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RP-TB7,therculosis: Pathogenesis. Protection, and Control

'^'^^AmericarTsMiMy for Microbiology. Washington. DC 20005

Chapter 24

Immune Mechanisms of Protection
John Chan and Stefan H. E. Kaufmann

Acquired resistance against tuberculosis oping active disease. Any later imbalance
paradigmatically rests on cell-mediated of the immune system will promote micro­
immunity, with the major factors being bial reemergence and ultimately result in
mononuclear phagocytes (MP) and T lym­ clinical disease. This chapter focuses on
phocytes. While the former cells act as the immune mechanisms involved in pro­
the principal effectors, the latter ones tective immunity against tuberculosis,
serve as the predominant inducers of pro­ with the awareness that in most cases the
tection. At the same time, however, MP immune response activated during infec­
provide the preferred biotype for the eti- tion with M. tuberculosis may be remarkologic agent "of tuberculosis, Mycobacte- ably powerful yet insufficient.

rium tuberculosis, and hence play a dual
role in tuberculosis, promoting not only
protection against the disease but also
survival of the pathogen. Similarly, T
cells not only are indispensable for pro­
tective immunity but also contribute to
pathogenesis. A coordinated cross-talk
between MP and T cells, therefore, is
essential for optimum protection. Such
coordination is best achieved in the gran­
ulomatous lesion, which provides the tis­
sue site for defense against tuberculosis.
Even in the face of coordinated T-cell-MP
interactions, full eradication of the patho­
gen is frequently not achieved, so that the
individual remains infected without devel-

John Chan • Department of Medicine, Montefiore
Medical Center, Albert Einstein College of Medicine,
Bronx, New York 10467.
Stefan H. E. Kauf­
mann • Department of Immunology, University of
Ulm, Albert-Einstein-Allee 11, D-89070 Ulm, Ger­

A HISTORICAL NOTE
In his epoch-making description of the
etiologic agent of tuberculosis in 1882, R.
Koch noted the intracellular location of
M. tuberculosis within giant cells (end­
stage-differentiated MP) in granulomatous
lesions (Koch, 1882). In his endeavor to
develop an active vaccination
protocol for
.
"
’
’ t
treating tuberculosis, Koch jound
after administration of glycerin extracts of
M. tuberculosis culture supernatants, the
lesions of tuberculous guinea pigs became
heavily necrotized (Koch, 1890). In these
necrotic reactions, many microorganisms
died because of nutrient and oxygen defi­
ciencies. Although Koch had already
noted that M. tuberculosis organisms can
be disseminated from such neciotizing
lesions to other tissue sites, he underrated

many.
389

390

f
I'

I

iL>r

IF

i
Mb

bF

Chan and Kaufmann

the detrimental
consequences of this effeet, which soon brought therapeutic vaccination with fubercultn to an end. E
Metchnikoff, a
contemporary but not a
close friend of Koch,
realize the importancewas
of the
MP first
i- to fully'
tenal immunity in general
i in
• antibacagainst tuberculosis in n t
i ‘n defense
nikoff, 1905) Thp
part,cular (Metch-

IN VITRO ACTIVATION OF

A
macrophage
antimycobacterial functions
Evidence has long existed tk .

ni urin,
■acteria
(Lurie
1969). Eartransferring strated that these cells'3 wh”*10"ics
68 dem°nprotection against tnv.r.
□ . ------ “*<
activated
.
b„.e. vitro by supernatants ’ twhen actlv
ated in
from immune animals stimulated lymphocytes f |‘mHmUnological|y
prompted numerous scientists
•
to attempt grees of antimycobacteri’ ,had. vanous dePassive vaccination L
agamst tuberculosis son and Youmans tin
(Pattera^e/tha'uch5:^-’ however ■----------re.’ Youmans, 1973^™
Klun and'
Youmans, 1973a, b;
Illh
,
.
—
197
^b;MuroakaetS
protection against t'-^ a'led ‘O transfer
- oumans,
access in this direction00 °S,SkThC firSt m a’” 1976)’ Soon’ hyc1976a, b; Turcotte
this directior —
hydrogen peroxide
1909 to 1910 by H Helmholtt °^ned in
One of the reactive c-~
- -• Helmholtz and O.
independently
succeed h °'Bai'' d'
(R0I) SCncra!ed -byoxygen
r- interme/ macrophages
tively transferring delaved
dr"1 ad°P‘ dUnn1f ‘he oxidati''e burst (Sbart
- — ra and KarS'tivity to tuberen
hypersen- n°vsky, 1959; Iyer et al ~
1961; Klebanoff,
(containing leukocytes^’111 Wh°,e b,°od 198?)’ WaS identified as the
.^“bacteriocidal effeas'of MP

Chase In 19« (aase7«!0MdL

E_ Suter independently found^hm nr

M'

'■ 1981). This finding
renting the significaVe "7 Ro?T h°n'
defense against
,
n host
gamma interferon (IF?/
°^s- Later,
the kev on 3
Was found to be

■rigged & TiVatin8 age"‘

-erSa-- r-d

1«3; Lune, 1964). AhhoCgh th^e sTuder'

I
f,

immune
contest Xnalive,n.VeSti8at°rS did no'
realized that "t
stra'cgies when they

lha'aXtion'oT

Macka"^ tolhow

rophag {u e"o°nfsa"'""^bacteria! mac
Phocytes (MaX aTd"^'^ by 'ymThat this activation is afftd a"!0"' 1967)'

murine macrophages^tlT't™1 effeC‘S °f
Hesch and Kaufmann ^7) 7 at\I986;
better-defined system (compie^oXs!

nlated lymp™esr^Obhain1,ed fr°m S,in”
'be antimycobacterial effects 'of6^™'”6
Phages. Recent remarkable advancesmacro3 made
ductionXut Characteriza(ion, and probinant DNA TecX0710^68 by recomsimilar in vitro exner 85, haVe facilita'ed
explore the potential oVih"0" deS'8ned '0
molecules in host Hof
hCSe ,nterest,ng
culosis. Thus tumo/1156 a8ainst M-

Chapter 24

TION OF
GE
L FUNCTIONS

ted that murine
'timycobacterial
systems (Lurie
ess, 1969). Earratories demonien activated in
mmunologically
ad various detetivity (Patter0; Klun and
and Youmans,
6a, b; Turcotte
ogen peroxide
xygen intermemacrophages
barra and Kar61; Klebanoff,
molecule that
effects of MP
• This finding
•h debate conROI in host
dosis. Later,
is found to be
ng agent that
al effects of
et al., 1986;
furnishing a
ed to one us­
'd from stim1 to examine
• of macro­
dances made
■n, and pros by recome facilitated
designed to
interesting
st M. tuberactor alpha
when used
to induce
ine macrovaufmann.

1990a). TNF-a also appears to play a criti­
cal role in the control of BCG infection in
vivo, although its direct effect on the antimycobactcrial capacity of macrophages
has not been addressed in this model. Nev­
ertheless, when TNF-a-specific monoclo­
nal antibodies were used to probe the sig­
nificance of this cytokine in defense against
mycobacteria, deficient TNF-a resulted in
poor granuloma formation and dissemi­
nated BCG infection in mice (Kindler el al.,
1989). The significance of TNF-a in granu­
loma formation has been demonstrated in
other infectious disease models (Chensue et
al., 1989; Amiri et al., 1992). More impor­
tantly, preliminary studies suggest that anti-TNF-a antibodies markedly exacerbate
disease progression in murine experimental
tuberculosis (Flynn et al., personal commu­
nication).
Other cytokines have been implicated in
macrophage defense against M. tuberculo­
sis, although their roles are not as well
established as those of IFN-7 and TNF-a.
In vitro, interlcukin-4 (IL-4) and IL-6 have
the ability to induce macrophage antimycobacterial activity (Kaufmann et al., 1989;
Flesch and Kaufmann, 1990a, b) by mech­
anisms presently undefined. Infection of
the human myelomonocytic cell line THP-1
with M. tuberculosis enhances production
of IL-6 (Friedland et al., 1993) compared to
that in cells infected with Toxoplasma gon­
dii, an intracellular protozoan known to
elicit little inflammatory response even in
immunocompetent patients. In the murine
system, BCG or its subcellular components
are capable of inducing production of IL-6
by splenocytes (Huygcn et al., 1991). The
antimycobacterial effects of IL-4 and IL-6
(Flesch and Kaufmann, 1990a, b) in the in
vitro macrophage system are seen only
when these cytokines are added to macro­
phage cultures after, but not before, the
establishment of BCG infection. This phe­
nomenon sharply contrasts with the ability
of IFN-'y to induce antimycobacterial activ­
ity in macrophages, which is markedly

Immune Mechanisms of Protection

391

blunted if it is given after initiation of infec­
tion (Flesch and Kaufmann, 1990a). The
mechanism and the significance of this ob­
servation are currently obscure, but it illus­
trates well the complexity of the interaction
between macrophages, cytokines, and the
organisms as well as the limitations of ex­
isting in vitro systems in dissecting the
likely complex cytokine network involved
during tuberculous infection. Thus, it is
known that THP-1 cells produce IL-8 in
response to M. tuberculosis infection in
vitro, but the role of this cytokine in host
defense in tuberculosis is completely un­
known (Friedland et al., 1992, 1993). Nev­
ertheless, it has been postulated that IL-8
plays a role in granuloma formation by
virtue of its ability to act as a chemotactic
agent for T cells (Larsen et al., 1989; Fried­
land et al., 1992). IL-1 (Kobayashi et al.,
1985; Dunn et al., 1988; Kasahara et al.,
1988), IL-2 (Mathew et al., 1990; Cheever
et al., 1992), IL-4 (Mclnnes and Rennick,
1988; Chensue et al., 1992), and IFN-y
(Squires et al., 1989; Chensue et al., 1992)
may similarly contribute to resistance
against M. tuberculosis, since these cyto­
kines have been implicated in granuloma­
tous reactions in various in vitro systems,
including a murine schistosomiasis model.
Recently, IL-10 (Bermudez and Champsi,
1993) and transforming growth factor betal
(TGF-pi) (Denis and Ghadirian, 1991; Ber­
mudez, 1993) have been shown to be asso­
ciated with diminution of macrophage an­
timycobacterial effect in vitro and with
disease exacerbation in mice infected with
M. avium. In contrast, preliminary studies
(Flynn and Bloom, personal communi­
cation) indicate that administration of re­
combinant IL-12, a recently characterized
heterodimeric glycoprotein produced by
various immune cells including macro­
phages (D'Andrea et al., 1992; Schoenhaut
et al., 1992; Gazzinelli et al., 1993), may
confer resistance to tuberculosis in mice.
IL-12 has recently been shown to play an
important role in resistance to Leishmania

1
o

392

Chan and Kaufmann

^ajor, T. gondii, and Listeria monocytoge­
nes (Gazzinclli ct al., 1993; Heinzel et al
1993; Locksley, 1993; Tripp et al., 1993)'
I he events triggered by IL-12 help identify
natural killer (NK) cells as a critical cellular
component in defense against M. tubercu­
losis By virtue of their ability to produce

.’Ln response to ,L-12 (Kobayashi et
989; Wolf et al., 1991), NK cells can
rapidly activate macrophages to express
microbicidal functions during the early
nommmune” phase of tuberculous infec­
tion, before the expansion and differentia­
tion ol specific T lymphocytes. As cyto­
kines are being examined in experimental
mycobacterial infection, it is becoming
antimycobacterial effector
clear that these molecules interact dynami­ I UNCTIONS
OF MACROPHAGES: HOW
cally to form a highly coordinated network
does m. tuberculosis survive?
that is configured by both host- and patho­
The mononuclear phagocyte constitutes a
gen-specific factors, which together influ­
ence disease outcome and progression.
potent antimicrobial component of cell-me­
Compared to the murine system, much diated immunity. The precise mechanisms
ess is known about the activation of an- bV which these cells mediate killing or
timycobacterial activity in human macro­ inhibition of bacterial pathogens are, how­
phages. While it is clear that IFN-7 has the ever,
€
not clearly understood. Nonetheless
capability to induce significant antimyco- m this section, some of the best-char­
bactenal activity in murine macrophages, acterized antimicrobial effector functions
its role m the human system is unsettled.’ of macrophages—phagosome-lysosome fu­
Thus, reports of the effect of IFN-7-treated sion, generation of ROI by the oxidative
human macrophages on the replication of burst, and production of reactive nitrogen
M. tuberculosis ranges from being inhibi­ intermediates (RN1) via the L-arginine-detory (Rook et al., 1986) to enhancing (Dou- pendent cytotoxic pathway—will be dis­
vas et al., 1985). This inconsistency had cussed in the context of tuberculous infec­
cast considerable doubts on the antimyco- tion together with the possible evasion
bactenal capability of human mononuclear mechanisms employed by the tubercle ba­
phagocytes until the demonstration that cillus to escape killing by activated macro­
1,25-dihydroxy vitamin D3 |1,25-(OH)1DJ, phages (Fig. 1).
alone or in combination with IFN--/and
TNF-ot, was able to activate macrophages
Phagosome-Lysosome Fusion
to inhibit and/or kill M. tuberculosis in the
The lysosome is a highly complex or­
human system (Crowlc et al., 1987; Rook,
ganelle
containing numerous enzymes
1988; Denis, 1991b). Interestingly, IFN-7
within
its
own limiting membrane that are
stimulates human (Adams and Gacad, 1985capable
of
degrading a whole range of macKoetfler et al., 1985; Reichel et al., 1987)
romolecules
but not murine (Rook, J1990)
---- macrophages tiaux [1966], (reviewed in de Duve and WatBainton
and Kornfeld
to produce 1,25-(OH)2D3. probably via
To provide optimal conditions for
inducuon of 25(OH)D
3
-l
a
.hydroxyla
S
e
25( OH )D3-la-hydroxy la:
the functioning of these degradative en­
the enzyme that converts 25(OH)D, to the
zymes, the intralysosomal milieu is main-

1

fl

4'

if

JI

I?
),

k
if

1,25(OH)2D3 to affect antimycobacterial ac
tivity in the murine system. This difference
m 1,25(OH)2D3 metabolism between mu­
rine and human macrophages should serve
as a reminder that species variations exist
and a caution against the occasional readi­
ness with which cross-species extrapola­
tions of experimental results are made The
value of existing in vitro and in vivo murine
models in understanding tuberculosis must
however, not be understated.

Chapter 24

dihydroxylated
he inability of
'cobacterial acThis difference
between mus should serve
ariations exist
casional readiies extrapolaare made. The
n vivo murine
rculosis must,

XphI 0
Arginine

Op X

S? J/

NO

2FFECTOR
AGES: HOW
SURVIVE?
■ constitutes a
■nt of cell-memechanisms
te killing or
ns are, howNonetheless,
e best-charor functions
ysosome fuie oxidative
ive nitrogen
arginine-dewill be disulous infecble evasion
ubercle ba­
ited macro­

sion

implex orenzymes
ne that are
ge of maceand WatI Kornfeld
Jitions for
iative enJ is main-

nadph\
h2oJ
OH XSoq

Citrulline

—

\

7

J
NADP

NH®

®?tL /
10

tained at a relatively acidic state (pH ~5) by
an ATP-dependent proton pump (Ohkuma
and Poole, 1978; Ohkuma el al., 1982). It is
generally accepted that certain microorgan­
isms, sequestered within the phagosome
upon ingestion by phagocytic cells includ­
ing macrophages, are subject to degrada­
tion by the various lysosomal digestive en­
zymes transferred into this subceliular
compartment as a result of phagolysosomal
fusion (Cohn, 1963). This fusion process, a
highly regulated event, most likely consti­
tutes a significant antimicrobial mechanism
of phagocytes. Examination of the interac­
tion between isolopically labeled bacteria
and macrophages, using the generation of
acid-soluble radioactive materials as an in­
dicator of degradation, suggests that certain
organisms are degraded extensively within

Immune Mechanisms of Protection

393

Figure 1. Antituberculous macrophage activities and
evasion mechanisms. Accumulating evidence suggests
that M. tuberculosis enters macrophages via specific
binding to cell surface molecules of phagocytes. It has
been reported that (he tubercle bacillus can bind
directly to the mannoase receptor via the cell wallassociated. mannosylated glycolipid LAM (1) or indi­
rectly via complement receptors of the integrin family
(CR1, CR3) or Fc receptors (2). Phagocytosis (3),
triggered by engaging certain cell surface molecules
such as the Fc receptor, stimulates the production of
RO1 via activation of the oxidative burst (4). Experi­
mental data indicate that M. tuberculosis can interfere
with the toxic effect of RO1 by various mechanisms.
First, various mycobacterial compounds including gly­
colipids (GL). sulfatides (ST), and LAM can downregulatc the oxidative cytotoxic mechanism (5; see text
for details). Second, uptake via CR1 bypasses activa­
tion of the respiratory burst. Cytokine-activated mac­
rophages produce RNI that, at least in the mouse
system, mediate potent antimycobacterial activity (6).
The acidic condition of the phagolysosomal vacuole
can be conducive to the toxic effect of RNI (7).
However, NH/ production by M. tuberculosis may
attenuate the potency of the L-argininc-dependent
antimycobacterial mechanism and that of lysosomal
enzymes (8), which operate best at an acidic pH. In
addition, mycobacterial products such as sulfatides
and NH/ may interfere with phagolysosomal fusion
(9). Finally, the tubercle bacillus may evade the highly
toxic environment by escaping into the cytoplasm via
the production of hemolysin (10).

2 h after having been phagocytized (Cohn,
1963). Also, electron microscopic studies
indicate that the cell wall of Bacillus subtilis
is degraded extensively within 30 min after
phagocytosis by polymorphonuclear leuko­
cytes (Cohn, 1963). How, then, does M.
tuberculosis survive the hostile environ­
ment of phagolysosomes?
M. tuberculosis has the ability to produce
ammonia in abundance (Gordon ct al.,
1980). This volatile weak base accumulates
in M. tuberculosis culture filtrates in con­
centrations of up to 20 mM and is thought to
be responsible for the inhibitory effect of
culture supernatants of virulent mycobac­
teria on phagolysosome fusion (Gordon ct
al., 1980). In addition, ammonium chloride
(NH4CI) has been shown to affect the sal­
tatory movement of lysosomes (D'Arcy

p

I

ii*

r

IIIlIll
i

394

Chan and Kaufmann

Hart et al., 1983) and to alkalinize the
fects of macrophages. This issue could perintralysosomal compartment (D’Arcy Hart
haps be addressed more rigorously and
et al., 1983). Thus, by virtue of its ability to
definitively by direct i
immunohistochemical
produce a significant amount of ammonia,
labeling of vacuolar membranes enclosthe tubercle bacillus can potentially evade
mg intracellular M. tuberculosis with
the toxic environment within the lysosomal
antibodies specific tto lysosomal glycoprovacuole by (i) inhibiting phagosome-lyso ­
teins (Joiner et al.,
' , ]1990) or by using the
some fusion and (ii) diminishing the po­
"trap-resistant” ionic
---■j i._
impermeant fluors
tency of the intralysosomal enzymes via
(lucifer yellow, lissamine
alkalmization. This latter attribute of rais­
Ir , ,
- rhodamine, and
sulforhodamine) as
as alternative
alternative lysosomal
lysosomal
ing intralysosomal pH might also be protec­
(Goren et al.’, —
1987a, b). JFinally,
it
•
...
Hid
tive against the RNI cytotoxic mechanism markers
IS
likely
(hat
like
is
hkely
that
virulent
tubercle
bacilli
of macrophages (see below).
certain intracellular pathogens, including
Another
mycobacterial
i
.
*
UiUULIH
product
thought rickettsiae (Winkler,
^aVe.lhe abllity t0 inhibit phagolysosoa"d shi8cnac (Sansonetti
15 the
‘he sulfatides
SU'fatides (Goren et
« al
al.,
. 9Z.6b’’ dtr,vatives of multiacylated treha- et al., 1986), evade killing by escaping from
lose 2-sulfate,. a lysosomotropic polyan- phagocytic vacuoles into the cytoplasm (for
ionic
■ •■‘/Cw8lyCOhpid Produced by M. tuberculo- a review, see Falkow et al. [1992]). Hemo­
lytic activities capable of lysing vacuolar
(Middlebrook et al., 1959; Goren eta?
membranes
are thought to be the common
1976a). Because of the ability of certain
virulent
determinant
that enables success­
polyanionic compounds to entrap comful parasitization of the cytoplasm (Falkow
moniy used lysosomal markers
-------- 3 employed et al., 1992). Indeed, the translocation of
to •'
' phagolysosome
•
study
fusion, artifactual
"inhibition” of this process can occur and M. tuberculosis from within phagocytic
vacuoles into the cytoplasmic compartment
has spawned much
.---------- controversy (Goren et
has been reported (Myrvik et al 1984al., io«7
iy«/a, b). These entrapment phenomena could be secondary to the formation of McDonough et al., 1993). These observa­
gelatinous, •sluggishly moving hydrocol- tions are reinforced by the presence of a
hemolytic activity in the tubercle bacillus
loids that physically retain lysosomalI mark­
King et al., 1993). Also, the cytoplasmic
ers or to ionic interaction with cationic
location
made possible by this potential
makers such as acridine orange. Although
evasion
mechanism
could, in theory, facil­
su fatides do not form hydrocolloids, the
itate
the
routing
of
mycobacterial compo­
polyanionic nature of these glycolipids
nents into the major histocompatibility
poses questions concerning their ability to
±S„V (.^HC 1) !’athway of
presen

^a,‘bcXXX'of^V';1'-; “
,tatiOn’ ,hUS
“ - ------------------------- in part the

these ^-(ipidsXk ^

appears to be warranted. Regardless of the
chemical components of the tubercle bacil­
lus that contribute to the inhibition of
Phagolysosomal fusion, this phenomenon
controversy notwithstanding) has been extensively studied (Armstrong and D’Arcv
Hart, 1971, 1975; Goren et al . W6b Myr
vik et al., 1984; D’Arcy Hart et al '1987)
and is certainly a mechanism bv which
y otoxic et

importance of MHC I molecules and CD8 +
1 cells in defense against M. tuberculosis
(Kaufmann, 1988; Flynn et al., 1992).

The Respiratory Burst
That ROI nlav « • n
defcn ‘ R01V s'8nificant ro|e in host
fied hv -h*8?1"' m'Cr°beS is best e“mPbl',a'cluent infectious complication
xperienced by chronic granulomatous dis-

[1988]), whose phagocytes cannot mount an

1

Chapter 24

issue could perngorously and
lohistochemical
branes encloslosis with anti0131 glycopror bV using the
-rmeant fluors
lodamine, and
,ve lysosomal
• b). Finally, it
le bacilli, like
ns, including
listeriae (Bieae (Sansonetti
escaping from
ytoplasm (for
992]). Hemo­
sing vacuolar
the common
bles successasm (Falkow
islocation of
phagocytic
:ompartment
•t al., 1984;
-se observaesence of a
cle bacillus
-ytoplasmic
is potential
>eory, facilial compo>mpatibility
gen presen­
in part the
and CD8 +
^berculosis
992).

»e in host
exempliiplication
itous disist et al.
mount an

oxidative burst (Sbarra and Karnovsky,
1959; Iyer et al., 1961; Klebanoff, 1980).
The significance of these toxic oxygen spe­
cies in defense against M. tuberculosis,
however, remains controversial. Since the
report that H2O2 produced by lymphokineactivated murine macrophages kills M. mi­
croti (Walker and Lowrie, 1981), much ef­
fort has been focused on testing the role of
the oxygen radical-dependent killing mech­
anism in defense against M. tuberculosis.
Such effort, however, provided evidence
indicating that oxygen radicals may not be
sufficient to inhibit and/or kill M. tubercu­
losis (Flesch and Kaufmann, 1987, 1988;
Chan et al., 1992). The validity of these
findings has been reinforced by the demon­
stration of evasion mechanisms employed
by the tubercle bacillus to elude the toxic
effect of ROI. Of these mechanisms, those
that are mediated by mycobacterial compo­
nents lipoarabinomannan (LAM) and phe­
nolicglycolipid I (PGL-1) are among the
best studied and characterized (for reviews,
see Brennan [1989] and Brennan et al.
[1990]).
LAM, a major cell wall-associated, phos­
phatidylinositol-anchored complex lipo­
polysaccharide, is produced by M. tubercu­
losis in large amounts (15 mg/g of bacteria)
(Hunter et al., 1986; Hunter and Brennan,
1991). Immunogold staining has demon­
strated that LAM exists in a capsular
sheath encasing M. tuberculosis (Hunter
and Brennan, 1991). This strategic location
places LAM at the frontline of attacks
directed by the various antimicrobial mech­
anisms of macrophages. It has now been
shown that LAM can incapacitate the oxy­
gen radical-dependent antimicrobial mech­
anism at at least two levels: (i) studies using
electron spin resonance spectroscopy and
spin-trapping have shown that LAM is an
effective ROI scavenger (Chan et al., 1991);
and (ii) LAM can downregulatc the oxida­
tive burst by inhibiting protein kinase C
(Chan et al., 1991), an enzyme that plays an
important role in activation of the oxidative

•

Immune Mechanisms of Protection

395

burst in phagocytic cells (Gennaro et al.,
1985; Pontyremoli et al., 1986; Wilson et
al., 1986; Gavioli et al., 1987). In addition,
since IFN-7 is a major factor for macro­
phage activation (Hamilton et al., 1984;
Hamilton and Adams, 1987; Fan et al.,
1988) and has the ability to enhance ROI
production by phagocytic cells, it is possi­
ble that LAM, by virtue of its ability to
inhibit transcriptional activation of IFN-7inducible genes (Chan et al., 1991), is able
to block the expression of an as yet uniden­
tified factor(s) inducible by this cytokine
that is required for the oxidative burst.
These results are in keeping with the find­
ings that mouse peritoneal macrophages
treated with LAM or infected with M. lep­
rae (a LAM-producing pathogenic myco­
bacterium) are not responsive to IFN-7
activation as assessed by microbicidal and
tumoricidal activities, O2- production, and
surface la antigen expression (Sibley et al.,
1988; Sibley and Krahenbuhl, 1988) and
may partially explain the inability of IFN7-stimulated macrophages from both hu­
mans and mice to effectively kill M. tuber­
culosis in vitro (Rook et al., 1986; Flesch
and Kaufmann, 1987).
Other mycobacterial components that in­
terfere with the oxygen radical-dependent
antimicrobial mechanism of macrophages
arc PGL-I and the sulfatides. PGL-I is an
oligoglycosylphenolic phthiocerol diester
with its species-specific trisaccharide moi­
ety glycosidically linked to a phenyl group
that in turn is attached to the branched
glycolic chain, phthiocerol; two hydroxyl
functions of the phthiocerol are estcrificd
by mcthyl-branched fatty acids (mycocerosates) (Hunter and Brennan, 1981; Hunter
et al., 1982). Although universally distrib­
uted among M. leprae, the expression of
PGL-I in the various strains of M. tubercu­
losis is much restricted (Daffe et al., 1987;
Brennan, 1989; Brennan et al., 1990). In
contrast, the sulfatides, derivatives of mul­
tiacylated trehalose 2-sulfate (Middlebrook
et al., 1959; Goren et al., 1976a), are widely

396

Chan and Kaufmann

expressed among different strains of M
tuberculosis (Middlebrook et al
1959’
Goren et al., 1974, 1976a). Because of its
restricted distribution among tuberculous
isolates, the significance of PGL-1 in the
pathogenesis of tuberculosis remains to be
determined. Nonetheless, both PGL-1 and
the sulfatides have the capacity to down•egulate ROI production in in vitro macro-

the tubercle bacillus does not
disease
not equal
equal disease
the host must be equally sophisticated I,
evolvmg effective defensive strategic,
against this formidable invader. It follows
then that there must exist antimicrobial
mechanisms to which the bacillus suc­
cumbs.

offTo^'p k SyStemS (Nci" and K,eba"-

Reactive Nitrogen Oxides

1

Ji

r
!
F
lK

e

f
!

Ct aL’ 1988; Vachu,a * a'-

The
L-arginine-dependent
cytotoxic
1989; Brozna et al., 1991), and PGL-I di­
rectly scavenges oxygen radicals in a cell- pathway of activated macrophages consti­
free system (Chan et al., 1989). Another tutes an important antimicrobial mecha­
against intracellular parasites (for re
mechanism by which M. tuberculosis could nism
™
evade the toxicity of ROI is to avoid bind­ views, see Nathan and Hibbs (19911 I few
ing to macrophage cell surface compo­ and Cox [1991], and Nathan [1992]). The
nents such as Fc receptors, that would cytotoxic effect of this pathway is mediated
piovoke an oxidative burst. Instead, the through nitric oxide (NO) and related RN1
tubercle bacillus parasitizes MP via com­ generated from the substrate L-arginine via
plement receptors CR1 and CR3. molecules the action of the inducible form of the enw.h .'nle!rin family whose interaction Zy?CunuuC °Xide synthase («NOS) (Nathan
with na ligand
WIth
igand does not trigger ROI produc- and Hibbs, 1991; Nathan, 1992). Recent
tion (Wright and Silverstein, 1983), in rest­ studies have demonstrated an association
between the antimycobacterial effect of cy­
ing macrophages (Schlesinger et al., 1990)
tokine-activated
murine macrophages and
I hus, as in other parasites (for reviews, see
the
activation
of
the L-arginine-dependent
sbeig [1991] and Falkow et al. [1992])
cytotoxic
pathway
(Denis, 1991b- Flesch
1a'|ClU1wm Bo^de'ella P^ssis (Reiman et
and Kaufmann, 1991; Chan et al 1992)
0), Histoplasma capsulatum (Buithus the capability of macrophages actilock and Wright, 1987), Legionella pneuva
cd by IFN-7 and Escherichia coli lipo­
mophila (Payne and Horwitz, 1987), and
polysaccharide
or TNF-a to kill and/or
19R7/Z/p/,Z/?/nSPP’ (M°SSer and Edclson,
inhibit
the
virulent
Erdman strain of M
987, Russell and Wright, 1988; Talamastuberculosis
correlates
well with RNI proRohana et al., 1990), exploitation of integnn receptors may be a common scheme of ouclion. and nitrogen oxides generated by
acid'fication of nitrite are also mycobactclnvasion among pathogenic mycobacteria
Although these in vitro data provide sub- ncidal Chan et al., 1992). Deletion analy­
mlpl,V?tbVldenCe tO SU8gest Path°genetic ses of the 5 flanking promoter sequence of
roles of the various mycobacterial glycolip­ murine iNOS indicate that IFN-y alone is
msufhcient for transcriptional activation of
ids, their in vivo significance is ppresently this gene
(Xii
' ,e el aI-’ 1993). The synergistic
undefmed and awaits rigorous genetic anal­
effect of IFN-y and TNF-a in inducing
yses. Nonetheless, it is undeniable that
mactophage antimycobacterial function via
Mycobacterium spp. are extremely well
<NI production underscores the impor­
adapted to the hostile environment of
tance
of these cytokines in defense against
Phagocytic cells, their deftness reflected by
M.
tuberculosis.
Indeed, IFN-y and IFN-y
the alarming morbidity and mortality
receptor
‘
-knockout
” mice that arc defi­
caused by tuberculosis worldwide (Murray
cient
in
mounting
an
RNI response to infec­
et al., 1990). However, since infection with
tion with the tubercle bacillus experience a

1

I

Chapter 24

report that the
to RNI varies
>93) further sigesistant myco■' such mutants
oecause of the
the mammalian
production,
on the precise
mediate antimyrgets that these
complexity of
1992). Nevcrrea of research
ificantly to the
^mistry in vivo
ns of virulent
■ly lead to new

>n of virulence
echanisms as
survival of M.
iy of iron meillus deserves
iship between
nown for dc1944; Weinset al., 1980),
i metabolism
tion are not
=nd characterng molecules
le variety of
(Nielands,
considerable
‘en reported
both in vitro
! in the pres:in hemogloences therecoli in a rat
ganisms are
icmoglobin.
>f this heme
ptaglobin, a
icmoglobin
rganic iron
t as hemo­

I

I

globin, a direct connection of iron and infec­
tion is made (Eaton et al., 1982). In human
diseases, the mortality rate of Vibrio vulnifi­
cus is markedly increased in patients suf­
fering from iron overload as a result of
conditions such as hemochromatosis and
alcoholism (Brennt et al., 1991; Bullen et al.,
1991). These experimental data thus suggest
a possible role of siderophores in bacterial
virulence.
Mycobactins, a group of iron-chelating
growth factors of mycobacteria, have been
considered a possible virulence factor of M.
tuberculosis (Snow, 1970). These hydroxamate derivatives chelate ferric ions with a
stability constant exceeding IO30 (Snow,
1970). Thus, mycobactins compete favor­
ably for chelating Fe3+ with human ferritin
and transferrin, the major iron storage and
iron-transporting proteins, respectively.
The significance of these mycobacterial
iron-binding agents in the pathogenesis of
tuberculosis, however, remains to be estab­
lished. Recently, the L-arginine-NO path­
way has been reported to participate in
posttranscriptional regulation of the ex­
pression of ferritin, transferrin receptor,
and 5-aminolevulinate synthase (a rate-lim­
iting enzyme in erythroid heme synthesis)
in macrophages (Drapier et al., 1993; Weiss
et al., 1993). It is fascinating that the very
same pathway that produces potent antimycobacterial activities in macrophages par­
ticipates also in the regulation of the metab­
olism of iron, whose availability is essential
to the optimum growth of M. tuberculosis.
Dissecting this likely complex tangle may
uncover additional roles for the NO path­
way in tuberculous infection and shed light
on the significance of iron in the pathoge­
nicity of M. tuberculosis.
DOES M. TUBERCULOSIS INVADE
CELLS OTHER THAN PROFESSIONAL
PHAGOCYTES?

There is little doubt that M. tuberculosis
has the ability to establish infection in and

Immune Mechanisms of Protection

399

replicate inside of a wide variety of mam­
malian cells in vitro (Sheppard, 1958). Yet
in infected tissues, the tubercle bacillus is
to be found only in polymorphonuclear
leukocytes and MP (Filley and Rook, 1991).
The findings by Filley and Rook that endo­
thelial cells and fibroblasts infected by M.
tuberculosis exhibit increased sensitivity to
the cytolytic effect of TNF have led to the
hypothesis that this cytokine contributes
significantly to the immunopathology of tu­
berculosis (Filley and Rook, 1991). The
enhanced susceptibility of nonphagocytic
cells to TNF upon mycobacterial infection
may also partially explain the difficulties
encountered in identifying such target cells
in vivo. It is also possible that these non­
phagocytic cells serve as a reservoir for
bacterial multiplication and thus aid in dis­
ease dissemination upon lysis by TNF. Re­
search in these areas is just beginning to
draw attention and is likely to help provide
insight into the pathogenic strategies of M.
tuberculosis. Finally, unlike the processes
of other pathogenic bacteria such as the
enteric shigellae and salmonellae and the
gram-positive listeriae (for reviews sec Isberg 11991] and Falkow et al. [1992]), the
processes of adhesion and invasion by
which M. tuberculosis enters host cells are
just beginning to be understood. M. tuber­
culosis gains entry into MP via cell surface
molecules, including the integrin family
CR1 and CR3 complement receptors (Schles­
inger et al., 1990) and the mannose receptor
(Schlesinger, 1993). Recently, M. avium
has been shown to enter macrophages via
avp3, another molecule of the integrin fam­
ily (Rao et al., 1993). Parasitization of
phagocytes via the CR1 and CR3 recep­
tors by various pathogens avoids triggering
the oxidative burst (Wright and Silverstein,
1983). Whether the same advantage is
gained by engaging the mannose receptor
or the avp3 integrin is presently unclear.
Since the cytoplasmic domain of p subunit
of integrin is coupled to the cytoskeleton
(Albclda and Buck, 1990), it is possible that

■
400

Chan and Kaufmann

S-«8to‘Onit^theCp'

r~

don by the host cell (Isbe^w^)" D

^WbaGterial invasin
(Arruda et al.
—- ■
’ y93) b,nd also to
to integrin
integrin
receptors? Comprehensj'
Jension of these adhesion and invasion events
ln advancing our --"'s is very important
pathogenicity of M. understanding of the
tuberculosis.
CONTRIBUTION OF T CEILS To

wtichtsp" ^'eCU,CS- and C“

by the MHC class I / Pep,,des Present

bacte™m.spe^rc8DTPTr<t"ShM-

have been identified eon.-

’^Phocyt

« X“F"" “

acquired resistance
11

mann, unpublished data)
P Tcells
cells car.
further divided into CD4a/T
wh
ofXcerh'8en,Cpepddes'n <h ccont<

of protecdo^ TheyredobnSr°ry mCdialors
-ust interact wither Tehsonb0
mune system tn n uc s ° the imtance.
All
T-cell
nn
Opt,nium
cells, CDS Lb TP lPn'a,,OnS
(CD4 resisT­

»

Conversely adonZ’ Pedrazz'ni « al.. 1987)
tuberculosis and BCC?'60!'011 a8a'nst "•
Ransfer of selecl^njT r,

contribute to protection Th^
T CeHs) Co,,ins
Collins.’^84;
1984; C
Orme 1987) c & (Orme and
these find
finding
T iytnphocytes has ^enlXl
'ngS' muta"‘”u : Consistent with
experiments showing that
P fi,cd b>' ?ency ■" the MHC classmice with a defimtce suffer more several.,
"" a"d scids devoid °f functionallv a.,’ H gene that are
lonally active CD4 T cells
tai M. i'«^3oXVearndy BCG6-Pferimen‘ r'h h BCG
ir control counterpart f“ daU) and M- and Kaufmann, unpubthan their
r <F'ynn C1
(Shcret al., al., unpublished
unpublished obseXaZr
oh«
^75, Jzzo and North
North, 1992)
conclusion, these exne
lnfect'°ns. In
Point to an essential role of^m
T-Cell Populations
T cells expressing an a/B-T-cell
constitute more f
P
;han 95% Of
cells in peripheral
contrast, 7/8 T ceUs organs and blood. In
are a minority at these
sites but they are more
A substantial role for CDX t
„ ■
prominent in mu
Protection against tnbrr iD8- f CC,,S ,n
cosal tissues such r •
asthejung. Forma! proof b7~ ^'"/^berculosi;
that a/p T cells are crucial r .
by several lines of ev
°S1S ’S ,ndicated
vMed3"06 against
a8ainS! ‘uberculosis0" acquired Depletion of CDS
CD8 T
T Pe
PC"
fr'menlal
mcnlal studies.
all a/p Tren's7 In 'th mUtanl m'Ce
coding the T-cell
m'Ce’ the 8ene en‘
been deleted by h"m .CePl°r P
had
(Mombaerts et al 1^?°S.^‘’'"bination
o/P-T-celi-deficient’ mice'm"’'1™8'’ these
sistant to sublethal R(-r fre a"vely reIhe first 4 weeks if r
eC"On duri"8

BCG rnartedry tc^as" :^"’
ultimately the a/P-T-cell-defidlT^’ and
CUmb t0
infection ILadel'^l^

~T'Xbodies ^SsT^

CDS T cells trarKf Jn.T,Ce- and se,ected
transfer
adoptive
against luberculo
8^(o
d°PtlVC proteclion
I’W; Muller et al 198^ o' and C
°"ins’
Collins,
drazzini et al., 1987)
°™e- 1987i peThese findings
have
becn further substantiated
■
--J recently by apnt mice in which the p2-

quiredforMHCcla it ^Se |32m is re'
‘
t mu
SUrfaCeexPression,
P2m-deficienl
mUtant m,ce are devoid of

Chapter 24

^3 T cells can be
} r cells, which
cs in the context
cs, and CD8 T
ptides presented
Products. Mycor lymphocytes
stently in experilosis (Kaufmann
off et al., 1988;
thermore, CD4
monoclonal annental infection
!osis and BCG
<ini et al., 1987).
ction against M.
ely depends on
-ells (Orme and
Consistent with
with a defiI gene that are
e CD4 T cells
fmann, unpublosis (Flynn et
) infections. In
ents strongly
2D4 T cells in
is. Consistent
-n as a result of
irus infection
uberculosis in
’8 T cells in
S is indicated
-ntal studies,
with specific
bates M. mand selected
e protection
»nd Collins,
e, 1987; Po­
ndings have
‘ntly by apn'ch the 32een deleted
32m is re­
expression,
devoid of

functionally active CD8 T cells. These mice
die rapidly from M. tuberculosis but not
from BCG infection. Impressive as these
studies are, it should be kept in mind that
p2m not only serves to stabilize MHC class
I surface expression but may also perform
other functions that could influence sur­
vival of M. tuberculosis in p2m-deficient
mice. Furthermore, mycobacterium-spe­
cific CD8 T cells have been isolated from
M. tuberculosis- and BCG-immune mice
(DeLibero et al., 1988). In contrast, such
mycobacterium-specific CD8 T cells were
rarely identified in patients suffering from
human tuberculosis (Rees cl al., 1988). CDS
T-cell lines derived from M. tuberculosisand BCG-immune mice arc MHC class I
restricted, thus raising the question of how
M. tuberculosis and BCG proteins gain
access to the MHC class I processing path­
way (DeLibero et al., 1988). Although it is
generally assumed that M. tuberculosis re­
mains in the cndosomal compartment, clear
evidence for escape of M. tuberculosis
from phagolysosomes into the cytoplasm
has been presented (Leake et al., 1984;
McDonough et al., 1993). Microbes resid­
ing in the cytoplasm could then produce
proteins that contact MHC class I mole­
cules, as has been clearly shown for Liste­
ria monocytogenes. Alternatively, it can be
assumed that during persistent replication
within the phagosome, mycobacterial pro­
teins or peptides are translocated into the
cytoplasm, where they contact the MHC
class I processing machinery. Recent evi­
dence indicates that MHC class I process­
ing can occur independently of microbial
egression into the cytoplasm (Pfeifer et al.,
1993).
Besides conventional MHC class I-rcstrictcd CDS T cells, T cells that arc appar­
ently MHC class I nonrestricted have been
described (DeLibero et al., 1988). Similar T
cells have been identified in the listeriosis
system, where these T lymphocytes are
focused on peptides containing the Nformylmethionine (A-fMet) sequence pre-

•

Immune Mechanisms of Protection

401

sented by nonconventional MHC class lb
molecules (Kaufmann et al., 1988; Kurlander et al., 1992; Pamer et al., 1992). The
N-fMet sequence probably serves as a se­
cretion signal in prokaryotic cells. In mam­
mals, the 7V-fMet sequence is present only
in proteins encoded by the mitochondrial
genome (probably of prokaryotic origin).
Furthermore, nonconventional MHC class
lb gene products are highly conserved and
vary in only few mouse strains. Thus, it
appears that a subset of bacterium-specific
CD8 T cells is focused on (i) conserved
bacterial peptides and (ii) nonpolymorphic
presentation elements. If these observa­
tions can be generalized to human tubercu­
losis, important consequences for peptide
vaccination against bacteria with few pep­
tides and independent of human lympho­
cyte antigen polymorphism can be envis­
aged.
A contribution of 7/8 T cells to protection
is suggested by indirect evidence. They
have been identified in reversal reactions of
leprosy patients and in tuberculous lymph­
adenitis lesions (Falini et al., 1989; Modlin
et al., 1989). No evidence for increased 7/8
T cell numbers, however, has been ob­
served in lymph node granulomas of tuber­
culosis patients (Tazi et al., 1991). In mice,
7/8 T cells accumulate early at the site of
BCG replication, in draining lymph nodes
after immunization with complete Freund’s
adjuvant, and in the lung after aerosol im­
munization with mycobacterial compo­
nents (Augustin et al., 1989; Janis et al.,
1989; Inoue et al., 1991). Furthermore, the
progressive BCG infection in scid mice
compared to nulnu mice and mice depleted
of CD4 and CDS T cells has been taken as
evidence for a role of 7/8 T cells (Izzo and
North, 1992). Direct proof, however, has to
await experiments with mutant mice devoid
of 7/8 T cells. The 7/8 T cells from healthy
individuals proliferate vigorously in re­
sponse to mycobacterial components (Kabelitz et al., 1990; Munk et al., 1990).
Although preferential 7/8-T-cell expansion

402

Chan and Kaufmann

by mycobacteria is caused to a large degree
by low-molecular-weight nonproteinaceous
components that act in a superantigen-like
fashion, 7/8 I cells also appear to be stim­
ulated by M. tuberculosis antigens (Munk
et al., 1990; Pfeffer et al., 1990). Thus far,
the kind of antigens and presentation mol­
ecules required for 7/8-T-cell stimulation
remain virtually unknown. Evidence from
other systems indicates that the relevant
peptides are presented by nonconventional
MHC molecules (Pamer et al., 1993). Per­
haps the MHC class lb molecules involved
in CDS T-cell stimulation also participate in
7/8-T-cell stimulation.

J

*

cells appear sequentially in the following
order: PNG. NK cells, 7/8 T cells, a/B T
cells.
Evidence has been presented elsewhere
that T-cell lysis of BCG-infected macro­
phages causes bacterial growth inhibition in
vitro (DeLibero et al., 1988). Perhaps target
cell lysis promotes discharge of toxic mac­
rophage products that inhibit mycobacterial
growth. This in vitro observation may be
taken as evidence for a direct protective
effect afforded by cytolytic T cells. More
iimportantly, a coordinated interplay be­
tween macrophage activation by IFN-7
(probably in conjunction with additional
mediators) and target cell lysis appears to
be required for optimum protection
T-Cell Functions
(Kaufmann, 1988). M. tuberculosis is ex­
Various in vitro studies of the human and tremely resistant to macrophage killing.
mui inc systems show that mycobacterium- The persistence of M. tuberculosis in
reactive CD4 T cells are potent IFN-7 pro­ healthy individuals for years without caus­
ducers (Emmrich et al., 1986; Kaufmann ing disease indicates that the immune sys­
and Flesch, 1986). IFN-7 is also produced
tem generally fails to stcrilely eradicate this
by murine CDS T cells with mycobacterial pathogen and must rely on mycobacterial
specificity (DeLibero et al., 1988). As de­ containment and growth inhibition. Not
scribed above, this cytokine is the principal only prior to but also after IFN-7 stimula­
mediator of antituberculous resistance. tion, macrophages are largely abused as
Mycobacterium-reactive CD4 T cells and habitat. Lysis of such macrophages pro­
CD.8 7 ccl,s also exPress specific cytolytic motes bacillary release from a shelter. Proactivities; i.e., they lyse macrophages vided that the microorganisms
—.3 are taken up
primed with mycobacterial antigens or in- 'by more efficient phagocytes soon after
fected with BCG or M. tuberculosis (De­ their liberation, this mechanism should im­
Libero et al., 1988; Ottenhoff et al., 1988).
prove host defense against tuberculosis.
It appears that these two functions not only Such an interplay between lysis and activa­
are demonstrable in vitro but also contrib­ tion of MP would best be controlled in
ute to protection in vivo. Besides the well- productive granulomas (see below). At the
characterized a/p T cells, other cells also same time, target VV11
cell lysis causes tissue
produce IFN-7 and express cytolytic activ- damage, affect^organ functions
------- 3, and, in the
ities, suggesting their participation in acqui­ absence of phagocytosis, promotes micro­
sition of resistance. In particular, both NK bial dissemination. Lysis of infected MP,
cells and 7/8 T cells produce IFN-7 and lyse therefore, i
• is a double-edged sword that,
mycobacterium-pulsed target cells (Munk depending on the general situation, has
a
et al., 1990; Bancroft et al., 1991; Follows beneficial or a detrimental outcome.
et al., 1992; Molloy et al., 1993). In ad­
dition, polymorphonuclear granulocytes
T-Cell Antigens
(PNG) produce highly proteolytic enzymes
causing tissue liquefaction (Weiss, 1989).
At least two characteristics of M. tuber­
At the site of M. tuberculosis growth, these culosis and BCG influence the type of anti-

Chapter 24

W. tuber'e of anti-

403

cines requires use of appropriate adjuvants
or viable carriers capable of targeting both
the MHC class I and the MHC class II
pathway. As long as MP fail to kill signifi­
cant numbers of intracellular M. tuberculo­
sis, secreted proteins and metabolically
produced peptides are the main, if not the
sole, source of antigens. Later, when M.
tuberculosis and M. bovis die in the acti­
vated macrophage, somatic proteins be­
come a major source of T-cell antigens. The
less metabolically active bacteria are, the
lower the relative proportion of secreted
protein antigens will be. Dormant tubercle
bacilli without significant metabolic activity
but resisting macrophage killing will be an
ineffectual source of any antigen. Both fea­
©
tures may be relevant to the low effective­
MHCH
MHCI \1
MHCII MHCI
ness of the only vaccine against tuberculo­
sis available, BCG. First, BCG seems to
primarily activate CD4 T cells (Pedrazzini
et al., 1987). While this seems to be suffi­
cient for protection against BCG, it appears
to be insufficient for effective vaccination
lAg|
somatic
against tuberculosis. Perhaps the shorter
secreted
intracellular survival of BCG together with
a deficiency in cytolysins restricts access of
BCG-derived proteins to the MHC class I
T (Months to years)
pathway. Second, owing to the shorter sur­
Figure 2. Relationship between intracellular persis­
vival time of BCG, somatic antigens will
tence of M. tuberculosis, antigen type, and 1-cell
predominate early after infection. Early
subset activation. (1) M. tuberculosis replicating in the
recognition of M. tuberculosis-infected
phagosome secretes proteins that are degraded into
macrophages, however, primarily depends
peptides and then translocated to the cell surface by
on T cells that recognize secreted proteins.
MHC class 11 molecules. (2) MHC class 1 molecules
capture M. tuberculosis peptides derived from se­
Thus, the preponderance of CD4 T cells
creted proteins in the cytoplasm. Either the proteins or
and somatic antigens may explain, at least
peptides had been translocated from the endosomal
in part, the insufficient protection against
into the cytoplasmic compartment, or they were se­
M. tuberculosis afforded by BCG vaccina­
creted into the cytoplasm by M. tuberculosis after its
evasion of the phagosome. Later, M. tuberculosis is
tion.

gens that are recognized by protective T
cells. First, the intracellular location (phago­
some versus cytosol) dictates processing
via the MHC class I or class II pathway.
Second, the intracellular viability of the
pathogen determines availability of poly­
peptides for processing (Fig. 2). MHC class
1 versus MHC class II processing has been
discussed above. Because soluble protein
antigens are not introduced into the MHC
class I pathway, the design of subunit vac-

e lollowing
ells, a/p T
elsewhere
ed macronh bition in
haps target
toxic macycobacterial
'On may be
protective
ells. More
erplay beby IFN-7
additional
appears to
protection
-losis is exige killing.
rculosis in
■thout causimune sysadicate this
cobacterial
•ition. Not
-7 stimulaabused as
hages pro■iclter. Proe taken up
soon after
should im'erculosis.
id activairolled in
■v). At the
ses tissue
and, in the
■les micro“Cted MP,
•'ord that,
■on, has a
ne.

Immune Mechanisms of Protection

i

i y x I

(i) ^) (|) (j) (±)

I

killed and degraded, thus giving rise to somatic pro­
teins. (3) Peptides derived from M. tuberculosis killed
in the phagosome contact MHC class II molecules. (4)
Peptides from somatic proteins present in the cyto­
plasm are charged to MHC class 1 molecules. (5)
Neither the source of peptides nor the presentation
molecules involved in 7/8 T-cell stimulation are fully
understood. This sequence of events leads to a first
wave of T cells with specificity for secreted proteins
followed by a second wave of T cells with specificity
for somatic proteins. Ag. antigen.

THE IN VIVO SITUATION

In tuberculosis, the port of entry as well
as the major organ of disease is the lung.
After being inhaled, the pathogen is en­
gulfed by alveolar macrophages that appear
to be insufficiently equipped tor microbial

404

II!

1

i

i

Chan and Kaufmann

killing. Probably these
alveolar macromay promote granuloma liquefaction
phages transport the pathogen ii
into the lung
and
rupture into the bronchoalveolar and
parenchyma and into P
’
vasdraining lymph
fecte'd’ mahere Hhe miCrObe rep,icates- In- ekv'X?S,hThe CellU,ar detritus
evalcd pO;, thus arising provide an excel­
tec ed macrophages produce chemokines
at cause the extravasation of additional lent medium for M. tuberculosis that favors
ts uncontrolled multiplication. Rupture of
P agocytes (Oppenheim et al., 1991- Fried
lhe
granuloma promotes microbial disse
land et al., 1992). These inflammatory
msecreuy? 4™° and bl°Od mon^yfes) mation through the bronchoalveolar system
Crete significant amounts of proteolytic into the environment and through the vasenzymes generating an exudatfve tesfon
cular system to other tissue sites.
Activated MP also secrete TNF which

™ Wi®?1"0"'3 f°rma,io" (Kindler et

r

a .,1989 . Eventually, T cells activated in
draining lymph nodes as well as NK cells
tO ,he Site Of “flammation
Although NK cells and 7/8 T lymphocytes
seem to precede «/p T cells, the former two

WHY DO WE NEED MORE THAN ONE

T-cell population for
PROTECTION?
Given that in vitro CD4 T cells CD« t
ce ls, and 7/8 T cells are so ffighly “flar

are soon outnumbered by the last. The a/B
T cells and 7/8 T cells interact with MP that Wi ! respect to their functional competext^of m.yCObacterial Peptides in the con­ s“sCfor y, ° We need Several T-Ce" ^fl­
text of adequate MHC molecules They sets for optimum protection to occur’ At
produce IFN-7, as do NK cells, which in the moment, this question cannot be fully
and^/S T fl'8' adVantage of CD8 T celll
act'vates tuberculostatic macrophage
and
7/8 T cells over CD4 T cells is their
high e'll '1 Produc,ive granuloma with a
restriction by MHC class I moleculeT
are confi11^ •tUrn°Ver develoPSi bacteria >
are confined in it, and their growth is re- which are expressed on virtually all host
e trictL 6 MHC C,aSS " “Passion is
t;™" ^.Although these granulomas effec­
tively inhibit bacterial replication, they are Al hXh
Cexa'n h°S‘ Ce"S such as Mpgeaerally unable to sterilely eradicate the Although M. tuberculosis preferentially re­
pathogens. In particular, the multinucle- sides m MP, a few parenchyma cells, typisXTb CeI1S warb°r M' ,ube™l°™ and These"1
ce1|l'e
become infected,
nese cells remain unnoticed by CD4 T
cXla
a™
t0 eradicate ,heir in‘racells and are identified only by CDS T cells
cellu|ar predators. Lysis of such cells
(and
perhaps 7/8 T cells). Second, the three
herefore, may contribute to protection by
allowing uptake by more efficient phagol vadoi Pk°PU at,ons ™y differ in their acti­
vation kinetics, with 7/8 T cells probably
marbec3'6'’ ‘he productive granuloma
°f mycobacterial
wall anlT enCapsula,ed by a fibrotic growth8 Th' at
wall, and the center of the granuloma may growth. Thus, 7/8 T cells may perform
"n fibO
SeemS tO P'ay a nolafl|e role essential effector functions before a/p T
“fibrotic encapsulation and central necro- cells do. Although 7/8 T cells may be lesl
l’ ‘heir faS'er kinetics °f “obili^
sis (Vassal!., 1992). Encapsulation further tiol
contributes to microbial containment, and iron and activation may give them some
ow Partlal °2 pressure (pO.) in the mardX
H 'heSe T’Ce" poPula"'™»
necrotic center provides
undea
ln
.
“'°r functions lhas far
unfavorable
"Clear, e.g„ m their capacity to leave the
growth conditions for M ituberculosis.
’
Uncontrolled cell destruction•' Iby cytolytic T vascular bed or in their responsiveness to
cells, NK cells, activated MP, and/or'PW 1ndaTTryni8na,S' F°Urlh' a/P T ce“s
and 7/8 T cells vary remarkably in their

Chapter 24

‘Quefaction and
veolar and vasdetritus and the
ovide an exccllosis that favors
■ion. Rupture of
crobial dissemalveolar system
lrough the vassites.

!E THAN ONE
>N FOR
9

cells, CDS T
highly similar
ional compeal T-cell subto occur? At
mnot be fully
f CDS T cells
cells is their
I molecules,
'ally all host
xpression is
such as MP.
-rentially rea cells, typine infected.
by CD4 T
CDS T cells
■id, the three
1 their actils probably
cobacterial
iy perform
fore a/p T
lay be less
1 mobilizahem some
■opulations
s thus far
leave the
veness to
3 T cells
v in their

1

tissue distributions. In mucosal tissues, in­
cluding the lung, as preferred port of entry
and site of disease manifestation in tuber­
culosis, the percentage of 7/8 T cells is
markedly higher than in peripheral blood
and central lymphoid organs. Finally, reg­
ulatory interactions between these T-cell
subsets may be required. In support of this
last possibility, evidence has been pre­
sented that 7/8 T cells control activation of
a/p T cells not only in vitro but also in vivo
(Kaufmann et al., 1993). Most impres­
sively, in the model of experimental listeri­
osis of 7/8 T-cell-deficient mutant mice,
huge, abscess-like lesions develop that are
strikingly different from the granulomatous
lesions at the site of listerial implantation in
healthy controls (Mombaerts et al., 1993).

GENETIC DETERMINANTS FOR
SUSCEPTIBILITY AND RESISTANCE
IN TUBERCULOSIS

While there is little formal genetic evi­
dence in humans, data obtained from epi­
demiological investigations suggest that
susceptibility to many infectious diseases,
including tuberculosis, is under some ge­
netic control (Motulsky, 1979; Skamene,
1986). The annual death rate from tubercu­
losis reached 10% when the disease first
became prevalent in the Qu’appelle Valley
Indian Reservation in Canada, eliminating
half the Indian families in the first three
generations; yet 40 years later, the annual
death rate had dwindled to 0.2%, suggest­
ing selection for host resistance (Goodman
and Motulsky, 1979). Clearly, it is conceiv­
able that different genetic strains of the
same pathogen cause diseases in different
geographical regions, so that with contin­
ued passage, as could be in the case of
tuberculosis in the Qu’appelle Valley, at­
tenuated virulence and thus in a drastic
drop in death rate over time result. While
this confounding factor is difficult to rule
out, nonetheless, the higher degree of con-

•

Immune Mechanisms of Protection

405

cordance of tuberculosis among monozy­
gotic than dizygotic twins (Comstock, 1978)
and the tragic incident of Lubeck in 1927
(Anonymous, 1935), in which infants inad­
vertently immunized with a single viable
virulent M. tuberculosis strain displayed
marked differences in susceptibility ranging
from death to recovery, argue for a genetic
basis for resistance to mycobacterial dis­
eases.
In contrast to work with the human sys­
tem, experimental studies on the genetics
of resistance to an enormous variety of
infectious agents (salmonellae, ieishmaniae, mycobacteria, murine leukemia vi­
ruses, rickettsiae, etc.) in inbred strains of
mice are abundant (Skamene, 1985). In the
case of resistance to Salmonella typhimurium, Leishmania donovani, and BCG,
compelling experimental evidence obtained
from backcross linkage analyses (Skamene
et al., 1982) suggests that resistance against
these three pathogens is under monogenic
control. This allele has been designated Ity,
Lsh, and Beg in the resistance models of 5.
typhimurium, Leishmania donovani, and
BCG, respectively. Through typing for re­
sistance and susceptibility to BCG among
recombinant inbred mouse strains together
with linkage analyses and detailed dissec­
tion of a 30-centimorgan segment on murine
chromosome 1, the cloning of the cDNA for
the Beg gene, designated Nramp (natural­
resistance-associated macrophage protein),
has recently been achieved (Vidal et al.,
1993). Sequence analysis of the Nramp
cDNA reveals a 1,452-nucleotide open
reading frame that encodes a 484-aminoacid protein with structural homology to a
eukaryotic nitrate transporter. Analysis of
Nramp cDNAs from seven Bcgr and six
Bcgs mouse strains indicates that BCG sus­
ceptibility is the result of a G-to-A transi­
tion at position 783 associated with a non­
conservative substitution of Asp-105 for
Gly-105 within a predicted transmembrane
domain of Nramp. Comparison of amino
acid sequences of the murine Nramp and a

406

i

Chan and Kaufmann

human homolog deduced from a partial
which dismutates to generate NO (Shank et
cDNA clone reveals 89% homology be­
al., 1962) and other more reactive and per­
tween the two species. Nucleic acid se­
haps more toxic reactive nitrogen species
quence analysis indicates that Gly-105 of
such as the nitrogen dioxide radical. A
murine Nramp is conserved in the human
corollary of this possibility is that ammonia
sequence.
production by M. tuberculosis (Gordon et
While it is known that the BcgT gene
al., 1980) is a means by which generation of
confers resistance against mycobacteria by
toxic RNI could be intercepted via alkalinacting early during the nonimmune phase of
ization of the phagolysosomal content. The
infection in mice (in contrast to the MHC
existence of a human homolog of Nramp, at
genes, which appear to be associated with least by cDNA analyses (Vidal et al., 1993)
recovery after infection), the precise bio­
together with the presence on human chro­
chemical and molecular mechanisms of
how Nramp regulates resistance and sus­ mosome 2q of a region syntenic to the
30-centimorgan segment on murine chro­
ceptibility to infection remain to be defined
(reviewed in Skamene (1986]). Experimen­ mosome 1 that contains the Beg allele
(Schurr et al., 1990) should presage opti­
tal evidence strongly suggests that the
Nramp phenotype is mediated via macro­ mism in unraveling the genetic basis for
phages. It has been demonstrated that the resistance and susceptibility to mycobacte­
rial diseases, at least at the early phase of
cell type expressing the Nramp phenotype
infection. It is hoped that the elucidation of
is derived from the bone marrow and is
relatively radioresistant. In addition, the one aspect of this difficult question will
form a firm springboard for understanding
phenotypic expression of Nramp can be
other as yet unknown genetic factors, e.g.,
inactivated by chronic exposure of mice to
the MHC molecules (Skamene, 1986), that
silica, a macrophage poison (Gros et al.,
aid in determining the outcome of myco­
1983). Finally, Nramp mRNAs are prefer­
bacterial infection.
entially expressed in the reticuloendothelial
system, particularly in macrophages. The
recent finding that RNI generated via the
CONCLUDING REMARKS
macrophage L-arginine-dependent cyto­
Around the world, as many as 60 million
toxic mechanism is effectively antimycopeople
suffer from tuberculosis. This high
bacterial (Denis, 1991a; Flesch and Kauf­
figure may lead to the false conclusion that
mann, 1991; Chan et al., 1992) and the
demonstration of marked structural resem­ protective immunity is totally insufficient
blance of Nramp protein to a eukaryotic for control of this disease. The figure, how­
ever, is clearly qualified by the even higher
nitrate transporter (Vidal et al., 1993) lend
number of more than 1.7 billion infected
support to the hypothesis that regulation of
individuals, i.e., one-third of the world pop­
RNI trafficking in macrophages might be
ulation, illustrating that in the vast majority
one way by which the resistance phenotype
of infected individuals, disease does not
of this gene is expressed. It is thus possible
develop in the face of an ongoing infection.
that Nramp participates in the L-argininedependent antimycobacterial pathway by Hence, protective immunity is extraordi­
narily inefficient in terminating infection
transporting NO2", a relatively stable and
and, at the same time, highly efficacious in
nontoxic nitrogen oxide formed via the ox­
idation of nitric oxide in the aqueous phase, preventing disease. Because the relation­
ship between M. tuberculosis and host im­
into the phagolysosomal compartment,
munity
underlying infection is a labile one,
whose acidic environment is requisite to
any
diminution
of protective immunity will
and allows the formation of nitrous acid.
cause progression into clinical disease.

I

Chapter 24

(Shank et
and pern species
adical. A
ammonia
3ordon et
deration of
ia alkalinntent. The
Nramp, at
al., 1993),
man chro­
nic to the
rine chroBcg allele
■sage optibasis for
tycobactephase of
nidation of
-•stion will
erstanding
tors, e.g.,
L986), that
of myco-

<S
60 million
This high
■usion that
isufficient
ure, howen higher
i infected
orld popt majority
does not
infection.
:xtraordiinfection
acious in
relation­
host imbile one,
inity will
jase.

•

Immune Mechanisms of Protection

407

human macrophages is associated with unrespon­
siveness to IFN-7. J. Immunol. 150:1838-1845.
Bermudez, L. E., and J. Champsi. 1993. Infection
with Mycobacterium avium induces production of
interleukin-10 (IL-10), and administration of anti1L-10 antibody is associated with enhanced resis­
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German Ministry of Science and Technology (BMFT).

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