Review

TRENDS in Immunology

Vol.25 No.9 September 2004

IL-4 in tuberculosis: implications for

vaccine design
Graham A.W. Rook1, Rogelio Hernandez-Pando2, Keertan Dheda1 and
Geok Teng Seah3
1

Centre for Infectious Diseases and International Health, Windeyer Institute for Medical Sciences, Royal Free and
University College Medical School, 46 Cleveland Street, London, UK, W1T 4JF
2
Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga 15, Tlalpan 14000, Mexico City, Mexico
3
Department of Microbiology, National University of Singapore MD4, 5 Science Drive 2, Singapore, 117597

Current attempts to find a vaccine for tuberculosis (TB)

are based on the assumption that it must drive a Th1
response. We review the evidence that progressive disease might not be due to absence of Th1, but rather to
the subversive effect of an unusual Th2-like response,
involving interleukin-4 (IL-4) and IL-4d2. This Th2-like
response can impair bactericidal function and lead to
toxicity of tumour necrosis factor-a (TNF-a) and to pulmonary fibrosis. If this is important, effective vaccines
will need to suppress pre-existing Th2-like activity.
Such vaccines are feasible and are active therapeutically
in mouse TB.
Immunity to tuberculosis (TB) in mice is mediated by Th1
lymphocytes. These cells activate macrophages, increase
release of tumour necrosis factor-a (TNF-a) and enhance
formation of cytotoxic T lymphocytes (CTLs) [1]. The same
cytokines are crucial in humans. Children with defective
receptors for interferon-g (IFN-g) or interleukin-12 (IL-12)
are susceptible to mycobacterial disease [2,3] and TB can
be activated when patients are treated with neutralising
antibodies to TNF-a [4]. However, rapid induction of an
IFN-g response to antigens of Mycobacterium tuberculosis
is an almost universal consequence of exposure to the
infection [5]. Why then, in some individuals, is the disease
not controlled? Here we review the possibility that
Th2-like or regulatory mechanisms undermine the efficacy
of the protective response.
Inappropriate cytokine release in human TB
Before the year 2000, some authors reported that, in
addition to Th1 cytokines, there was also increased expression of IL-4 in TB [6,7]. However, other authors failed to
detect raised IL-4 and the issue remained controversial [8].
New data indicate that these earlier failures are attributable to a series of technical problems (outlined in Box 1).
Now, there is therefore a substantial body of evidence that,
in addition to Th1 cytokines, there is an IL-4 response in
human TB, whether the patients are in Europe [9,10] or
in Africa [11,12], although a survey of the literature
(G.A.W. Rook et al., unpublished) suggests that IL-4 levels
Corresponding author: Graham A.W. Rook (g.rook@ucl.ac.uk).
Available online 26 June 2004

could be higher in patients close to the equator, possibly as

a consequence of simultaneous infection with helminths
[13]. For instance, before bacillus Calmette Guerin (BCG)
vaccination, more healthy Malawians than UK adolescents made IL-5 in response to purified protein derivative
(PPD). There was a tendency for greater IL-5 production
with increasing numbers of helminth infections in the
Malawians (G.F. Black et al., pers. commun.). TB patients
have several IL-4-dependent phenomena, including IgE
antibody to M. tuberculosis [14], increased expression of
DC-specific intercellular adhesion molecule (ICAM)-grabbing non-integrin (DC-SIGN) [15,16], and antibody to
cardiolipin [17]. Similarly, both IL-4 mRNA, and T cells
containing IL-4, are increased in human pulmonary TB,
and correlate significantly with serum IgE, serum soluble
CD30 and the extent of cavitation [9 11,18]. Interestingly,
CD8 T cells also make IL-4 in TB [10,19], and CD8 cells
secreting IL-4 correlated with cavitation in the work of van
Crevel et al. [10]. IL-4 expression can also be detected in
some pulmonary lesions by in situ hybridisation [20].
Source and significance of IL-4 in TB
A variety of different cell types can make IL-4 [T cells,
eosinophils, basophils, mast cells, natural killer (NK) cells
and some antigen-presenting cells (APCs)]. Attention has
been focused recently on the neglected observation that
IL-4 can be involved in driving Th1 responses [21] and
CTLs [22]. This can be IL-4 administered externally or
IL-4 from CD11bCD11clo APCs, which are present at an
early stage of Th1 response induction [23]. IL-4 present at
later stages downregulates Th1 responses [21], and this
must be happening in TB, where at least some of the Th2
cytokines are coming from T cells [10,19].
Box 1. Why is assay of interleukin-4 (IL-4) so difficult?
(i) The low concentrations and low mRNA copy numbers at which IL-4
is active [59]
(ii) Short half-life of mRNA encoding IL-4 in clinical samples (an hour
or less) [60]
(iii) The existence of a splice variant of IL-4, which lacks exon 2 [61]
(Box 2)
(iv) Pre-stimulation of cells causes secretion of interferon-g (IFN-g)
and rapid reciprocal downregulation of Th2-cell proliferation, further
depleting an already low copy number cytokine (IL-4)

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A further possibility is that IL-4 in TB has a necessary

regulatory or anti-inflammatory role. Under some circumstances, regulatory T cells express IL-4 in addition to
classical regulatory cytokines, such as IL-10 and transforming growth factor-b (TGF-b) [24]. However, the
evidence is against this role for IL-4. In progressive
disease, IL-4 causes increased, rather than diminished,
immunopathology [25]. The presence of specific IgE
antibody [14] and of T cells that release IL-4 in vitro
when driven by TB antigen [11] indicates a genuine
antigen-specific Th2 lymphocyte response.
Nevertheless, the increased IL-4 expression in TB is
different from that seen in the partly Th2-mediated
disorder, allergic asthma [26]. In the latter disease, the
increase is mostly due to IL-4 itself, rather than to the
splice variant. By contrast, in TB patients, levels of mRNA
encoding IL-4d2, an inhibitory splice variant, were found
to rise as well [26] (Box 2). Therefore, we refer to this small
IL-4 component in TB as Th2-like.
Bovine TB
Infection of cattle with Mycobacterium bovis (a strain
derived from the human M. tuberculosis) generates both
IFN-g and IL-4 responsiveness to PPD and to ESAT-6
(early secreted antigenic target-6 kDa) [27]. Similarly, the
extent of lung pathology correlates with the production of
IL-4 by peripheral blood mononuclear cells in vitro in
response to PPD [28]. Interestingly, when cattle were
immunised with a DNA vaccine, and then 3 weeks and
6 weeks later, vaccinated again with the protein encoded
by the DNA vaccine emulsified in oil, they were more
susceptible than non-immunised controls. This increased
susceptibility correlated with an increased IL-4 response
to subsequent challenge [28].
Mouse models
Because the increased expression of IL-4 in human
(and bovine) TB is no longer in dispute, the relevant
mouse models are those in which, as in humans, IL-4
production is observed during the progressive phase of the
disease. Studies of Th1 and Th2 cytokine expression reveal
that in some, but not all, murine models of TB there are at
Box 2. Interleukin-4d2 (IL-4d2): a neglected cytokine
IL-4d2 is a splice variant of IL-4 that lacks exon 2 (48 base pairs)
[61,62]
IL-4d2 mRNA is found in primates, rabbits and woodchucks but not
in rats or mice [63]
Recombinant IL-4d2 functions as a natural antagonist of IL-4 [61,62]
Final proof that the IL-4d2 protein is produced in vivo has not yet
been obtained
Because antibodies used in flow cytometry or ELISA assays can
bind either, or both, existing work on IL-4 in humans using these
assays could be uninterpretable
IL-4d2 is easily distinguished by RT PCR [64], however, most
published work uses primers that will amplify both cytokines
IL-4d2 might be the dominant form of IL-4 mRNA in human lungs,
and associated with fibrosis [39]. Possibly an agonist on fibroblasts
IL-4d2 rises little in atopic asthma, in which mRNA encoding IL-4
itself rises 1000x more [26]
IL-4d2 mRNA does rise to a variable extent in tuberculosis [9] and in
contacts
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Vol.25 No.9 September 2004

first high IFN-g levels, followed later, in the progressive

phase, by a superimposed IL-4 response [29,30]. Confusion
about this point arose from the study of the effects of IL-4
gene knockout in mouse strains C57Bl 129SvJ [31] and
C57Bl [32], in which IL-4 secretion is not characteristic of
the infection. These are elegant studies, however, C57Bl
mice develop progressively increasing lung granulomas
and eventually drown in Th1 cells, without the switch to
Th2 cytokine production that is seen in Balb/c mice and,
albeit to a lesser extent, in humans.
The Th2 response is cause rather than effect
Is the IL-4 response in TB an irrelevant consequence of
the infection, or is it significantly involved in reducing
protection and increasing immunopathology? Simple
experiments where mice were pre-immunised, so that
they had small Th2 responses to one or several components
of M. tuberculosis before infection, have shown that
pre-existing Th2 activity leads to increased severity of
infection and to death [33,34]. Wangoo et al. demonstrated
that a pre-existing Th2 response to a single 16 amino
acid epitope expressed within the challenge strain of
M. tuberculosis was sufficient to undermine the efficacy of
the dominant Th1 response, and to aggravate weight loss
and lung fibrosis [35]. Further studies in Balb/c mice with
knocked out IL-4 genes proved that the absence of IL-4 led
to diminished bacterial growth, and confirmed that IL-4 is
directly involved in TNF-a-mediated immunopathology
and fibrosis in this strain [25] (see later). Interestingly,
although other authors have not studied IL-4 knockout
Balb/c mice, it was found that STAT6 (signal transducer
and activator of transcription 6) knockout Balb/c respond
normally to aerosol infection with M. tuberculosis [36].
This implies that the relevant functions of IL-4 (fibrosis
and enhanced toxicity of TNF-a) might be signaled
through insulin receptor substrate-2 (IRS-2) or IRS-1
rather than via STAT6 [37].
Fibrosis is a major cause of lung dysfunction in TB
Fibrosis in TB is a major cause of permanent respiratory
dysfunction, however, it has never been satisfactorily
explained. The dominant cytokine in TB, IFN-g, opposes
fibrosis in humans and in mice [38]. Interestingly, in
human diseases characterised by pulmonary fibrosis,
there is usually marked expression of type 2 cytokines
[systemic sclerosis, idiopathic pulmonary fibrosis, radiationinduced pulmonary fibrosis, chronic lung allograft rejection (reviewed in Ref. [38])]. These observations have led to
the type 2 cytokine hypothesis of fibrosis [38]. Fibrosis
in human TB might therefore be related to the presence
of the Th2-like response (Figure 1). Interestingly, pulmonary fibrosis in systemic sclerosis is associated with
CD8 cells secreting IL-4 and IL-4d2 [39], reminiscent of the
CD8 cells expressing either IL-4 or IL-4d2 noted previously
in human TB [10,19].
TNF-a is a crucial protective molecule but it is toxic in TB
when IL-4 is present
TNF-a-mediated immunopathology is the other phenomenon associated with IL-4 in the mouse models. In IL-4
gene knockout tuberculous Balb/c mice, there is not only

Review

M. tuberculosis

TRENDS in Immunology

Th1

FAILED Th1-MEDIATED
IMMUNITY
BACTERIAL
PROLIFERATION

Environmental
mycobacteria
BCG?

PROPHYLACTIC
VACCINE

Th2-like
IL-4
IL-13
IL-5
(IL- 42)

IMMUNOPATHOLOGY
(+TNF-)
FIBROSIS
APOPTOSIS

THERAPEUTIC
VACCINE
TRENDS in Immunology

Figure 1. The roles of the subversive Th2-like component of the immune response
in progressive human tuberculosis. This might be primed by bacillus Calmette
Guerin (BCG) vaccination or by environmental mycobacteria but it is then
enhanced following exposure to Mycobacterium tuberculosis. A vaccine, even
if prophylactic, might need to eliminate any pre-existing Th2 component.
Abbreviations: IL-4, interleukin-4; TNF-a, tumour necrosis factor-a.

diminished bacterial proliferation, but also a complete

absence of TNF-a-mediated toxicity following TNF-a
challenge [25]. This observation can explain the paradoxical role of TNF-a in TB. TNF-a is crucial for protection
against TB in animals [40] and in humans [4]. Nevertheless, in progressive human disease, as in progressive
TB in the Balb/c mouse [25], it is toxic (Figure 1), and drugs
that shorten the half-life of the mRNA encoding TNF-a
lead to weight gain and symptomatic relief [41]. Because
IL-4 levels are raised in progressive human disease, it is
likely that, as in mice, IL-4 is involved in the toxicity of
TNF-a. Examples of interactions between IL-4 and TNF-a
in other diseases, and possible mechanisms for proinflammatory interactions between these cytokines, are
described in Box 3.
Most existing prophylactic TB vaccine candidates fail as
therapeutic vaccines
Most known vaccine candidates (i.e. effective as prophylactic vaccines in mice) are ineffective as therapeutic
vaccines [42,43]. This is also true of BCG and of various
preparations of M. tuberculosis itself [43]. The history of
this observation goes back to Robert Koch, who observed
necrosis in tuberculous guinea-pigs given injections of
Old Tuberculin (the Koch Phenomenon), and then
repeated the experiment, with the same result, in humans
(reviewed in Ref. [44]). The role of TNF-a in the immunopathology triggered by BCG in tuberculous mice was
highlighted again recently [43].
Vaccines that downregulate IL-4 are active
therapeutically in murine TB
Interestingly, there are two preparations that are exceptions to the rule that protective vaccines are not therapeutic. One is a DNA vaccine based on heat-shock
protein 65 (hsp65) of Mycobacterium leprae, which, in
contrast to a different construct used in an aerosol
model [45], will treat ongoing TB after intravenous
infection, and while doing so, dramatically reduces IL-4
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485

Box 3. Interleukin-4 (IL-4), decreased immunity and

increased immunopathology in tuberculosis (TB)
There are several reasons why immunity to TB might be particularly
susceptible to detrimental effects of IL-4, even when Th1 cytokines,
such as interferon-g, dominate.
Further examples of immunopathology attributable to Th1 1 Th2 1
tumour necrosis factor-a (TNF-a)
In Trichinella spiralis infection, immunopathology in the gut is
attributable to TNF-a in a mixed Th1 Th2 response. No immunopathology in IL-4 knockout mice [65]
The mixed Th1/Th2 granulomata of Schistosomiasis are exquisitely sensitive to systemic cytokine release triggered by lipopolysaccharide (LPS) [66,67]
Possible mechanisms of increased toxicity of TNF-a in mixed
Th1 1 Th2 lesions
Induction of cortisol-resistance in T cells exposed simultaneously
to Th1 and Th2 cytokines [68]
Synergistic induction of vascular cell adhesion molecule-1
(VCAM-1) on endothelial cells leading to increased recruitment of
inflammatory cells [69]
Downregulation of TRAF-2 (TNF receptor-associated factor-2)
biases functions of TNF-a away from activation and towards cell
death, which was observed in CD30 T cells incubated with TB
antigen [50]
Why is expression of IL-4 detrimental to immunity to TB?
Inappropriate type of macrophage activation [57]
Decreased expression of, and signalling through, Toll-like receptor 2
(TLR2) [56]
Downregulated inducible nitric oxide synthase (iNOS) [55];
iNOS might be fundamental to the induction and maintenance of
latency [70]

ELISPOT T-cell numbers [46]. In the same experiment, a

BCG vaccine run in parallel neither treated the disease
nor reduced IL-4 production [46]. The other exception,
heat killed Mycobacterium vaccae, is also active as an
immunotherapeutic in mice, in which it strikingly reduces
IL-4 expression when administered 60 days after intrapulmonary infection [30]. Its major mode of action is not
the induction of Th1, but rather, suppression of Th2 by
driving CD4CD45RBlow regulatory T cells [47]. Thus,
although these are only correlations, effective therapy
might relate to downregulation of IL-4 (Figure 1).
Efficacy of BCG vaccine varies in different geographical
areas
BCG vaccine is a deletional mutant of M. bovis derived by
repeated in vitro culture. Like M. bovis itself [27], BCG
primes not only Th1 but also IL-4 responses both in cattle
[48] and in mice [49]. Most workers fail to notice this
induction of IL-4, because it is far weaker that the
concomitant induction of IFN-g, and as discussed earlier,
more difficult to measure. Moreover, we do not know if
BCG induces an IL-4 response in humans, but it is likely to
because peripheral blood mononuclear cells from healthy
BCG-vaccinated donors release IL-4 in response to antigens of M. tuberculosis [50]. Not only does BCG not downregulate Th2 responses in murine TB [46], but BCG itself
evokes IL-4-secreting cells [49].
If this IL-4 component to the response evoked by BCG is
detrimental, then one would expect the efficacy of BCG to

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be variable, particularly in areas close to the equator [51]

where the Th2 response to M. tuberculosis is likely to be
particularly high [13]. Similarly, repeat BCG vaccination
might be hazardous in the same environments because it
might boost the IL-4, rather than the IFN-g? component.
Repeat BCG vaccination of cattle reduced the protective
efficacy of a single vaccination, and the effects of repeat
vaccination in humans are variable, as reviewed in a paper
on cattle [48].
IL-4 and IL-4d2 in contacts of TB patients
Is expression of IL-4, or of the potential antagonist IL-4d2,
increased in latent TB, and if so, how does this differ from
what happens in those in whom the disease progresses?
Recent studies have focused on healthy individuals in
The Gambia and Ethiopia, whose peripheral blood mononuclear cells give IFN-g responses to the TB-specific
antigen, ESAT-6. These people have latent TB and it has
emerged that they have significantly raised levels of
mRNA encoding IL-4d2 in their unstimulated peripheral
blood mononuclear cells [52,53]. In The Gambia, expression of IL-4 is also increased [53]. These findings fuel
speculation that the balance of the agonist (IL-4) and
potential antagonist (IL-4d2) might be relevant to whether
or not the disease progresses, and current studies are
addressing this question. Crucial questions include whether
IL-4d2 is present only as mRNA or also active protein, and
which cell types are expressing these mRNAs in the
latently infected contacts [52]. These experiments are
currently in progress. Meanwhile, a study of healthcare
workers in Portugal has revealed that those individuals
whose peripheral blood mononuclear cells synthesised
IL-4 in response to M. tuberculosis in vitro went on to
develop overt TB within 2 4 years [54]. The methods used,
however, will not have distinguished between IL-4 and
IL-4d2. Further work will be required to discover whether it
is high IL-4, rather than IL-4d2, that predisposes to disease.
Conclusions
A Th1 response to M. tuberculosis develops rapidly following exposure and diagnostic methods detect this Th1
activity [5]. Why, then, does the disease progress? And why
do the Th1-inducing vaccine candidates that have been
tried as therapeutic vaccines cause necrosis and make the
disease worse? We have reviewed evidence that the Th1
response fails because a smaller Th2 response (IL-4, partially opposed by the antagonist, IL-4d2) is undermining its
efficacy and driving immunopathology in synergy with
TNF-a. We have also considered the more conventional
views of the role of IL-4 in TB, which are that it is needed to
drive CTLs, that it has a useful anti-inflammatory role, or
that it is merely a late consequence of progressive disease.
We have shown that recent findings make all of these
views very difficult to sustain.
IL-4, with its ability to downregulate inducible nitric
oxide synthase (iNOS) [55], Toll-like receptor 2 (TLR2) [56]
and macrophage activation [57], is potentially an early
player in the events that determine whether the infection
becomes latent or progressive. Recent studies of contacts
with latent disease [52], and of healthcare workers who
subsequently developed the disease [53,54], lend support
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Vol.25 No.9 September 2004

to this possibility. Now that we can distinguish IL-4 from

the antagonist IL-4d2, the role of IL-4 can be more precisely delineated. If this role is important, effective vaccines against TB might need to concentrate on elimination
of the IL-4 response, rather than on boosting the Th1
response, which develops spontaneously as a consequence
of infection (Figure 1). This principle might be equally
applicable to pre-exposure vaccines, post-exposure vaccines and therapeutic vaccines, because in each case a preexisting IL-4 response will exist, whether induced by BCG
vaccination, exposure to M. tuberculosis or cross-reactive
organisms in the environment.
How might such vaccines be achieved? The components
of M. tuberculosis responsible for driving the IL-4-secreting
T cells could be identified. It has been shown, using IL-4
ELISPOTs, that ESAT-6 and peptides derived from it do
not do so [58]. The components that do drive IL-4 can be
identified by ELISPOT or by monitoring their ability to
drive expression of IL-4 mRNA in vitro. Such antigens are
present in crude sonicated M. tuberculosis [50]. IL-4driving components could be combined with adjuvants
capable of converting the response to Th1 or of driving a
regulatory T-cell response.
To be a pathogen, an organism can either fail to evoke
the protective response or it can drive the protective
mechanisms and then deliberately sabotage them. Recent
data suggest that this might be how M. tuberculosis operates. If this proves to be correct, radically new approaches
to vaccine design will be needed, and this train of thought
will deserve to be extended into the fields of HIV and
certain parasitic diseases.
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Immunology online
General information
An Immune System Glossary: http://www.molbiol.ox.ac.uk/pathology/tig/gloss.html
AMADEO: Free Medical Literature Guide: http://www.amedeo.com/
Immunology Index: http://www.keratin.com/am/
Glossary of Immunology: http://www-micro.msb.le.ac.uk/MBChB/ImmGloss.html
Molecular Immunology: http://www.mi.interhealth.info/
Spiders and Immunology: http://www.xs4all.nl/~ednieuw/
Societies
British Society for Immunology: http://www.immunology.org/
American Academy of Allergy, Asthma and Immunology: http://www.aaaai.org/
Clinical Immunology Society: http://www.clinimmsoc.org/
Society for Mucosal Immunology: http://www.socmucimm.org/
Australasian Society for Immunology: http://www.wehi.edu.au/collegiate/ASI/
Canadian Society of Allergy and Clinical Immunology: http://csaci.medical.org/
International Society for Interferon and Cytokine Research: http://www.isicr.org/
Federation of Clinical Immunology Societies: http://www.focisnet.org/
Discussion groups:
BioWWW.net: http://biowww.net/
Nomenclature
CD: http://www.immunologylink.com/cdantigen1.htm
HLA: http://www.ebi.ac.uk/imgt/hla/
KIR: http://www.gene.ucl.ac.uk/nomenclature/genefamily/kir.html
http://www.ncbi.nlm.nih.gov/prow/guide/679664748_g.htm
LILR: http://www.gene.ucl.ac.uk/nomenclature/genefamily/lilr.html
Immunoglobulins
Immunoglobulin Structure/Function: http://www.path.cam.ac.uk/~mrc7/mikeimages.html
Porcine Immunology Resources: http://eis.bris.ac.uk/~lvkh/welpig.htm
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