(Received for publication, December 13, 1994; and in revised form, January 26, 1995)
From the
Tumor necrosis factor (TNF) is a pleiotropic cytokine which has
both cytotoxic and proliferative effects. HuT 78, a T-cell line derived
from a Sezary lymphoma, is resistant to the cytotoxic effects of TNF,
suggesting that TNF may be a growth factor for this cell line. The aim
of this study was to determine whether autocrine TNF production could
function as a growth factor for HuT 78. Resting HuT 78 and K-4 cells, a
protein kinase C--deficient clone of HuT 78, both produced
significant amounts of TNF compared with Jurkat cells. Thymidine
incorporation by HuT 78 and K-4 cells was inhibited by 90.5 and 73.2%,
respectively, with addition of a neutralizing monoclonal antibody to
TNF
, suggesting that TNF is an autocrine growth factor for these
cells. HuT 78 and K-4 cells also expressed high levels of
constitutively active NF
B, unlike Jurkat cells, which expressed
high levels only upon activation with TNF or phorbol 12-myristate
13-acetate. p50 was the major component in the NF
B complexes in
HuT 78 and K-4 cells. Anti-TNF
antibody dramatically decreased
levels of NF
B in both HuT 78 and K-4 cells. As the TNF gene has an
NF
B binding motif, an autocrine loop involving TNF induction of
NF
B is therefore likely in these cells. These findings in a
neoplastic T-cell line suggest that therapy directed against TNF could
be effective in a subset of T-cell lymphomas.
Tumor necrosis factor (TNF
) (
)is a potent
cytokine with a wide range of biological activities(1) . It was
initially described in serum of endotoxin-treated mice as the mediator
of the necrosis of some transplantable tumors(2) . It has since
been reported to be cytotoxic to certain transformed cell lines (3, 4, 5) and may act either as a mediator in
beneficial processes of host defense, immunity, and tissue homeostasis
or in the pathogenesis of infection, tissue injury, and
inflammation(6) . In vitro, TNF has been shown to be
involved in mediating a wide range of biological activities, including
differentiation, antiviral responses, and proliferation. TNF stimulates
proliferation of many cell types, both lymphoid and non-lymphoid,
including thymocytes(7) , fibroblasts (5) ,
chondrocytes(8) , and some human cancers, including chronic
lymphoid leukemia and ovarian cancer(9, 10) . TNF is
synthesized by a broad range of cells, including macrophages/monocytes,
T and B lymphocytes, NK cells(1) , mast cells(11) ,
some transformed cell lines (12, 13) , and breast and
epithelial tumor cells(14, 15) . The activated
macrophage is considered to be the main producer of TNF in
vivo(16) . The most potent inducer of TNF production in
these cells is lipopolysaccharide (LPS) derived from the cell wall of
Gram-negative bacteria(1) . Transformed myeloid cell lines such
as U937 and HL60 can also be induced to produce TNF with LPS and such
agents as phorbol esters and the calcium ionophore
A23187(17, 18) . Similarly, T lymphocytes produce TNF
in response to phorbol esters and calcium ionophore or
mitogens(19) . Certain transformed cell lines produce TNF
constitutively(12, 13) , although the basis for such
constitutive production has not been determined.
The regulation of
production of TNF is complex. There are likely to be four levels of
control: the transcriptional level, the mRNA abundance level, the level
of translation and, finally, posttranslationally. Most attention has
been focussed on regulatory elements responsible for transcriptional
activation. Elements responsive to the transcription factor NFB
are especially important to confer LPS inducibility(20) .
Determining how NF
B becomes activated in cells is therefore likely
to be important for our understanding of TNF induction. The NF
B
family of proteins currently comprises three main groups of molecules.
The first includes NFKB1 p105/p50 and NFKB2
p100/p52(21, 22) . p105 and p100 represent precursor
proteins which are proteolytically cleaved to form p50 and p52,
respectively, which are transcriptionally
active(23, 24) . The second subclass includes RelA
(formerly p65)(25) , c-Rel (26) , v-Rel(27) ,
and RelB(28) , none of which require processing in order to be
active. The third subclass includes I
B
(29) ,
I
B
(30) , and Bcl-3 (31) which regulate the
cytoplasmic retention, DNA binding, and transcriptional activity of
NF
B complexes. The predominant form of NF
B in cells appears
to comprise a heterodimer of p50 and RelA complexed to I
B
,
although other arrangements of NF
B occur and are likely to play a
role in NF
B-driven gene expression(32) . NF
B is
constitutively present and active in the nucleus only in a restricted
subset of cells such as B cells (33) and certain T-cell
lines(34) . Latent NF
B is the predominant form in other
cell types where it is maintained in the cytoplasm complexed to an
inhibitory subunit I
B(35) . Upon activation with agents
such as LPS, interleukin 1 (IL1), and TNF, I
B dissociates from
NF
B, which then translocates to the nucleus where it binds its
decameric DNA recognition sequence. Phosphorylation and proteolysis of
I
B have been implicated in the dissociation process (36, 37) .
Here, we describe for the first time the
constitutive production of TNF and high levels of constitutively active
NFB in a human T lymphoma cell line, HuT 78, which is resistant to
cytotoxicity by TNF. In addition, we demonstrate that a monoclonal
antibody to TNF blocks proliferation of the cells and constitutive
NF
B. The results suggest that an autocrine loop involving TNF
production and NF
B activation may be operating in the cells which
leads to proliferation.
TNF was also measured with a commercially available ELISA
(Innogenetics NV, Antwerp, Belgium). The detection limit for this assay
is 4 pg/ml.The intraassay and interassay coefficients of variation are
5.5 and 7.9% respectively.
Figure 1: TNF production by T-cell clones. HuT 78 and K-4 cells were incubated for 48 h in medium and TNF levels in supernatants measured by L929 bioassay (A) or ELISA (B). Mean ± S.E. of three experiments.
IL2 was not detected in supernatants of resting HuT 78, K-4, or Jurkat cells (data not shown).
Figure 2:
Inhibition of proliferation of T-cell
clones with a monoclonal anti-TNF antibody. HuT 78 and K-4 cells
were incubated for 24 h in medium alone, with 3 ng/ml TNF
, with
anti-IE (1/20 dilution of 3.5 mg/ml stock solution), an irrelevant
control antibody, and with anti-TNF
(1/20 dilution of 3.5 mg/ml
stock solution). Proliferation was assessed by
[
H]thymidine incorporation as described under
``Experimental Procedures.'' Representative of three
experiments, mean ± S.E.
Figure 3:
Time course of activation of NFB in
Hut 78, K-4, and Jurkat T lymphomas. Hut 78 (A), K-4 (B), or Jurkat (C) T lymphomas were incubated with
IL1, PMA, or TNF
(all at 10 ng/ml) or left untreated (lanes
C) for the indicated times and then assessed for NF
B as
described under ``Experimental Procedures.'' NF
B-DNA
complexes are shown.
All of the complexes detected in each of the cell types
appeared to constitute protein components which interacted specifically
with the NFB recognition sequence, as their formation was
inhibited by addition of unlabeled oligonucleotide, containing the
NF
B binding site, to nuclear extracts before incubation with
labeled probe (Fig. 4A). An unlabeled oligonucleotide
containing the OCT1 sequence motif failed to exhibit any such
inhibitory effect. We also examined the extracts for the presence of
p50, RelA, and c-Rel, as shown in Fig. 4B. Samples
prepared from TNF-treated Jurkats were shown to contain p50 and RelA
but no c-Rel, as indicated by the ability of p50 and RelA antisera to
cause a further retardation in probe mobility (Fig. 4B), lanes 2 and 3). In HuT 78
and K-4, a similar result was obtained, although much larger amounts of
p50 were detected than RelA (Fig. 4B, lanes 6 and 7 for HuT 78 and lanes 10 and 11 for K-4). Again,
no c-Rel was detected in either cell type.
Figure 4:
Analysis of NFB in HuT 78, K-4, and
Jurkat T lymphomas. A, 2 µg of nuclear extract protein
prepared from Jurkat cells stimulated with TNF
(10 ng/ml) for 1 h
or unstimulated Hut 78 and K-4 were incubated in the absence (lanes
0) or presence of unlabeled oligonucleotides (1.75 pmol)
containing the NF
B or OCT1 consensus sequences prior to assaying
for NF
B binding activity as described under ``Experimental
Procedures.'' NF
B-DNA complexes are shown. B, 2
µg of nuclear extract protein prepared from Jurkat cells stimulated
with TNF
(10 ng/ml) for 1 h or unstimulated Hut 78 and K-4 were
incubated with the indicated antisera on ice prior to assaying for
NF
B binding activity as described under ``Experimental
Procedures.'' NF
B-DNA complexes are shown. Supershifted
complexes corresponding to p50 and RelA are
indicated.
Figure 5:
Effect of a neutralizing monoclonal
antibody to TNF on NF
B in HuT 78 and K-4. A, Hut 78
cells and K-4 were washed twice in RPMI and were then incubated in
medium alone(-) or 125 µl TNF
antibody (3.5 mg/ml stock
solution) (+) for 24 h, 48 h, or 72 h as indicated. NF
B
binding activity was measured as described under ``Experimental
Procedures.'' NF
B-DNA complexes are shown. B, Hut 78
cells and K-4 were washed twice in RPMI and were then incubated in
medium alone(-) or 125 µl of control antibody anti-IE IgG1
(3.5 mg/ml stock solution) (+) for 24 h, 48 h, or 72 h as
indicated. NF
B binding activity was measured as described under
``Experimental Procedures.'' NF
B-DNA complexes are
shown.
TNF is a pleiotropic cytokine, which is produced mainly by
activated macrophages and lymphocytes in response to many stimuli,
including bacterial toxins and viruses, as well as cytokines, including
TNF itself(1) . It is also produced by a number of transformed
and neoplastic cells(12, 13, 15) . In this
study, we demonstrated that HuT 78, a T-cell lymphoma, and K-4, a PKC
-deficient clone of HuT 78, produce TNF spontaneously but Jurkat,
another T-cell line, however, does not.
TNF is a growth factor for many normal lymphoid and non-lymphoid cell types, including thymocytes, fibroblasts, chondrocytes, and lymphoid cells (T and B lymphocytes) (5, 7, 46, 47) as well as certain neoplastic cells, including ovarian cancer (10) and B-cell chronic lymphoid leukemia(9) .
HuT 78 and K-4 are resistant to the cytotoxic effects of TNF, even at concentrations of up to 30 ng/ml TNF, much higher concentrations than that needed to kill L929 cells (data not shown). Jurkat, which do not produce TNF, are sensitive to TNF-mediated killing(48) . TNF production by other cell types which are resistant to cytotoxicity by TNF have previously been reported(14, 43, 49) , suggesting that TNF may be an autocrine growth factor for certain cell types.
A
monoclonal anti-TNF antibody significantly inhibited proliferation
of HuT 78 and K-4, suggesting that TNF was a growth factor for these
cells. Recently, Pimentel-Muinos et al.(50) suggested
that PMA-induced TNF was an autocrine growth factor for T-cells,
stimulating IL 2 secretion, which then stimulated proliferation.
However, this does not seem to occur in HuT 78 or K-4, as IL2 was not
detected in the supernatants of resting cells after 48 h, suggesting
that IL2 is not involved here (data not shown). Also, K-4 produce
significantly less IL2 than HuT 78 upon stimulation with
PMA(38) , yet the anti-TNF
antibody inhibited
proliferation of K-4.
The identification of two TNF receptors on cells suggested initially that individual receptors might mediate individual functions of killing and proliferation(51) . However, both receptors have since been found to mediate both proliferation and cytotoxicity(52) , therefore both may trigger proliferation in HuT 78.
Once TNF binds to receptors on cells, its
intracellular signaling pathways are complex. TNF has been reported to
activate multiple protein kinases and transcription factors, including
NFB(1) . We detected high levels of constitutive NF
B
in HuT 78 and K-4 cells. In most cell types, NF
B exists in a
latent form in the cytosol complexed to an inhibitory subunit
I
B(35) . Only a limited number of lymphoid cells have been
found to contain constitutively active nuclear NF
B. These include
mature B cells (33) and a few T lymphomas(34) . In
these cells, however, the constitutive activation of NF
B is only
partial as stimulation of the cells results in a further activation.
This does not appear to be the case in HuT 78 and K-4 as stimulation of
the cells with agents which potently activate NF
B in other cells
failed to cause increased activation. The mechanism here appears to
involve the constitutive production of TNF by the cells as a
neutralizing monoclonal antibody to TNF completely blocked the
appearance of NF
B in the nucleus of the cells. As NF
B is
known to play a key role in the control of TNF gene
transcription(20) , an interesting autocrine loop may therefore
be operating in the cells. The role of activated NF
B in the
proliferation of the cells through the induction of other genes is
unresolved. For example, NF
B is known to regulate the c-myc gene(53) , which has a well established role in growth
control. Kitajima et al.(54) have previously
demonstrated that inhibiting NF
B causes an inhibition of
tumorigenesis, in experiments using antisense technology. Hence,
NF
B could have indirect effects on growth control regulation
through interaction with either proto-oncogenes or growth factor genes
involved in tumor growth and proliferation.
Although lymphotoxin is
detected by the L929 bioassay(55) , it is unlikely to be
relevant here as an autocrine growth factor for HuT 78, as the
monoclonal antibody producing virtually complete inhibition of cell
proliferation was specific for TNF. Also, Ware et al.(56) reported that lymphotoxin is not produced by HuT 78
and recently, it has been reported that lymphotoxin does not activate
NF
B(57) .
Attempts to identify proteins within the
NFB complex in the cells revealed the presence of both p50 and
RelA. Two recent studies have shown that HuT 78 nuclei contain high
levels of both p52 (58) and p50 (59) . Two additional
proteins of p84 and p85 were also detected and represented aberrantly
processed p100. The basis for these observations was found to be a
rearrangement of the NF
B2 gene in the cells, resulting in a
protein which had lost an ankyrin repeat domain in its C terminus.
Because ankyrin domains on p100 and p105 are important for cytoplasmic
retention, the authors suggest that the lack of the C-terminal ankyrin
domain results in a protein which is not processed in the normal way
and escapes cytoplasmic retention, thereby localizing to the nucleus.
p85 and p52 were also shown to be transcriptionally
active(58) . The studies did not explain why normal p52 is
found in the nucleus of the cells as this would be expected to be
retained in the cytoplasm complexed to I
B. Our results indicate
that constitutive TNF production by the cells is critical for the
nuclear localization of both p52 and p85, along with p50 and RelA, as a
neutralizing antibody to TNF dramatically decreased nuclear NF
B
levels. This suggests that the missing ankyrin repeat in p85 would not
be sufficient to cause nuclear translocation, the five remaining
repeats being sufficient to retain the protein in the cytoplasm. It is
likely, however, that once the aberrant NF
B has translocated to
the nucleus in response to TNF, a dysregulation in TNF gene expression
must occur which results in a lack of feedback inhibition. In normal
cells, NF
B activation leading to TNF production is self-limiting
through mechanisms likely to involve, at least in part, I
B
induction which acts to neutralize transactivating NF
B. Such
mechanisms appear to be absent in HuT 78. This is most likely due to
the presence of abnormal NF
B2 proteins altering the functioning of
the NF
B system in the cells.
An autocrine role for TNF in
NFB activation has also been demonstrated in a study involving
PMA-stimulated T lymphocytes, where it was shown that activation of
NF
B by PMA required the induction of TNF as neutralizing
antibodies to TNF blocked the response(48) . Our results with
HuT 78 are analagous except that no stimulus was needed to trigger TNF
production. Both studies confirm the importance of autocrine regulation
in NF
B activation in cells and suggest that once TNF is induced,
it will feed back on the cells and activate NF
B. However, the
current study differs significantly from studies on peripheral blood
T-cells. HuT 78 is a malignant T-cell clone derived from a cutaneous
T-cell lymphoma. Inhibition of TNF
blocked proliferation of this
malignant cell line, suggesting the possibility that pharmacological
interventions involving inhibition of TNF production may have potential
effects.
In conclusion, this study demonstrates that proliferation
of HuT 78 is stimulated by the autocrine production of TNF. This also
results in the activation of NFB in the cells, which is likely to
lead to further TNF production. The HuT 78 T-cell line is derived from
a cutaneous Sezary type T-cell lymphoma. The finding that proliferation
of this cell line responds to inhibition of TNF production suggests the
intriguing possibility that anti-TNF strategies could be employed in
the treatment of these lymphomas in the clinical environment.