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TUMOR NECROSIS FACTOR ALPHA (TNF-a) 307
TUMOR NECROSIS FACTOR ALPHA (TNF-a)
S J Levine, National Institutes of Health, Bethesda,
MD, USA
Published by Elsevier Ltd.
Abstract
Tumor necrosis factor alpha (TNF, TNFSF2) is an important
multifunctional cytokine that regulates inflammation, host defense, immune responses, and apoptosis. TNF exists as a membrane-bound 26 kDa, type II integral membrane protein, and
a 17 kDa soluble homotrimeric TNF that is generated via
proteolytic cleavage by TNF-a converting enzyme (TACE,
ADAM17). Both the membrane-bound and soluble forms of
TNF can interact with either the 55 kDa, type I tumor necrosis
factor receptor (TNFRSF1A, TNFR1) or the 75 kDa, type II
TNF receptor (TNFRSF1B, TNFR2). TNFR2, however, may be
preferentially activated by membrane-bound TNF rather than
soluble TNF, which may lead to qualitatively distinct TNF responses. The main biologic function of TNF is to induce inflammation via the upregulation of gene transcription, primarily
through the NF-kB and AP-1 signaling pathways, which leads to
the expression of a large number of genes. TNF plays an important role in innate immunity and host defense against
bacterial, fungal, and parasitic pathogens and is particularly
important for host defense against intracellular organisms, such
as Mycobacterium tuberculosis. TNF has also been implicated
in the pathogenesis of pulmonary diseases, such as idiopathic
pulmonary fibrosis, emphysema, sarcoidosis, acute lung injury,
bacterial pneumonia, and asthma.
Introduction
Tumor necrosis factor alpha (TNF, TNFSF2) is
the prototypical member of a large superfamily of
at least 17 related cytokines that play key roles in
regulating inflammation, host defense, adaptive immunity, apoptosis, autoimmunity, and organ development. Although the antitumor activity of TNF was
initially recognized in the nineteenth century when
tumor regression was observed in the setting of acute
bacterial infection, it was not until 1984 that the
protein was purified and the cDNA was cloned. TNF
is recognized as an important mediator of inflammatory and immune responses as well as apoptosis.
TNF also plays a central role in the pathogenesis of
several inflammatory diseases, including rheumatoid
arthritis and inflammatory bowel disease. Furthermore, the proinflammatory effects of TNF have
served as the rationale behind the introduction of
anti-TNF therapies for the treatment of rheumatoid
arthritis and Crohn’s disease, which have included a
recombinant soluble human TNFR2-Ig fusion protein and an anti-TNF monoclonal antibody.
Structure of the TNF Gene and Protein
The TNFSF2 gene resides on human chromosome
6p21.3 and on murine chromosome 17, where it is
preceded by the TNFSF1 gene that encodes lymphotoxin-a (LTA, LT, TNF-b). Human TNFSF2 is a single-copy gene of 3.6 kb that is composed of four
exons and three introns. Expression of the TNFSF2
gene can be regulated at both the transcriptional and
the translational level. The TNFSF2 promoter is
complex and contains multiple transcription factor
binding sites, including those for nuclear factor
kappa B (NF-kB), activator protein 1 (AP-1), AP-2,
specificity protein-1 (SP-1), Eq6-transformation
specific (ETS), nuclear factor of activated T cells
(NF-AT), activating transcription factor-2 (ATF-2),
and Krox-24. A cyclic AMP response element (CRE),
a purine-rich box (PU.1), and a MHC class II-like ‘Y
box’ are also present. Translational regulation may
also occur via the UA-rich sequence in the 30 untranslated region of human TNF mRNA, which may
destabilize the transcript.
The crystal structure for soluble TNF protein was
initially solved in 1989 and has been obtained at high
resolution. TNF exists as a compact trimer in the
shape of a cone or bell that is composed of three
monomers, which are intimately associated about a
threefold axis of symmetry. Each of the TNF monomers exists as two elongated, antiparallel b-pleated
sheets, each with eight, antiparallel b-strands that
form a ‘jelly-roll’ topology. Further insights have
been gained by analyses of the interaction between
lymphotoxin and the extracellular domain of
TNFR1, which forms a complex composed of three
receptor molecules bound symmetrically to one
lymphotoxin trimer. The receptors, which exist in a
rod-shaped configuration, align along their long parallel axis and interact with the lateral grooves of the
trimeric ligand. Although TNF receptors were
thought to undergo ligand-dependent trimerization,
pre-ligand-binding assembly domains have been
identified in the extracellular domains of TNFR1
and TNFR2 that mediate specific ligand-independent
assembly of receptor trimers.
Regulation of TNF Production and Activity
Macrophages are a primary source of TNF production. TNF can also be produced by many cell types,
including T and B cells, mast cells, fibroblasts,
keratinocytes, osteoblasts, smooth muscle cells,
308 TUMOR NECROSIS FACTOR ALPHA (TNF-a)
Cellular sources
of TNF
Mediators of increased
TNF production
Infection
LPS
Bacterial
Viral
Fungal
Parasitic
Inflammation
IL-1
IL-2
IFN-
GM-CSF
M-CSF
TNF
C5a
Immune complexes
Subtance P
Injury
Macrophages
T lymphocytes
B lymphocytes
Mast cells
Eosinophils
Epithelial cells
Fibroblasts
Smooth muscle cells
Tumor cells
Disease pathogenesis
Acute lung injury
Asthma
Emphysema
Pneumonia
Pulmonary fibrosis
Sarcoidosis
TNF target
genes & proteins
Adhesion molecules
Apoptosis
CC10
Cytokines
Chemokines
Colony stimulating factors
Enzymes
Extracellular matrix
Growth factors
MHC class I and II
Mucins
Transcription factors
Proteinases
Proteinase inhibitors
Receptors
Signaling proteins
Figure 1 The role of TNF in respiratory disease.
endothelial cells, epithelial cells, and tumor cells
(Figure 1). TNF production can be stimulated by a
wide variety of mediators, including bacterial products (e.g., lipopolysaccharide), cytokines (e.g., IL-1,
TNF, and interferon-g), viruses (e.g., HIV and influenza A), parasites (e.g., Plasmodium and Entamoeba), complement (e.g., C5a), immune complexes,
phorbol ester, calcium ionophore, and UV irradiation. In particular, large quantities of TNF can be
produced in response to bacterial lipopolysaccharide,
which contributes to the pathogenesis of septic shock.
Alternatively, suppressors of TNF production include
cytokines (e.g., IL-4, IL-6, IL-10, IL-13, and TGF-b),
cyclic AMP, cyclic nucleotide phosphodiesterase
inhibitors (e.g., pentoxifylline and rolipram), cyclosporine A, dexamethasone, and viruses (e.g., Epstein–
Barr virus).
Human TNF exists in either membrane-bound or
soluble forms. TNF is initially expressed as a 26 kDa,
type II integral membrane protein that is arranged as
a stable homotrimer. The 17 kDa soluble homotrimeric TNF is generated from the membranebound form via proteolytic cleavage between Ala
76 and Val 77 by TNF-a converting enzyme (TACE,
ADAM17), which is a member of the ADAM (a
disintegrin and metalloprotease) family. ADAMs are
a large family of type I transmembrane proteins that
contain multiple functional domains, including a
prodomain (that maintains the enzyme in an inactive
state via a cysteine switch mechanism), a zinc-dependent catalytic domain, a disintegrin–cysteine-rich
domain, an epidermal growth factor-like repeat, a
transmembrane domain, and an intracytoplasmic
tail. Both the membrane-bound and the soluble
forms of TNF can interact with either the 55 kDa,
type I tumor necrosis factor receptor (TNFRSF1A,
TNFR1) or the 75 kDa, type II TNF receptor
(TNFRSF1B, TNFR2). TNFR2, however, may be
preferentially activated by membrane-bound TNF
rather than soluble TNF, which may lead to qualitatively distinct TNF responses.
Biological Functions of TNF
TNF is a multifunctional, proinflammatory cytokine
that mediates pleiotropic biological functions, especially host defense. The broad spectrum of TNFmediated biological functions is a direct consequence
of its ability to induce the expression of a large
number of gene products. Genes or proteins that can
TUMOR NECROSIS FACTOR ALPHA (TNF-a) 309
be induced by TNF include transcription factors
(e.g., c-fos and c-jun), cytokines (e.g., IL-1, TNF,
IL-6, and IL-8), chemokines (e.g., eotaxin), growth
factors (e.g., platelet-derived growth factor and
granulocyte-macrophage colony-stimulating factor),
adhesion molecules (e.g., ICAM-1, VCAM-1, and
E-selectin), receptors (e.g., IL-2Ra and EGF receptor), complement (e.g., C3), major histocompatibility
complex proteins (e.g., MHC class I and MHC class
II), acute phase reactants (e.g., haptoglobin), and
enzymes (e.g., nitric oxide synthase, manganese
superoxide dismutase, and matrix metalloproteases).
TNF was initially identified via its ability to induce
cytotoxicity against tumor cell lines and to mediate
tumor necrosis in certain animal models. TNF was
also found to be identical to the protein cachectin,
which mediates fever and muscle wasting in cancer
patients and weight loss, anorexia, and lethality in
mice. TNF has been identified as playing an important role in innate immunity and host defense against
bacterial, fungal, and parasitic pathogens. In particular, TNF plays a key role in host defense against
intracellular pathogens, such as Mycobacterium
tuberculosis, Leishmania major, and Listeria monocytogenes. This is exemplified by the association
between anti-TNF therapy and the reactivation of
latent tuberculosis. Similarly, anti-TNF therapy has
been associated with infections caused by Histoplasma capsulatum, Aspergillus fumigatus, and L. monocytogenes. Tissue macrophages and T lymphocytes
produce TNF in response to invasion by intracellular
pathogens, which then mediates cytokine and
chemokine production and the subsequent recruitment of activated leukocytes to the site of infection.
In later stages of infection, TNF enhances antigen
presentation and T-cell costimulation, thereby promoting the induction of acquired immunity.
Another important function of TNF is the induction of apoptosis, which typically occurs in the absence of concomitant signaling by NF-kB. This is
exemplified by transgenic mice deficient in NF-kB
signaling, which undergo embryonic lethality as a
consequence of TNF-mediated liver cell apoptosis.
The biologic role of TNF-mediated apoptosis, however, requires further clarification.
Insights into the biological function of TNF have
also come from studies utilizing transgenic models.
TNF knockout (TNF / ) mice develop normally
and have no structural or morphological abnormalities. TNF / mice are highly susceptible to challenge with infectious agents (e.g., Candida albicans
and L. monocytogenes), are resistant to lethality
from minute doses of lipopolysaccharide following
sensitization with D-galactosamine, demonstrate
low toxicity to TNF, have defective granuloma
formation, do not form germinal centers after immunization, and display reduced immunoglobulin
responses to thymus-dependent antigens. Furthermore, the function of macrophages, neutrophils, and
T cells derived from TNF / mice is normal. TNF
has been implicated in the pathogenesis of diabetes
and may be an important mediator in obesity because TNF / mice are protected from obesityinduced insulin resistance.
Insights into TNF function have also been derived
from studies of transgenic mice lacking the two TNF
receptors, TNFR1 and TNFR2. For example, TNF
may play an important role in the regulation of central nervous system injury and function. TNF may
have a neuroprotective function because neuronal
damage secondary to ischemic and epileptic injury is
exacerbated in TNFR1/TNFR2 double knockout
mice. Similarly, in a model of experimental autoimmune encephalitis, TNF / mice develop severe
autoimmune-mediated demyelination, which can be
attenuated by TNF administration.
TNF Receptors and Signaling Pathways
The main biologic function of TNF is to induce inflammation via the upregulation of gene transcription, primarily through the NF-kB and AP-1
signaling pathways. This is accomplished by signaling through two members of the TNF receptor
superfamily, TNFR1 and TNFR2. TNFR1 is constitutively expressed by most cell types, whereas
TNFR2 has a highly regulated pattern of cellular
expression. Binding of TNF to TNFR1 mediates the
translocation of TNFR1 to lipid rafts, where it recruits the adaptor protein, TNF receptor-associated
death domain (TRADD), via a direct interaction with
the TNFR1 death domain. TRADD then serves as
an assembly platform to mediate the recruitment of
other adaptor proteins, such as receptor-interacting
protein (RIP) and TNFR-associated factor-2
(TRAF2), which in turn recruit I-kB kinases and
thereby activate the NF-kB pathway (Figure 2).
TNFR1 and RIP within the lipid rafts are then
ubiquitinated, which leads to their degradation via
the proteasome pathway. TNFR1 signaling through
TRAF2 can also activate the c-Jun N-terminal kinase
(JNK) pathway through the MAPK kinase, mitogenactivated protein kinase kinase-7 (MKK7), which results in the phosphorylation of c-Jun, with resultant
increases in AP-1 activity.
TNF signaling can also induce apoptosis, primarily
via TNFR1-mediated signaling. TNF-mediated apoptosis requires the blockade of the NF-kB pathway
because NF-kB activation induces the expression
of several antiapoptotic genes, including c-FLIP
310 TUMOR NECROSIS FACTOR ALPHA (TNF-a)
TNF
TNFR1
Plasma membrane
Inter
naliz
TRADD
TRAF2
ation
RIP1
MKK7
IKK- IKK-
JNK
IKK-
c-Jun
NF-B
TNF
TNFR1
TNF
receptosome
TRADD
FADD
Caspase 8
Inflammation
Apoptosis
Figure 2 TNFR1 signaling pathways. TNF binding to TNFR1 mediates the translocation of TNFR1 to lipid rafts, where it recruits the
adaptor proteins TRADD, RIP1, and TRAF2, which can activate the NF-kB pathway via an I-kB kinase complex composed of I-kB
kinase-a, I-kB kinase-b, and I-kB kinase-g (NEMO). Alternatively, TNFR1 signaling through TRAF2 can activate the c-Jun N-terminal
kinase (JNK) pathway through the MAPK kinase, MKK7, which results in the phosphorylation of c-Jun, with resultant increases in AP-1
activity. Both the NF-kB and JNK pathways stimulate inflammation. Furthermore, activation of the NF-kB pathway inhibits apoptosis as
a consequence of the increased expression of antiapoptotic genes (i.e., c-FLIP, cIAP1, and cIAP2). TNFR1 signaling can induce
apoptosis in the absence of NF-kB activation. A model proposed that the formation of a TNFR1-associated death-inducing signaling
complex (DISC) is dependent on receptor internalization within endocytic vesicles, termed TNF receptosomes. Internalized receptors
recruit TRADD, FADD, and caspase-8 to form a TNFR1-associated DISC. This results in the rapid activation of caspase-8, which
initiates apoptosis. TNF receptosomes can fuse with trans-Golgi vesicles to form multivesicular endosomes, allowing for the caspase8-mediated activation of acid sphingomyelinase and cathepsin D cascades, which also induce apoptosis via Bid cleavage and caspase-9 activation (not shown). Other signaling pathways that can be activated by TNF include the stress-activated protein kinase
(SAPK) cascade, the p38 mitogen-activated protein kinase (MAPK) pathway, and the neutral sphingomyelinase/ceramide pathway (not
shown).
(cellular FADD-like interleukin-1-converting enzyme-inhibitory protein) and the cellular inhibitor
of apoptosis proteins (IAPs), cIAP1 and cIAP2. Two
models have been proposed for TNF-mediated
apoptosis. One model proposes two sequential signaling complexes, with the initial formation of a
TNFR1, TRADD, RIP1, and TRAF2 signaling complex I at the plasma membrane, followed by a cytoplasmic complex II composed of TRADD, RIP1,
FADD (Fas-associated death domain), and caspase-8
that mediates cell death in the absence of NF-kB
activation. The second model proposes the liganddependent formation of a TNFR1-associated deathinducing signaling complex (DISC) that is dependent
on receptor internalization within endocytic vesicles,
termed TNF receptosomes. In this model, receptor
internalization allows the recruitment of TRADD,
FADD, and caspase-8 to form a TNFR1-associated
DISC within TNF receptosomes (Figure 2). Caspase8 is rapidly activated and initiates apoptosis. TNF
receptosomes then fuse with trans-Golgi vesicles
to form multivesicular endosomes, allowing the
caspase-8-mediated activation of acid sphingomyelinase and cathepsin D cascades, which can induce
apoptosis via Bid cleavage and caspase-9 activation.
The adaptor protein FAN (factor associated with
neutral Smase activation), via binding to a neutral
sphingomyelinase domain in TNFR1, can also induce
apoptosis via signaling through neutral sphingomyelinase and ceramide. TNFR2 does not contain intracytoplasmic death domains but can directly bind
TRAF2, leading to the activation of the JKK/NF-kB
and the JNK pathways. TNFR2-mediated TRAF2
degradation, however, may downregulate NF-kB activation and thereby promote apoptosis. Other signaling pathways that can be activated by TNF include
the stress-activated protein kinase (SAPK) cascade
and the p38 mitogen-activated protein kinase
(MAPK) pathway.
TNF and Pulmonary Disease
TNF has been implicated in the pathogenesis of a
variety of diseases, including rheumatoid arthritis,
TUMOR NECROSIS FACTOR ALPHA (TNF-a) 311
inflammatory bowel disease, endotoxin-mediated
septic shock, HIV infection, diabetes, graft-versushost disease, allograft rejection, and neoplastic disease. TNF may also play a role in the pathogenesis of
pulmonary diseases, including pulmonary fibrosis,
bacterial pneumonia, emphysema, sarcoidosis, acute
lung injury, and asthma. Evidence of a role for TNF
in pulmonary fibrosis derives from murine models in
which overexpression of TNF in the lung produced a
lymphocytic and fibrosing alveolitis that resembled
human idiopathic pulmonary fibrosis. Similarly, TNF
receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers. TNF
has also been shown to mediate anti-inflammatory
and antifibrotic effects in murine models of bleomycin-induced lung injury and fibrosis, consistent
with a complex role for TNF in the pathogenesis of
pulmonary fibrosis.
TNF knockout mice display impaired bacterial
killing and increased mortality, consistent with a role
for TNF in host defense against bacterial pneumonia.
TNF has also been implicated in the pathogenesis of
cigarette smoke-induced emphysema because TNFmediated processes were responsible for 70% of the
airspace enlargement in a murine model, which may
be a consequence of enhanced neutrophil recruitment. Similarly, overexpression of TNF in murine
type II alveolar epithelial cells has been associated
with severe airspace enlargement and pulmonary
hypertension. TNF may also contribute to weight
loss and cachexia associated with severe chronic obstructive pulmonary disease.
TNF has also been implicated in the pathogenesis
of pulmonary sarcoidosis and the acute respiratory
distress syndrome. Patients with progressive or severe
sarcoidosis demonstrate increased spontaneous TNF
production from alveolar macrophages, whereas elevated TNF release from alveolar macrophages may
correlate with disease progression. TNF may play an
important role in acute lung injury because TNF or
TNFR1 knockout mice demonstrated attenuated
lung neutrophil recruitment and microvascular permeability in a murine model of hemorrhage-induced
acute lung injury. Lastly, although clinical evidence
suggests a possible role for TNF in the pathogenesis
of asthma, investigations utilizing murine models of
allergic asthma have produced varied results. The
results of future clinical trials will clarify whether a
role exists for anti-TNF therapies in the treatment of
these pulmonary diseases.
See also: Acute Respiratory Distress Syndrome.
Apoptosis. Asthma: Overview. Chronic Obstructive
Pulmonary Disease: Overview. Human Immunodeficiency Virus. Leukocytes: Pulmonary Macrophages.
Pneumonia: Community Acquired Pneumonia, Bacterial
and Other Common Pathogens; Mycobacterial. Pulmonary Fibrosis. Systemic Disease: Sarcoidosis.
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