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We thank Rosenstiel et al for their interest in our paper
regarding the role of the chimeric anti-tumor necrosis factor (TNF)-alpha
antibody, infliximab, in reverting defective lamina propria T-cell
apoptosis in Crohn’s disease via a caspase-dependent pathway.
In their interesting letter Rosenstiel et al investigate the complex
TNF-alpha blocker machinery in Crohn’s disease...
In their interesting letter Rosenstiel et al investigate the complex
TNF-alpha blocker machinery in Crohn’s disease by analyzing the influence
of infliximab and recombinant TNF receptor/immunoglobulin G fusion
protein, etanercept, on p38 mitogen-activated protein kinase (MAPK)
activation, apoptosis and signal transducer and activator of transcription
3 (STAT3) tyrosine phosphorylation in lamina propria T-cells from active
Crohn’s disease patients. Infliximab, but not etanercept, has been shown
to result in p38MAPK activation and apoptosis, while both these agents
were able to reduce STAT3 phosphorylation in Crohn’s disease lamina
If data on the differences in mucosal T-cell apoptosis-inducing
capacity of infliximab and etanercept are in keeping with the results
obtained by van den Brande et al, the observation that infliximab
induces activation of a pro-inflammatory MAPK does not tally with the
evidence that the guanylhydrazone c-Jun N-terminal kinase/p38 inhibitor
CNI-1493 induces clinical improvement in steroid-resistant Crohn’s disease
patients. The reason for this discrepancy probably lies in the complex
and dichotomal role of p38MAPK in the inflammatory signal transduction.
p38MAPK inhibition is efficacious in chronic inflammatory disease but not
in acute experimental colitis, where it causes adverse effects [3,4] and
it shares differential actions in different T-cell subpopulations . In
naïve T cells, in fact, the p38 inhibitor SB203580 inhibits the pro-
inflammatory cytokine production, whereas in T helper type (Th) 1 cells it
does not affect the TNF-alpha release but strongly impairs the anti-
inflammatory IL-10 production, thus tilting the balance between Th1 and
Th2 cytokines to the former.
Since several lines of evidence have also suggested the pro-apoptotic
role of the p38 pathway, Waetzig et al hypothesized that the
increased phosphorylation of the p38MAPK downstream effector ATF-2, found
after infliximab treatment only in the responder Crohn’s disease patients,
could enhance the infliximab-induced apoptosis of immune cells in this
subgroup of patients. However, the similar proportion of lamina propria
immune cell apoptosis after infliximab treatment in both responders and
non responders suggests that differences in the signal transduction
downstream of p38MAPK are not related to the infliximab-induced immune
Disturbances in STAT signaling pathways, which trasduce the
immunomodulatory messages of most of cytokines/cytokine receptors, have
been shown to be involved in the pathogenesis of Crohn’s disease. In
particular, STAT3 and STAT4 proteins are constitutively activated in
lamina propria CD4+ T-cells from active Crohn’s disease patients. The
finding shown by Rosenstiel et al that both infliximab and etanercept are
able to reduce the in vitro STAT3 tyrosine phosphorylation of Crohn’s
disease lamina propria T-cells is probably related to the capacity of both
these agents to neutralize soluble TNF-alpha. Interestingly, the absence
of quantitative differences in the potential of infliximab and etanercept
in downregulating STAT3 signaling is consistent with the data of van den
Brande et al, who have shown that both drugs are able to neutralize
soluble TNF-alpha to a similar extent.
Taken together, these findings suggest that both the apoptotic and
non-apoptotic intracellular signalling pathways underlying the therapeutic
benefit of anti-TNF-alpha strategies in Crohn’s disease are multifacetted
and more complex than initially thought. Dissecting the MAPK signaling
cascades that are selectively activated in the abnormal immune response of
inflammatory bowel disease is critical in the identification of selective
targets and development of new and rational therapies for treatment of
(1) Di Sabatino A, Ciccocioppo R, Cinque B, et al. Defective mucosal T
cell death is sustainably reverted by infliximab in a caspase dependent
pathway in Crohn’s disease. Gut 2004;53:70-7.
(2) van den Brande JMH, Braat H, van den Brink GR, et al. Infliximab
but not etanercept induces apoptosis in lamina propria T-lymphocytes from
patients with Crohn’s disease. Gastroenterology 2003;124:1774-85.
(3) Hommes D, van den Blink B, Plasse T, et al. Inhibition of stress-
activated MAP kinases induces clinical improvement in moderate to severe
Crohn’s disease. Gastroenterology 2002;122:7-14.
(4) ten Hove T, van den Blink B, Pronk I, et al. Dichotomal role of
inhibition of p38 MAPK with SB 203580 in experimetal colitis. Gut
(5) Waetzig GH, Rosenstiel P, Nikolaus S, et al. Differential p38
mitogen-activated protein kinase target phosphorylation in responders and
nonresponders to infliximab. Gastroenterology 2003;125:633-4; author reply
(6) Wada T, Penninger JM. Mitogen-activated protein kinases in
apoptosis regulation. Oncogene 2004;23:2838-49.
(7) Lovato P, Brender C, Agnholt J, et al. Constitutive STAT3
activation in intestinal T cells from patients with Crohn’s disease. J
Biol Chem 2003;278:16777-81.
There is growing evidence that the efficacy of anti-TNF-a therapies in
Crohn’s disease (CD) may critically depend on the binding of the
transmembrane precursor of TNF-a (mTNF-a), thus eliciting complex
intracellular signaling events, a process described as ‘reverse
In their recent paper, Di Sabatino et al. have shown
that infliximab reverts defective peripheral and la...
In their recent paper, Di Sabatino et al. have shown
that infliximab reverts defective peripheral and lamina propria lymphocyte
apoptosis in CD patients via a caspase-dependent mechanism, which further
corroborates the findings of prior studies.[1-3,5] It has also been
suggested that the failure of another TNF-binding agent, etanercept
(Enbrel® ;a recombinant TNFR2:Fc fusion protein), to induce peripheral and
lamina propria lymphocyte apoptosis, provides a possible molecular
explanation for the lack of efficacy of etanercept in a randomized,
placebo-controlled trial in active CD.
However, the authors do not discuss other signaling pathways that are
activated via the ligation of transmembrane TNF-a by infliximab, e.g. we
have shown that infliximab also transiently activates p38 MAPK in
monocytes in vitro and in the lamina propria of CD patients in vivo 3.
Responders and non-responders to infliximab differ in the pattern of
mucosal p38MAPK target phosphorylation, but not caspase-3 activation,
further emphasizing the complex modulation of intracellular signaling
pathways beyond the mere neutralization of sTNF-a. To show if these
signaling pathways are also activated in primary T-cells, we analyzed the
influence of infliximab and etanercept on p38MAPK activation and apoptosis
in an established model of non-transformed, in situ activated T-
According to the findings of van den Brande et al, we observed
PARP cleavage as a molecular hallmark of apoptosis in cultures grown with
infliximab (Fig.1A), but not in the presence of etanercept. Whereas no
increase of phosphorylated p38MAPK could be detected after etanercept
stimulation, a significant activation (i.e. dual phosphorylation) of p38
MAPK 24 h after infliximab treatment was observed in 4/5 of the cell lines
derived from CD patients (Fig.1A).
[View Figure 1 A and B]Figure 1
The intestinal CD 4+ T cells were obtained from colonic biopsies from
five patients with Crohn’s disease (median age 34, range 18-49) with an
established diagnosis of Crohn’s disease according to histopathological
and endoscopical criteria. All patients had active disease at inclusion
treated with 5-ASA (2-4g/day, 5 patients), one patients was treated with
prednisone (20mg/day) and four patients with azathioprine (150-200mg/day).
None of the patients recieved anti-TNF-a treatment. Neither antigen nor
feeder cells were added to the intestinal T cells cultures, which
consisted of more than 97% CD3+ /CD4+ T cells.(A) Infliximab, but not etanercept activates p38a and induces apoptosis in
intestinal T-lymphocytes from CD patients. Levels of (phosphorylated) p38
MAPK and PARP in in situ activated intestinal T-lymphocytes were
investigated by Western blot 24h after the stimulation with the respective
TNF-a binding agent as described 3. (n=5, data are representative of
experiments performed in all patients)(B) STAT3 is activated in CD4+ cells in the intestinal mucosa in CD
patients in vivo and downregulated by both infliximab and etanercept in
intestinal T-cells from CD patients in vitro. Western blot and
immunofluorescence staining were performed as described previously.
Filled arrowheads, cells positive for phospho-Y-STAT3 (anti-phospho-Y-
STAT3, 1/100, Cell Signaling Technology) and CD4 (anti-CD4, 1/500, BD
PharMingen, San Diego, CA); open arrowheads, CD4 immunoreactivity only
(n=5 for immunofluorescence and Western blot, representative result for
We have demonstrated previously that constitutive tyrosine phosphorylation
of the transcription factor signal transducer and activator of
transcription 3 (STAT3) may represent a specific feature of intestinal T
cells from Crohn's disease. Tyrosine-phosphorylated STAT3 can be found
in CD4+ cells in the inflamed mucosa as shown by immunofluorescence
analysis. (Fig.1B). Surprisingly, infliximab and etanercept were able to
reduce STAT3 phosphorylation in T-cells from CD patients (cultured with IL
-2 and IL-4) to a similar extent (Fig.1B). It is tempting to speculate
that the previously reported downregulation of IFN-g/GM-CSF by both
infliximab and etanercept is also involved in the decrease of STAT3
tyrosine phosphorylation, as both cytokines are potent inducers of STAT3
activation via Janus kinases. Mechanistically, this common action of
infliximab and etanercept might be due to the neutralization of an
autocrine loop of constitutively released sTNF-a. Although the findings
suggest that inhibition of cytokine-dependent, inducible STAT3
phosphorylation could be dispensable for therapeutic efficacy in CD,
aberrant constitutive STAT phosphorylation in T-lymphocytes may still have
an important modulatory role in chronic intestinal inflammation 9.
These observations corroborate the hypothesis, that TNF-a binding
agents may exert distinct functions on lymphocyte activation and survival,
either by ‘reverse signaling’ via binding of mTNF-a resulting in p38MAPK
activation and apoptosis or by neutralization of sTNF-a, which in this
model may serve to inhibit STAT3 signaling. The individual properties of
TNF-a blockers to induce either of these complex molecular actions may be
differentially responsible for therapeutic success or failure in chronic
inflammatory disorders such as rheumatoid arthritis, psoriasis or CD. In
CD, the ligation of mTNF-a, subsequent apoptotic processes and MAPK
signaling seems to be critically required. Molecular dissection of mTNF-a
apoptotic and non-apoptotic signaling may have important implications for
the design of future therapeutic strategies in IBD.
1. ten Hove T, van Montfrans C, Peppelenbosch MP, van Deventer SJ.
Infliximab treatment induces apoptosis of lamina propria T lymphocytes in
Crohn's disease. Gut 2002;50:206-11.
2. Lugering A, Schmidt M, Lugering N, Pauels HG, Domschke W, Kucharzik T.
Infliximab induces apoptosis in monocytes from patients with chronic
active Crohn's disease by using a caspase-dependent pathway.
3. Waetzig GH, Seegert D, Rosenstiel P, Nikolaus S, Schreiber S. p38
mitogen-activated protein kinase is activated and linked to TNF-alpha
signaling in inflammatory bowel disease. J Immunol 2002;168:5342-51.
4. Di Sabatino A, Ciccocioppo R, Cinque B, Millimaggi D, Morera R,
Ricevuti L, Cifone MG, Corazza GR. Defective mucosal T cell death is
sustainably reverted by infliximab in a caspase dependent pathway in
Crohn's disease. Gut 2004;53:70-7.
5. Van den Brande JM, Braat H, van den Brink GR, Versteeg HH, Bauer CA,
Hoedemaeker I, van Montfrans C, Hommes DW, Peppelenbosch MP, van Deventer
SJ. Infliximab but not etanercept induces apoptosis in lamina propria T-
lymphocytes from patients with Crohn's disease. Gastroenterology
6. Sandborn WJ, Hanauer SB, Katz S, Safdi M, Wolf DG, Baerg RD, Tremaine
WJ, Johnson T, Diehl NN, Zinsmeister AR. Etanercept for active Crohn's
disease: a randomized, double-blind, placebo-controlled trial.
7. Waetzig GH, Rosenstiel P, Nikolaus S, Seegert D, Schreiber S.
Differential p38 mitogen-activated protein kinase target phosphorylation
in responders and nonresponders to infliximab. Gastroenterology
2003;125:633-4; author reply 635-6.
8. Agnholt J, Dahlerup JF, Kaltoft K. The effect of etanercept and
infliximab on the production of tumour necrosis factor alpha, interferon-
gamma and GM-CSF in in vivo activated intestinal T lymphocyte cultures.
9. Lovato P, Brender C, Agnholt J, Kelsen J, Kaltoft K, Svejgaard A,
Eriksen KW, Woetmann A, Odum N. Constitutive STAT3 activation in
intestinal T cells from patients with Crohn's disease. J Biol Chem