature of the peroxide generating system, the manner of its linkage to phagocytic stimulation and hencethe manner of its priming by lymphokinesare at presentobscure(REISS & ROOS, 1978; ANDREW et al., 1980; BELLAVITE et al., 1981). Further studiesby JACKETTet al. (1982) showed that neither opsonization nor the viability of the bacilli madeany difference to the peroxidereleaseper BCG bacillustaken up by macrophages from either normal or granulomatousguinea-piglungs, although opsonizationincreaseduptake (Table I). This finding was consistentwith the consensusview that specific antibody contributesnothing to resistanceto tubercuTable I-Release to phagocytosis
of hydrogen peroxide of BCG in V&O
by alveolar
macrophages
649
losis.However, it wasof particular interest because, aswill be discussedlater, tubercle bacilli, including BCG, inhibit fusion of lysosomeswith phagosomes and this ability is overcomeif the bacilli are either killed or antibody-coatedbefore phagocytosis(ARMSTRONG & HART, 1975; LOWRIE et al., 1980). Since neither killing nor antibody affected the hydrogen peroxide releasedper bacillus taken up it is evident that the two macrophageresponses,phago-lysosome formation and releaseof hydrogen peroxide, are substantiallyindependent. There is someevidencethat macrophageperoxide is important in the host’sdefenceagainsttuberculosis in man. Someof the subjectswho developeddisseminated BCG infection after routine vaccination may have had a defect in macrophageability to produce hydrogen peroxide analogousto that underlying chronic granulomatousdisease(MACKAY et al., 1980; URBAN et al., 1980). Also the most peroxidesusceptibletubercle bacilli (fully isoniazid-resistant SouthIndian strains)seemto be lessvirulent than the peroxide-resistantbacilli in man. This is indicatedby reduced ability to establish disease(MITCHISON, 1963; TRIPATHY et al., 1969; DEVADATTA et al., 1970),to cause rogressivediseaseduring inadequate chemotherapyPRAMAKRISHNANet al., 1961)or to disseminatesystemically(OESTREICHER et al, 1955). Nevertheless, the peroxide-susceptiblebacilli are capableof causingdiseasethat is just assevereasthat causedby the peroxide-resistantstrains(DEVADATTA et al., 1961;RAMAKRISHNANet al., 1962).Thus the roleof peroxidein manmight be moremodestthan in guinea-pigsbut, also, infected individuals might differ widely in their ability to mount or sustaina peroxide-dependentimmune response.Comparison of peroxide release from guinea-pig and human alveolarmacrophagessuggeststhat the speciesdiffer rather little in this respect, at least with phorbol myristate acetateas stimulus. This agent elicited on average80 mnol per lo6 cells per hour from guineapig alveolarmacrophages (JACKETTet al., 1981b) and 35 mnol per lo6 per hour from alveolarmacrophages from human subjectswith a history of recent lower respiratory tract infections(GREENING et al., 1981& unpublished.Nevertheless,it is unlikely that peroxide providesa comprehensiveexplanationof immunity to tuberculosis,evenin guinea-pigs.An antimicrobial systemasoxygen-dependentashydrogen peroxide production is probably ineffective within the closedgranulomatathat developlater in tuberculosis. Indeed, developmentof infection is impaired under from
normal
and BCG-vaccinated
Normalguinea-pigs Bacilli per macrophage
Stimuh.ts
DeadBCG Live BCG
Opsonized Opsonized Values for differences
;:;
guinea
pigs in response
Vaccinatedguinea-pigs
Hydrogen peroxide released (fmole/bacilludhour) 0.16
Bacilli per macrophage 5.8
0.14
6.6
Hydrogen-peroxide released (fmolelbacillusihour) 0.32 0.12
dead BCG 6.8 0.14 12.2 0.11 live BCG 6.1 0.15 12.4 0.24 peroxide release from phagocytosing macrophages were corrected for release from control macrophages and in bacillus uptake.
650
THE
MACROPHAGE
AND
MYCOBACTERIAL
INFECTIONS
Fig. 8. Electron micrographs of ultrathin-sections of mouse peritoneal macrophages showing (a) fusion of lysosomes containing heat-killed M. microti (HKM); (b) absence of lysosome fusion with phagosomes containing live M. micron’ lysosomes were labelled with electron-opaque gold particles.
(L) with phagosomcs (LM). The contents of
D.
conditions
where
oxygen
availability
B.
capacity to avoid exposurebut multipliesexclusively within lvsosomes(BROWNet al.. 1969). Attemnts have been made to understandthe mechanism‘of fusion inhibition by tubercle bacilli in the reasonable expectation that the bacillusbenefits from avoiding contact with lysosomalcontents(HART, 1982).Three distinct, but possiblycomplementary,mechanisms of fusion Inhibition have been proposed: bacterial releaseof polyanioniccell wall components(GORENet al., 1976),releaseof ammonia(GORDONet al., 1980) and either releaseof cyclic AMP or stimulation of macrophagesynthesisof cyclic AMP (LOWRIEet al.,
is restricted
(DIJBOS,1955; CHANDLERet al., 1965) and it is unlikely that this can be accountedfor entirely by the oxygen dependenceof tubercle bacillusmetabolism. Attention has beenfocussedon macrophagelysosomes as an alternative
to peroxide
since the demon-
stration by ARMSTRONG & HART (1971) that living tubercle bacilli have the ability to inhibit (or fail to induce) phagosome-lysosome fusion whereasdead onesdo not. This property is shared by M.’ microti (Fig. 8). M. Zeprue also avoids exposure to lysosomal
contentsbut doesso by escapinginto the cytoplasm (LEVYet al., 1975) whereas M. lepraemurium showsno Table
II-Effect
of modification
of phagosome-lysosome
Normal
fusion response Fusion Non-fusion
Organism M. M.
lepraenwium tuberculosis
microti Non-fusion *Table modified from HART (1982).
M.
20
651
LOWRIE
1975, 1979b, 1980). In cell-free preparations, fusion
Agent added Polyanion Polyanions Antibody coating Lipophilic amines Antibody coating
on mycobacterial
phago-
fate*
Modified fusion response Fusion inhibited Fusion inhibited Fusion promoted Fusion promoted Fusion promoted
Modified fate Multiplication increased Multiplication increased Unaltered Killing or stasis Unaltered or stasis
Catalase
kacetyl-P-glucosaminidase :““‘: : .
10
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.05
1.10
1.15
1.20
1.25
1.30
1.35
Density Fig. 9. Selective phagolysosome formation among lysosome subpopulations in rabbit alveolar macrophages infected with BCG: demonstration by analytical subcellular fractionation on sucrose density gradients then assay of gradient fractions for BCG colony-forming units and macrophage organ&e marker enzyme activities. Macrophages were obtained by broncho-alveolar lavage of granulomatous lungs from rabbits intravenously vaccinated with BCG in Freund’s adjuvant 3 weeks earlier. The cells were disrupted and fractionated after they had adhered overnight as monolayers and then phagocytosed BCG (--I or antibody-coated BCG ( -). Controls (. .) were macrophages without BCG and BCG without macrophages.
652
THE
MACROPHAGE
AND
MYCOBACTERIAL
some-lysosome fusion is resistantto inhibition by at leasttwo of theseagents(ammoniaand poly-glutamic acid)sothat a simpleeffect on phagosome or lysosome membranes seems to be unlikely (GEISOW et al., 1982; P.D’A. Hart, unpublished). The significanceof avoidanceof lysosomecontents by fusioninhibition remainsunknown for a variety of reasons.Notable amongtheseare: (i) it hasnot been establishedif or to what degreefusion is inhibited in viva; (ii) when fusion inhibitors enhanceinfection in vitro or in viva it is not certain that phagolysosome formation is the only host defencesysteminhibited and (iii) fusion promotion has inconsistently antimycobacterialeffects(Table II); (iv) althoughvarious anti-mycobacterialsubstancescan be found in macrophagelysosomes (GINSBURG, 1979; LOWFSE, 1983) their significancein resistanceto infection is not established;(v) an associatonbetweenvirulence and high content of fusion-inhibitory polyanionic lipids (GRANGE et al.. 1978)hasbeenweakenedbv studies of morestrainsbfM. iuberculosis (GOREN et ai., 1982); (vi) subpopulationsof lysosomesexist with different enzymecontents(LOWRIEet al., 1979~)and microscopical assessments of fusion inhibition have not allowed for the possibility of selective effects on different subpopulations. Fig. 9 showsthe results of an experiment using analytical subcellularfractionation to test whether lysosomesubpopulationsdiffer in their tendency to fusewith phagosomes containingBCG bacilli (D. B. Lowrie & P. W. Andrew, unpublished).In this type of experiment macrophagesare gently disrupted beforeor after infection andthe subcellularorganelles are centrifugedinto sucrosedensity gradientssothat each organellecomes to rest at a position in the gradientswhere the densitiesof the organelleand sucroseare equal. Each organellehasa characteristic and often distinctive density distribution profile and this can be revealedby assessing the distribution of a marker enzymeactivity known to be localizedto that organelle.Since tubercle bacilli and lysosomesshow peaksat different densitiesin control gradients, the formation of phagolysosomes in infected cellsis most simply revealedby the appearanceof a peak of both lysosomalenzyme activity and bacilli at a new commondensity. Acid phosphatase activity (Fig. 9) moved to the new peak of bacilli and did so to the sameextent with opsonizedor non-opsonizedbacilli; lysozyme and N-acetyl-B-glucosaminidase activities shifted more substantiallyto this position with the opsonizedthan with the unopsonizedbacilli; cathepsin D activity did not shift with either opsonizedor unopsonizedbacilli. Since the addition of bacilli to cell homogenates before centrifugation did not affect any distributions, these results suggest that the different lysosomesubpopulationsindicated by the different enzymeactivities (LOYVRIEet al., 1979~)did indeedhave different tendenciesto fuse with phagosomescontainingBCGandonly somewereinfhtenced by opsonization. Since macrophagescontain no myeloperoxidaseit wasalsonotablethat there wasno transferof catalaseto the positionof the bacilli. Either peroxidaseor catalasecan enhancethe toxicity of hydrogen peroxide for tubercle bacilli under appropriate circumstances(JACKETT et al., 1978b, 1980)but it is now doubtful that either is normally in contact with the bacilli in macrophages.
INFECTIONS
I
0
I
I
I
2
4
6 Days
I
a
1
I
10
12
Fig. 10. Inhibition of multiplication of M. microri in mouse peritoneal macrophagesby coa&g with antibody before phagocytosis. Monolayers were maintained for 1 week in serum-rich medium beforeinfection with opsonized (0) or non-opsonized (0) bacilli. No antibiotics were present at any stage; extracellular bacilli were removed by frequent rinsing.
Inhibition of multiplication of tubercle bacilli by macrophages might be at leastasimportant askilling in host defenceagainsttuberculosis.This is perhaps best illustrated in the bacteriostasisthat can be a prominent feature of acquired immunity against tuberculosisin mice (HART & REES, 1960; REES & HART, 1961). An indication that exposure to lysosomecontentscould significantlycontribute to macrophage-inducedbacteriostasiswas obtained when it wasfound that promotion of phagolysosome formation by antibody-coatingM. microti beforephagocytosiswould sometimesresult in bacteriostasis (Fig. 10). The basisof the variability of this finding is not known but it may be relevant that the mostfrequently reportedconsequence for tubercle bacilli of immunological activation of macrophageswas bacteriostasis. It is possiblethat different degreesof immunological activation result in different degreesof exposureto some growth-inhibitory lysosomal component. Lysozyme (OSHIMA et al., 1961)and the antibacterial factor describedby SHARMA& MIDDLEBROOK (1977) might be relevant in this context. The development of immunopharmacological agents that promote phagosome-lysosome fusion might be therapeuticallyusefulif either tuberculostasisis a usefulcomponentof host defenceor fusion in immunologicallyactivated cellsis lethal to the tubercle bacillusor if delivery or action of tuberculocidal drugs is thereby enhanced. However, it should be borne in mind that (i) such tuberculostasismight contribute to the developmentof persistentstatesof infection and thereby even further enhancethe risk of future reactivation and (ii) non-multiplying tubercle bacilli arelikely to be moreresistantto at leastsomeof the major antitubercular chemotherapeuticagents. Indeed, on thesetwo groundsa casemight be made for the oppositeapproach:that of trying to develop agents that prevent tuberculostasiswhile leaving tuberculocidal host defences intact. Such agents might reasonablybe usedin conjunction with potent tuberculocidal drugs.
D.
B.
Whilst it is now clearthat enhancementor repair of macrophagefunction should be the goal of immunopharmacologyin tuberculosis,the right targets for immunopharmacological agentsare aslikely to be found among the complex amplification and feedbackloopsin the cellularimmunesystemthat modify macrophagefunction asin the macrophageitself. As these targets becomeidentified and understood in biochemicalterms it will becomepossible,at leastin principle, to tailor agents for the precise function required. Other, usually lessdirect and even serendipitous, approachesto immunopharmacologyhave sofar yieldedagentsthat have beenrather disappointing (DREWS, 1982; GATNER & ANDERSON, 1982).
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OF THE ROYAL
SOCIETY
OF TROPICAL
The macrophage
MEDICINE,
Unit for Laboratory
77, No.
5, 646-655
(1983)
and mycobacterial D. B.
M.R.C.
VOL.
Studies of Tuberculosis,
LOWRIE
Royal Postgraduate W12 OHS
This paperdiscusses almostexclusively the interaction betweentubercle bacilli and macrophages,primarily because this has been studied far more extensivelythan interaction with Mycobacterium leprae or other pathogenicmycobacteria.But first I wish to indicatehow increasingunderstandingof that interaction might leadto far-reachingbenefitsvia improved tuberculosistreatment. It surprisesmost British people to learn that tuberculosiskills at least three million peopleevery year worldwide, a numberthat is increasing(STYBLO, 1980;WHO, 1981). They are also surprisedto find that tuberculosisis still the causeof many hundredsof deathsper annum in England andWales.,or about as many asall of the other notifiable infectiousdiseases added together (ENGLAND& WALES, 1982). The situationis still sobad, despitethe existenceof highly effective chemotherapy, becauseof two fundamentalpractical problems:(1)finding the cases,(ii) applying treatment. The first problem will not be consideredhere. The main componentof the second is that somuch chemotherapyis still requiredreliably to achievesterilization of an infection (a minimum of six months treatment even with the most potent rifampicin- and pyrazinamide-containingregimens, MITCHISON, 1980; Fox, 1981) that it is beyond practical reach on the scaleneededin the countries most affected. In the UK the difficulty increasingly arisesfrom reactivation of infection after many years of dormancy in the tissuesand late diagnosis(FOX, 1981). Treatment that also eliminated dormancy would therefore be useful. Whilst we can continue to hopefor new and better antimicrobialchemotherapeuticdrugs from the drug companiesand can aim further to improve our useof thosecurrently available,a moment’sreflectionon the intensity of interest, academicand commercial,in the development of immunopharmacological agents (see:- Abstracts, 1982)indicatesthat we would do well to consider using such agents as adjuncts to antimicrobialchemotherapy.If their useresultedin a substantial reduction in the length of treatment needed their impact on the tuberculosis problem would be enormous. Fortunately, from the kind of laboratory studiesof the macrophagewhich I will describe, and from parallel studiesof the cellsthat regulatemacrophage function, we are increasinglyin a position to define the kinds of immunopharmacoIogica1 agents that might be of value in tuberculosis and to devise appropriatein vitro teststo screenfor suchagentsor monitor their use. The foundation of thesedevelopmentsis the availability in the laboratory of systemsin which macrophagescan be activated so that they
infections
Medical
School, Ducane Road, London
acquire activity against pathogenic mycobacteria. Several such systemshave been described. Acquisition of bacteriostaticeffect againstphagocytosedmycobacteriahasbeena frequent finding when macrophageshave beentreated with lymphokinesin vitro. Lymphokineshavebeenpreparedin a variety of ways and used to activate various kinds of macrophages.Bacteriostasishas been conferred against Mycobacterium tuberculosis in resident and exudate mouseperitonealmacrophages(PATTERSON & YouMANS,1970;KLUN & YOUMANS,1973a,b; CAHALL & YOUMANS, 1975a, b; TURCO-REet al., 1976; MUROAKA et al., 1976a, b) and in macrophages differentiated from human peripheral blood monocytes (CROWLE& MAY, 1981). Lymphokine treatment also inhibited growth of M. lepraemurium and M. microti (vole acid-fast bacillus) in rabbit and human peripheral blood monocytes(GODAL et al., 1971) and of M. lepraemurium in mouseperitoneal macrophages (ALEXANDER& SMITH, 1978).In none of thesesystemswere macrophagesactivated to the point of showinga substantialor unequivocalcapacity to kill phagocytosedpathogenicmycobacteriain zlirro, increasingdoubts as to whether macrophagesever really killed these organismsat all, even in viva. The killing of pathogenic mycobacteria is now known with certainty to be within the capabilitiesof
1-j 0
,
1
3.5
24 Time
Fig. 1. Killing of intracellular mouse peritoneal macrophages. control macrophages.
(h /
M. microti by lymphokine-activated 0 = activated macrophages;
0
=
D.
B
macrophages, at least in one particular instance (WALKER & LOWRIE, 1981a). When resident mouse peritoneal macrophages were maintained as monolayers for three days in a serum-rich medium which was replenished daily they had little effect on phagocytosed M. microti. In contrast, if the maintenancemediumhad beensupplementedwith lymphokines from immunologicallyactivated mousespleen cells they killed >90% of the phagocytosedbacteria within 24 hours (Fig. 1). This finding wasobtained consistentlyin the absenceof antibiotics. This was important becausethe assumptionthat antibiotics do not interfere with testsof intracellularkilling because they do not penetratephagocytesadequatelyhasbeen shown to be erroneous(COLE & BROSTOFF, 1975; TULKENS
& TROUET,
1978;
LOWRIE
et al.,
5. 5
1979a,
b-b
5
4
2 1
0
15
3
?O
60 Tlme I minutes
90
.OWRIE
647
Usingthis systemit hasbecomepossibleto examine the mechanisms by which macrophages kill pathogenicmycobacteria.As anticipatedfrom studies by NATHAN et al. (1979), the mousemacrophages treated with lymphokinesm this way had acquireda substantialcapacity to releasehydrogen peroxide in respqnseto stimulation with phorbol myristate acetate, which is a convenient membraneperturbing agent, and in responseto phagocytosisof M. microti (Fig. 2). Direct evidencethat macrophageperoxide wasinvolved in killing M. microti wasobtainedwhen it was shown that catalaseadded to the system abolishedkilling (Fig. 3). This systemtherefore not only provides the basisfor directly relevant tests of lymphocyte and macrophagefunction, that will make it much easierto study the control of tuberculocidal immunity, but also has indicated that the control systemsfor induction and expressionof the hydrogen peroxide responsiveness of macrophagesmight be goodtargetsfor immunopotentiatingdrugsin tuberculosis. A rational approachto the possibilityof therapeutic enhancementof peroxide releasefrom macrophages that are either infected with, or encountering,tubercle bacilli dependson understandingthe underlying biochemistryof the macrophageand its responsivenessto environmentalsignals.Progressin this area seemslikely to follow from the discovery (L. Walker & D. B. Lowrie, unpublished)that normal mouse peritoneal macrophagesthat were maintained in a serum-less mediumwere thereby activated to kill M. microti (Fig. 4). This finding arosefrom an experiment to test the hypothesis that some degree of nutritional deprivation of the macrophagemight be necessaryfor tuberculocidalactivation in viva and in
120
I
Fig. 2. Release of hydrogen peroxide from lymphokine-activated macrophages. 0 = without stimulation; A = in response to phorbol myristate acetate; n = during phagocytosis of M. microfi.
100
20
IO 1 0
, 22.5
2.5 Time Ihl
Fig. 3. The protective effect of added catalse against killing of M. micrott by activated macrophages. 0 = catalase present; n = heat-denatured catalase present A = no added catalase.
Fig. 4. Killing of M. mirrori inside macrophages that have been activated by maintenance in a serum-less medium. Resident peritoneal macrophages were obtained from normal BALBc mice and maintained as monolayers for 3 days with daily medium changes in the serum-less medium of NEUMAN & TYTELL (1960) supplemented with human transferrin (5 pg ml-‘). After phagocytosis the macrophages contained an average of about 3 bacilli per cell. ExtraceUular bacilli were removed by frequent rinsing. No antibiotics were present at any time. 0 = Medium without iron supplement; 0 = medium supplemented with 2 pg Fe3+ ml-‘.
THE
648
MACROPHAGE
AND
MYCOBACTERIAL
INFECTIONS
own 0
30
60
90
120
MlllUkS
Fig. 5. Release of hydrogen peroxide from macrophages that had been maintained in serum-less medium with transferrin then stimulated with phorbol myristate acetate. 0 = Medium without iron supplement; 0 = medium supplemented with 2 pg Fe’+ ml-‘.
vitro. Indeed, consistentwith this hypothesis,increasing the iron content of the mediumfrom 0.2 pg per ml to a “phvsiological” level of 2 to 3 ug per ml reduced the subsequentkilling of phagocyiosedM. microti. When the abilitv of the cells to release hydrogen peroxide in responseto stimulation was tested they were found to be as activated as those activated -with lymphokine in serum-rich medium (Fin. 5). The enhancement of neroxideresnonsiveness by &intenance in serum-fr&medium waslessif the medium had been supplementedwith iron. The implication of thesefindings is that iron-deprivation might play a part in priming macrophageperoxidemediatedkilling in viva. However, the role of iron in infection is complex (KOCHAN, 1977; WEINBERG, 1978) and M. microti grown with iron asthe growthlimiting nutrient was found to have an increased resistanceto killing by lymphokine-treated macrophages(WALKER & LOWRIE, 1981b). Indirect evidencethat macrophagesusehydrogen peroxide to kill M. tuberculosis aswell asM. microti hasbeenaccumulatingfor almost30 years. After the tuberculocidaldrug isoniazidhad been in usefor a few years,isoniazid-resistant mutantsof M. tuberculosis were isolated that had a low virulence in the guinea-pig,were catalase-negative and were susceptible to killing by hydrogen peroxide (BARNETT et al., 1953; COHN et al., 1954; MITCHISON, 1954; MORSE et al., 1954;PEIZER 81 WIDELOCK, 1955).At that time hydrogenperoxide production by mammaliantissues had not even beendemonstratedbut the implication that peroxide might reach tuberculocidalconcentrations-insideleucocyteswasappreciatedby COLEMAN & MIDDLEBROOK (1956).This ideawasconsiderablv strengthenedin the early 1960swith reports that a substantialproportion of isolatesof M. tuberculosis from patientsin the Indian subcontinentwere of low virulence in the guinea-pigand peroxide-susceptible even though they had a normal catalasecontent and isoniazid susceptibility (SUBBAIAH et al., 1960; MITCHISON et al.. 1963). JACKETT; et al.. (198la, b) investigated whether oeroxide-mediatedkilling of isoniazid-resistanttuber&e bacilli in the guinia-pig could be attributed predominantly to immunologically activated mac-
0
3
6
Clays
Fig. 6. Effect of peroxide susceptibility of M. ~berculoris on the course of intravenous infection in the lungs of normal and BCGvaccinated guinea-pigs. l ,O = Parent M. ruberculosis strain H37Rv; A, a = peroxide-susceptible (iwniazid-resistant) mutant of H37Rv; solid symbols = cells from normal animals; open symbols = cells from vaccinated animals.
rophages. They compared the fates of isoniazidresistant (peroxide-susceptible)mutants and their parent strains in the organs of normal and BCGvaccinatedguinea-pigsand in parallel examinedthe capacity of macrophages obtained from the infected lungs to releaseperoxide. The mutants consistently survived lesswell than the parent bacilli (Fig. 6). In the normalanimalthe effect of the bacterialmutation to peroxide susceptibilitywasthe samebetweendays 1 to 3 after infection as between days 4 to 6, suggestingthat peroxideavailability wasthe samein the two periods.In contrast, in the vaccinatedanimal the effect of peroxidesusceptibilitvwasgreaterin the secondperiod suggestingan increasingavailability of oeroxide. Durine the first neriod the effect of peroxide suscepcbility was expressedequally in normal and vaccinated animals suggestingequal initial availability of peroxide in normal and vaccinated animals. These implicationsabout peroxide availability depend on the assumptionthat the only significant consequenceof the mutation to isoniazid resistance .wasacquisitionof peroxide susceptibilityand this was not necessarilythe case. Nevertheless,the implications were borne out by measurements of peroxide releasefrom macrophages obtainedfrom the infected lungs. Vaccination did not affect the releaseof peroxide from macrophagesthat were removed and tested immediately after infection of the animal. Phagocytosisof opsonizedtubercle bacilli enhanced peroxidereleaseand the incrementper bacillustaken up by the cells was at first modestand equal with normal and vaccinated donors. The increment then increasedwith time elapsedsince infection of the animal (Fig. 7). The increase in responsiveness occurredfaster in the vaccinatedthan in the normal animal,presumablyasa consequence of lymphokine generation.Surprisingly, this increasein responsivenesswas selectivein that the macrophagesdid not differ with time or between normal and vaccinated
D.
I
,
1
0
3
6
B.
LOWRIE
Days
Fig. 7. Increasein alveolartnacrophagehydrogenperoxide response to phagocytosisof tuber& bacilli during the 6 daysafterintravenous infection. 0 = cells from normal animals; 0 = cellsfrom vaccinated animals.
animalsin respectof peroxide releasedin responseto phorbol myristate acetate (JACKETT et al., 1981b). Thus a selectiveincreasein the efficiencyof linkageof phagocyticstimulationto peroxideresponsemay have contributed to an enhancedperoxide-mediatedkilling in macrophageswith the development of acquired immunity. Releaseof superoxidebroadly paralleled the release of peroxide but tubercle bacilli are resistantto this potentially toxic product (JACKETT et al., 1978a).The nature of the peroxide generating system, the manner of its linkage to phagocytic stimulation and hencethe manner of its priming by lymphokinesare at presentobscure(REISS & ROOS, 1978; ANDREW et al., 1980; BELLAVITE et al., 1981). Further studiesby JACKETTet al. (1982) showed that neither opsonization nor the viability of the bacilli madeany difference to the peroxidereleaseper BCG bacillustaken up by macrophages from either normal or granulomatousguinea-piglungs, although opsonizationincreaseduptake (Table I). This finding was consistentwith the consensusview that specific antibody contributesnothing to resistanceto tubercuTable I-Release to phagocytosis
of hydrogen peroxide of BCG in V&O
by alveolar
macrophages
649
losis.However, it wasof particular interest because, aswill be discussedlater, tubercle bacilli, including BCG, inhibit fusion of lysosomeswith phagosomes and this ability is overcomeif the bacilli are either killed or antibody-coatedbefore phagocytosis(ARMSTRONG & HART, 1975; LOWRIE et al., 1980). Since neither killing nor antibody affected the hydrogen peroxide releasedper bacillus taken up it is evident that the two macrophageresponses,phago-lysosome formation and releaseof hydrogen peroxide, are substantiallyindependent. There is someevidencethat macrophageperoxide is important in the host’sdefenceagainsttuberculosis in man. Someof the subjectswho developeddisseminated BCG infection after routine vaccination may have had a defect in macrophageability to produce hydrogen peroxide analogousto that underlying chronic granulomatousdisease(MACKAY et al., 1980; URBAN et al., 1980). Also the most peroxidesusceptibletubercle bacilli (fully isoniazid-resistant SouthIndian strains)seemto be lessvirulent than the peroxide-resistantbacilli in man. This is indicatedby reduced ability to establish disease(MITCHISON, 1963; TRIPATHY et al., 1969; DEVADATTA et al., 1970),to cause rogressivediseaseduring inadequate chemotherapyPRAMAKRISHNANet al., 1961)or to disseminatesystemically(OESTREICHER et al, 1955). Nevertheless, the peroxide-susceptiblebacilli are capableof causingdiseasethat is just assevereasthat causedby the peroxide-resistantstrains(DEVADATTA et al., 1961;RAMAKRISHNANet al., 1962).Thus the roleof peroxidein manmight be moremodestthan in guinea-pigsbut, also, infected individuals might differ widely in their ability to mount or sustaina peroxide-dependentimmune response.Comparison of peroxide release from guinea-pig and human alveolarmacrophagessuggeststhat the speciesdiffer rather little in this respect, at least with phorbol myristate acetateas stimulus. This agent elicited on average80 mnol per lo6 cells per hour from guineapig alveolarmacrophages (JACKETTet al., 1981b) and 35 mnol per lo6 per hour from alveolarmacrophages from human subjectswith a history of recent lower respiratory tract infections(GREENING et al., 1981& unpublished.Nevertheless,it is unlikely that peroxide providesa comprehensiveexplanationof immunity to tuberculosis,evenin guinea-pigs.An antimicrobial systemasoxygen-dependentashydrogen peroxide production is probably ineffective within the closedgranulomatathat developlater in tuberculosis. Indeed, developmentof infection is impaired under from
normal
and BCG-vaccinated
Normalguinea-pigs Bacilli per macrophage
Stimuh.ts
DeadBCG Live BCG
Opsonized Opsonized Values for differences
;:;
guinea
pigs in response
Vaccinatedguinea-pigs
Hydrogen peroxide released (fmole/bacilludhour) 0.16
Bacilli per macrophage 5.8
0.14
6.6
Hydrogen-peroxide released (fmolelbacillusihour) 0.32 0.12
dead BCG 6.8 0.14 12.2 0.11 live BCG 6.1 0.15 12.4 0.24 peroxide release from phagocytosing macrophages were corrected for release from control macrophages and in bacillus uptake.
650
THE
MACROPHAGE
AND
MYCOBACTERIAL
INFECTIONS
Fig. 8. Electron micrographs of ultrathin-sections of mouse peritoneal macrophages showing (a) fusion of lysosomes containing heat-killed M. microti (HKM); (b) absence of lysosome fusion with phagosomes containing live M. micron’ lysosomes were labelled with electron-opaque gold particles.
(L) with phagosomcs (LM). The contents of
D.
conditions
where
oxygen
availability
B.
capacity to avoid exposurebut multipliesexclusively within lvsosomes(BROWNet al.. 1969). Attemnts have been made to understandthe mechanism‘of fusion inhibition by tubercle bacilli in the reasonable expectation that the bacillusbenefits from avoiding contact with lysosomalcontents(HART, 1982).Three distinct, but possiblycomplementary,mechanisms of fusion Inhibition have been proposed: bacterial releaseof polyanioniccell wall components(GORENet al., 1976),releaseof ammonia(GORDONet al., 1980) and either releaseof cyclic AMP or stimulation of macrophagesynthesisof cyclic AMP (LOWRIEet al.,
is restricted
(DIJBOS,1955; CHANDLERet al., 1965) and it is unlikely that this can be accountedfor entirely by the oxygen dependenceof tubercle bacillusmetabolism. Attention has beenfocussedon macrophagelysosomes as an alternative
to peroxide
since the demon-
stration by ARMSTRONG & HART (1971) that living tubercle bacilli have the ability to inhibit (or fail to induce) phagosome-lysosome fusion whereasdead onesdo not. This property is shared by M.’ microti (Fig. 8). M. Zeprue also avoids exposure to lysosomal
contentsbut doesso by escapinginto the cytoplasm (LEVYet al., 1975) whereas M. lepraemurium showsno Table
II-Effect
of modification
of phagosome-lysosome
Normal
fusion response Fusion Non-fusion
Organism M. M.
lepraenwium tuberculosis
microti Non-fusion *Table modified from HART (1982).
M.
20
651
LOWRIE
1975, 1979b, 1980). In cell-free preparations, fusion
Agent added Polyanion Polyanions Antibody coating Lipophilic amines Antibody coating
on mycobacterial
phago-
fate*
Modified fusion response Fusion inhibited Fusion inhibited Fusion promoted Fusion promoted Fusion promoted
Modified fate Multiplication increased Multiplication increased Unaltered Killing or stasis Unaltered or stasis
Catalase
kacetyl-P-glucosaminidase :““‘: : .
10
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.05
1.10
1.15
1.20
1.25
1.30
1.35
Density Fig. 9. Selective phagolysosome formation among lysosome subpopulations in rabbit alveolar macrophages infected with BCG: demonstration by analytical subcellular fractionation on sucrose density gradients then assay of gradient fractions for BCG colony-forming units and macrophage organ&e marker enzyme activities. Macrophages were obtained by broncho-alveolar lavage of granulomatous lungs from rabbits intravenously vaccinated with BCG in Freund’s adjuvant 3 weeks earlier. The cells were disrupted and fractionated after they had adhered overnight as monolayers and then phagocytosed BCG (--I or antibody-coated BCG ( -). Controls (. .) were macrophages without BCG and BCG without macrophages.
652
THE
MACROPHAGE
AND
MYCOBACTERIAL
some-lysosome fusion is resistantto inhibition by at leasttwo of theseagents(ammoniaand poly-glutamic acid)sothat a simpleeffect on phagosome or lysosome membranes seems to be unlikely (GEISOW et al., 1982; P.D’A. Hart, unpublished). The significanceof avoidanceof lysosomecontents by fusioninhibition remainsunknown for a variety of reasons.Notable amongtheseare: (i) it hasnot been establishedif or to what degreefusion is inhibited in viva; (ii) when fusion inhibitors enhanceinfection in vitro or in viva it is not certain that phagolysosome formation is the only host defencesysteminhibited and (iii) fusion promotion has inconsistently antimycobacterialeffects(Table II); (iv) althoughvarious anti-mycobacterialsubstancescan be found in macrophagelysosomes (GINSBURG, 1979; LOWFSE, 1983) their significancein resistanceto infection is not established;(v) an associatonbetweenvirulence and high content of fusion-inhibitory polyanionic lipids (GRANGE et al.. 1978)hasbeenweakenedbv studies of morestrainsbfM. iuberculosis (GOREN et ai., 1982); (vi) subpopulationsof lysosomesexist with different enzymecontents(LOWRIEet al., 1979~)and microscopical assessments of fusion inhibition have not allowed for the possibility of selective effects on different subpopulations. Fig. 9 showsthe results of an experiment using analytical subcellularfractionation to test whether lysosomesubpopulationsdiffer in their tendency to fusewith phagosomes containingBCG bacilli (D. B. Lowrie & P. W. Andrew, unpublished).In this type of experiment macrophagesare gently disrupted beforeor after infection andthe subcellularorganelles are centrifugedinto sucrosedensity gradientssothat each organellecomes to rest at a position in the gradientswhere the densitiesof the organelleand sucroseare equal. Each organellehasa characteristic and often distinctive density distribution profile and this can be revealedby assessing the distribution of a marker enzymeactivity known to be localizedto that organelle.Since tubercle bacilli and lysosomesshow peaksat different densitiesin control gradients, the formation of phagolysosomes in infected cellsis most simply revealedby the appearanceof a peak of both lysosomalenzyme activity and bacilli at a new commondensity. Acid phosphatase activity (Fig. 9) moved to the new peak of bacilli and did so to the sameextent with opsonizedor non-opsonizedbacilli; lysozyme and N-acetyl-B-glucosaminidase activities shifted more substantiallyto this position with the opsonizedthan with the unopsonizedbacilli; cathepsin D activity did not shift with either opsonizedor unopsonizedbacilli. Since the addition of bacilli to cell homogenates before centrifugation did not affect any distributions, these results suggest that the different lysosomesubpopulationsindicated by the different enzymeactivities (LOYVRIEet al., 1979~)did indeedhave different tendenciesto fuse with phagosomescontainingBCGandonly somewereinfhtenced by opsonization. Since macrophagescontain no myeloperoxidaseit wasalsonotablethat there wasno transferof catalaseto the positionof the bacilli. Either peroxidaseor catalasecan enhancethe toxicity of hydrogen peroxide for tubercle bacilli under appropriate circumstances(JACKETT et al., 1978b, 1980)but it is now doubtful that either is normally in contact with the bacilli in macrophages.
INFECTIONS
I
0
I
I
I
2
4
6 Days
I
a
1
I
10
12
Fig. 10. Inhibition of multiplication of M. microri in mouse peritoneal macrophagesby coa&g with antibody before phagocytosis. Monolayers were maintained for 1 week in serum-rich medium beforeinfection with opsonized (0) or non-opsonized (0) bacilli. No antibiotics were present at any stage; extracellular bacilli were removed by frequent rinsing.
Inhibition of multiplication of tubercle bacilli by macrophages might be at leastasimportant askilling in host defenceagainsttuberculosis.This is perhaps best illustrated in the bacteriostasisthat can be a prominent feature of acquired immunity against tuberculosisin mice (HART & REES, 1960; REES & HART, 1961). An indication that exposure to lysosomecontentscould significantlycontribute to macrophage-inducedbacteriostasiswas obtained when it wasfound that promotion of phagolysosome formation by antibody-coatingM. microti beforephagocytosiswould sometimesresult in bacteriostasis (Fig. 10). The basisof the variability of this finding is not known but it may be relevant that the mostfrequently reportedconsequence for tubercle bacilli of immunological activation of macrophageswas bacteriostasis. It is possiblethat different degreesof immunological activation result in different degreesof exposureto some growth-inhibitory lysosomal component. Lysozyme (OSHIMA et al., 1961)and the antibacterial factor describedby SHARMA& MIDDLEBROOK (1977) might be relevant in this context. The development of immunopharmacological agents that promote phagosome-lysosome fusion might be therapeuticallyusefulif either tuberculostasisis a usefulcomponentof host defenceor fusion in immunologicallyactivated cellsis lethal to the tubercle bacillusor if delivery or action of tuberculocidal drugs is thereby enhanced. However, it should be borne in mind that (i) such tuberculostasismight contribute to the developmentof persistentstatesof infection and thereby even further enhancethe risk of future reactivation and (ii) non-multiplying tubercle bacilli arelikely to be moreresistantto at leastsomeof the major antitubercular chemotherapeuticagents. Indeed, on thesetwo groundsa casemight be made for the oppositeapproach:that of trying to develop agents that prevent tuberculostasiswhile leaving tuberculocidal host defences intact. Such agents might reasonablybe usedin conjunction with potent tuberculocidal drugs.
D.
B.
Whilst it is now clearthat enhancementor repair of macrophagefunction should be the goal of immunopharmacologyin tuberculosis,the right targets for immunopharmacological agentsare aslikely to be found among the complex amplification and feedbackloopsin the cellularimmunesystemthat modify macrophagefunction asin the macrophageitself. As these targets becomeidentified and understood in biochemicalterms it will becomepossible,at leastin principle, to tailor agents for the precise function required. Other, usually lessdirect and even serendipitous, approachesto immunopharmacologyhave sofar yieldedagentsthat have beenrather disappointing (DREWS, 1982; GATNER & ANDERSON, 1982).
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