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*
Department of Paediatric Gastroenterology, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, London, United Kingdom;
Department of Paediatrics, Chelsea and Westminster Hospital, London, United Kingdom; and
Department of Molecular Biology, Genentech, South San Francisco, CA 94402
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Abstract |
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 Top Abstract IntroductionMaterials and Methods4ResultsDiscussionReferences |
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Ab therapy has been shown to be of benefit in the
treatment of active Crohn’s disease, but the tissue-injuring processes
in the gut mediated by TNF- that might be inhibited by neutralizing
Ab are unknown. In this work, we have used a p55 TNF receptor-human IgG
fusion protein (TNFR-IgG) to prevent the severe mucosal injury that
ensues when lamina propria T cells in explant cultures of human fetal
small intestine are directly activated with the lectin PWM. Following T
cell activation and associated with mucosal injury, there is a marked
elevation of soluble TNF- in organ culture supernatants and a large
increase in TNF- mRNA transcripts. The addition of TNFR-IgG at the
onset of cultures greatly reduced PWM-induced tissue injury, without
inhibiting the increase in TNF- and IFN-
transcripts seen
following T cell activation. Mucosal injury in this model is mediated
by endogenously-produced matrix metalloproteinases (MMPs). When
TNFR-IgG was added to PWM-stimulated explants, there was a reduction in
MMPs in the explant culture supernatants, especially stromelysin-1.
Recombinant TNF- and IL-1ß added directly to mucosal mesenchymal
cell lines also caused an increase in MMP production, but only the
former was inhibited by the TNFR-IgG. These results suggest that one of
the ways in which TNF- causes tissue injury in the gut is by
stimulating mucosal mesenchymal cell to secrete matrix-degrading
metalloproteinases. Neutralization of this activity should help
maintain tissue integrity. |
Introduction |
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TopAbstractIntroductionMaterials and Methods4ResultsDiscussionReferences |
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is a pleiotropic
cytokine produced mainly by monocytes, macrophages, and T cells, which
plays a key role in host defense; however, it also contributes to
tissue injury in inflammatory and autoimmune diseases, including septic
shock and rheumatoid arthritis when produced in excess (1, 2, 3). There is
increasing evidence that TNF- is also important in inflammatory
bowel disease (IBD).3 In Crohn’s disease,
serum TNF- concentrations are moderately increased (4).
TNF--positive cells can be detected by immunohistochemistry
throughout the mucosa, and elevated numbers of TNF--secreting cells
are present in single-cell suspensions of mucosal biopsies (5, 6).
TNF- immunoreactive cells are also elevated in the gut in ulcerative
colitis, but in this case the increase is restricted to the mucosa (6).
High concentrations of TNF- can be detected in stools of children
with active IBD (7), and isolated mononuclear cells from patients with
IBD secrete more TNF- than cells from control patients (8).
Several studies have addressed the potential efficacy of
anti-TNF- treatment in Crohn’s disease (reviewed in 9 .
Treatment of patients with severe steroid refractory Crohn’s colitis
with a TNF- Ab (cA2) resulted in a rapid decrease in Crohn’s
disease activity index, remarkable healing of mucosal ulcers, complete
clinical remission, and few side effects (10). A single infusion of cA2
Ab therapy has been shown to be efficacious in inducing remission in
Crohn’s disease in a placebo-controlled, double-blind multicenter
trial (11). Work that has been reported in abstract form shows that
repeated administration of cA2 Ab can also maintain remission
(12).
The mechanism by which anti-TNF- therapy inhibits inflammation
in Crohn’s disease is unknown. The Abs may be neutralizing soluble
TNF- in the interstitial fluids. Alternatively, the Ab most widely
reported, cA2, is of the IgG1 isotype and may bind onto membrane-bound
TNF- and fix complement. Thus, there is a possibility that the
therapeutic effect is not due to TNF- neutralization, but is by the
cytotoxic killing of TNF--secreting T cells and macrophages, thereby
lowering the concentrations of all tissue cytokines. In this regard,
another TNF- Ab of the IgG4 isotype, which is unlikely to fix
complement, appears to be less effective in Crohn’s disease than the
cA2 Ab (13). Underlying all of this is the lack of knowledge of the
mechanisms by which increased concentrations of TNF- cause tissue
injury in the gut.
We have recently demonstrated that following activation of lamina
propria T cells in explant cultures of human fetal small intestine with
the lectin PWM there is complete destruction of the mucosa (14, 15).
This is a relatively acute model of tissue injury compared with the
chronic response seen in Crohn’s disease in patients, and obviously
lacks the complexity seen in vivo. Nevertheless, in both Crohn’s
disease and in the fetal gut system, Th1 cells appear to be of major
importance (15, 16, 17). The final mediators of tissue injury in the fetal
gut explant model appear to be matrix metalloproteinases (MMPs),
especially stromelysin-1. When stromal cells from fetal intestine are
stimulated in vitro with TNF- they produce extremely large amounts
of MMPs (15). Therefore a possible therapeutic effect of
anti-TNF- therapy is to interrupt this pathway of tissue injury
and prevent mucosal matrix degradation. In this work, we have therefore
used a soluble TNF- receptor-human IgG fusion protein to attempt to
inhibit injury in the fetal gut model. We show that the fusion protein
is highly effective in preventing injury and that this is related to a
down-regulation of MMP production.
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Materials and Methods4 |
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TopAbstractIntroductionMaterials and Methods4ResultsDiscussionReferences |
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TNFR-IgG fusion protein consists of the extracellular portion of
human p55 TNFR linked to the hinge, CH2 and CH3
domains of human IgG1 heavy chain (18). The TNF--binding affinity of
TNFR-IgG is 75 to 100 pM (18, 19). It has been shown that this TNFR-IgG
is more potent than anti-TNF- mAb in protecting against rat
endotoxic shock in vivo (18, 20) and that its ability to neutralize the
activity of endogenous TNF- in murine listeriosis is 10-fold that of
anti-TNF- mAb TN3-19.12 (21).
Organ culture of human fetal small intestine
Second-trimester human fetal small intestine was obtained within 2 h of surgical termination from the Medical Research Council Tissue Bank, The Hammersmith Hospital, London, U.K. All the specimens used in this study were aged between 16 and 18 wk gestation.
Culture of human fetal small intestine explants in serum-free medium was performed as previously described (22, 23). Mucosal T cells were activated by the addition of PWM (10 µg/ml, Sigma Chemical, Dorset, U.K.) to the explant culture. TNFR-IgG fusion protein (10 µg/ml, Genentech, South San Francisco, CA) was added together with PWM at the onset of culture. Human IgG (10 µg/ml, Sigma Chemical) was used as a control.
Fetal explants were cultured up to day 3 unless otherwise stated. Tissue explants were snap-frozen in liquid nitrogen-cooled 2-methylbutane and the culture supernatants were spun at 1200 x g for 10 min to remove cell debris and stored at -70°C for further analysis.
TNF- production in human fetal small intestine explant culture
Aliquots of 100 µl of explant culture supernatant from each
20-explant culture were assayed for TNF- by an ELISA (R&D Systems
Europe, Abingdon, U.K.). All assays were standardized using recombinant
protein and were done in duplicate. The lower limit of sensitivity of
the assay was 1 pg/ml. The production of TNF- was normalized to
explant tissue protein. Culture explants were homogenized in buffer
that contains 1% Triton X-100 in TBS. The protein content was
determined by the Bio-Rad Protein Assay.
Quantification of cytokine mRNA
To examine whether the TNFR-IgG fusion protein had any effect on
the T cell-mediated immunity of the intestinal explants, the expression
of IFN- and TNF- mRNA was examined by competitive reverse
transcriptase PCR according to the method of Jung et al. (24). A DNA
construct pHCQ1 was used that encodes the primer sites for various
cytokines, a kind gift from Dr. Martin Kagnoff (Department of Medicine,
University of California at San Diego, La Jolla, CA). This was
transcribed in vitro using T7 RNA polymerase (Promega, Madison, WI)
according to the manufacturers instructions to yield a synthetic RNA
encoding the cytokine primer sites identical to those on cellular
target RNA. A competitive PCR was then performed after the standard RNA
and test RNA were coreverse transcribed. PCR products were
electrophoresed in 1% agarose gels. Bands were visualized and their
intensities were quantified by densitometry (Seescan 1D gel analysis
package v1.00, Seescan, Cambridge, U.K.). The ratios of the band
intensities of the PCR products from the standard RNA and the target
RNA were plotted against the starting amount of standard RNA molecules
on a semilogarithmic scale. In this way, the point at which the
starting number of standard RNA transcripts is equal to the starting
amount of cellular target RNA transcripts can be determined. This
technique allows us to quantify the number of cytokine transcripts in a
tissue sample down to as little as 1,000 transcripts per microgram of
total RNA.
Morphologic and histologic assessment of fetal explants
The morphology of the living explants was assessed by inverted phase contrast microscopy and the changes in gross morphology were assessed by the criteria described previously (23). In brief, normal explants showed long villi and there was little surface debris. The explants showing adaptive changes had partial villus atrophy and crypt hyperplasia with extensive debris on the surface of the mucosa; the villi were short and of variable length but clearly identifiable. However, no villi were visible in those destroyed explants. The surface of the explants was covered with debris and when the cell debris was shaken off the avillous surface was clearly apparent. Frozen sections of explants were stained with hematoxylin and eosin (H&E) to view the histology.
Mucosal mesenchymal cells
Human fetal mesenchymal cells were isolated and characterized
according to the method described previously (15). A total of 5 x
105 cells were seeded into 25-cm2 area culture
flasks and maintained in MEM and 10% FCS until confluence. The cell
layer was then washed twice in serum-free MEM and cultured with
rTNF- (1 ng/ml; R&D Systems Europe) or IL-1ß (1 ng/ml; R&D Systems
Europe) in serial dilutions of TNFR-IgG for 48 h. Culture
supernatants were removed and spun at 1200 x g for 10
min to remove cell debris.
Detection of MMPs
To determine the effect of TNFR-IgG on MMP and TIMP production by explants and mucosal mesenchymal cells, Western blotting and substrate gel electrophoresis were conducted using the reagents and according to the methods described previously (15). In all cases, equivalent amounts of protein were loaded onto each lane of the gel. Computer-assisted scanning densitometry (SeeScan) was used to analyze the intensity of the immunoreactive bands.
Statistical analysis
Differences between groups were compared using either the Mann-Whitney U test, if the data were not normally distributed, or Student’s t test, if the observations were consistent with a sample from a normally distributed population.
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Results |
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TopAbstractIntroductionMaterials and Methods4ResultsDiscussionReferences |
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protein and mRNA in PWM-stimulated human
intestinal explants
Following activation of lamina propria T cells with PWM, there was
a 10-fold increase in TNF- protein secreted in the day 3
PWM-stimulated explant culture supernatants compared with those of
unstimulated controls (Fig. 1
). Using
competitive quantitative PCR, it is also clear that T cell activation
induces a significant increase in TNF- transcripts in the fetal gut
explants (a 1000-fold increase in the number of TNF- transcripts in
PWM-stimulated explants compared with controls), as well as a large
increase in IFN- transcripts (Fig. 2).
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In two separate experiments, PWM produced severe tissue injury
with complete mucosal destruction in 100% of the explants. If TNFR-IgG
was added at the onset of the cultures along with the PWM, about 93%
(37/40 explants) were morphologically normal (Table I). Control normal human IgG had no
effect on tissue injury. Histology showed that control explants had
normal villi, short crypts, and columnar epithelium, while in day 3
explants cultured with PWM, there was severe tissue injury with
epithelial cell shedding and only shreds of the mucosa remaining (Fig. 3). The addition of TNFR-IgG at 10
µg/ml dramatically inhibited tissue injury, and villus morphology was
retained even though there is some crypt hypertrophy (Fig. 3).
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transcripts and IFN- transcripts in the PWM-treated
explants cultured with TNFR-IgG compared with PWM alone, and that they
were markedly elevated above the values detected in control explants
and explants cultured with the fusion protein alone (Fig. 2
). TNFR-IgG fusion protein inhibits the production of MMPs
As we have shown that activation of lamina propria T cells results
in increased concentrations of MMPs in the organ culture supernatants
and that inhibition of their enzymatic activity ameliorates injury
(14), Western blotting was used to investigate the effect of TNFR-IgG
on PWM-induced MMP production. In control supernatants, collagenase,
stromelysin-1, gelatinase A and B, and TIMP-1 were detectable. T cell
activation with PWM resulted in increased amounts of both the inactive
and active forms of MMP-1, and MMP-3, increased amounts of the inactive
forms of gelatinase A and B, and increased TIMP-1. Normal human IgG did
not have any inhibitory effect on PWM-induced MMP production (Fig. 4); however, TNFR-IgG produced a slight
decrease in collagenase and gelatinase B, had little effect on
gelatinase A and TIMP-1 immunoreactivity, but markedly reduced
stromelysin-1. When MMP-specific bands (both latent form and active
form) were analyzed by quantitative scanning densitometry, the total
amount of interstitial collagenase was 1.7 times lower, stromelysin-1
was 16.8 times lower, and gelatinase-B was 7.8 times lower in explants
cocultured with PWM and TNFR-IgG fusion protein than in those cultured
with PWM and normal human IgG. When TIMP-1 bands were scanned, the
density was three times higher in PWM-stimulated cultures than in
control explant supernatants alone but was only slightly decreased in
explants cocultured with PWM and TNFR-IgG fusion protein.
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but not IL-1ß-induced production of
MMPs and TIMP-1 by mucosal mesenchymal cells
TNF- (1 ng/ml) causes an increase in MMP (especially
collagenase and stromelysin-1) and TIMP-1 production by mucosal
mesenchymal cells (Fig. 5A).
When graded doses of TNFR-IgG were added along with the TNF-,
significant inhibition of collagenase, stromelysin-1, gelatinase B, and
TIMP-1 was seen, even at 10 ng/ml TNFR-IgG. Gelatinase A was
unaffected. This was confirmed by zymography (Fig. 5B). As a
specificity control, IL-1ß was also investigated (Fig. 5, C and D). It also produced an increase in MMPs,
but had less effect on TIMP-1. However, in the presence of graded doses
of TNFR-IgG, IL-1ß-induced MMP production was unaltered (Fig. 5, C and D).
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Discussion |
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TopAbstractIntroductionMaterials and Methods4ResultsDiscussionReferences |
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There was no evidence that the TNFR-IgG had any unexpected
immunosuppressive effects since neither TNF- or IFN- transcripts
were reduced, and therefore we can attribute its effects to
neutralization of the TNF- within the tissue. Whether this occurs by
binding soluble TNF- trimers or membrane-bound molecules is unknown;
however, we can exclude complement-mediated destruction of
TNF-secreting cells since the explants were cultured in serum-free
medium.
Elevated concentrations of TNF- were present in organ culture
supernatants following activation of lamina propria T cells with PWM.
This TNF- likely originates from lamina propria T cells (25), or
tissue macrophages activated by T cell products. In patients with IBD,
the majority of the TNF--secreting cells are monocytes recently
extravasated from the blood (26); there is no a priori reason why
resident macrophages should not make TNF-, especially in the fetus
where there are marked differences between the class II+
MHC cells compared with postnatal intestine (27). In any regard, the
cellular source of the TNF- is probably less important than its
functional consequences, since in IBD, TNF- has been detected in
epithelial cells, fibroblasts, neutrophils, mast cells, eosinophils,
Paneth cells, and macrophages (28), but how many of these cells produce
meaningful amounts is unknown.
The effects of TNF- in the gut have been studied in some detail. In
a series of experiments, Hsueh and colleagues (29, 30) showed that
TNF-, synergizing with endotoxin, caused elevated mucosal
phospholipase A2 and increased local concentrations of
platelet-activating factor, resulting in bowel necrosis. TNF- has
also been reported to kill epithelial cells (31) and impair epithelial
barrier function (32). It can also up-regulate adhesion molecule
expression on human mucosal vascular endothelial cells in culture (33).
All of these pathways probably play a role in tissue injury in the gut.
However, since the lamina propria is a soft tissue consisting of
structural collagens, proteoglycans, and glycoproteins such as
fibronectin, glycosaminoglycans, and ground substance, we have been
interested in the role of matrix-degrading enzymes in tissue injury in
the gut.
TNF- markedly up-regulates interstitial collagenase and
stromelysin-1 production by mucosal mesenchymal cells (15), as it does
with mesenchymal cells from other tissues (34, 35, 36, 37, 38). In vitro
experiments using mesenchymal cell lines (Fig. 5, A and
B) showed that the addition of very low doses of TNFR-IgG
(10 ng/ml) were effective at inhibiting TNF--induced interstitial
collagenase and stromelysin-1 production. The increase in MMPs was also
seen in the culture supernatants of explants treated with PWM in which
there was extensive mucosal injury, confirming previous studies (15).
When the TNFR-IgG was added, there was a marked reduction in
stromelysin-1 and gelatinase B, but much less of a reduction in
collagenase and gelatinase A. We consider it highly probable that the
reduction in stromelysin-1 is the reason for the maintenance of
structural integrity in the PWM-stimulated explants cocultured with
TNFR-IgG. In previous studies, we have added recombinant gelatinase A
and B, stromelysin-1, and interstitial collagenase to explants and only
stromelysin causes tissue injury at low concentrations. In addition, a
stromelysin/gelatinase inhibitor is much more effective than a
collagenase inhibitor in preventing injury (15). We would therefore
suggest that a major role for TNF- in gut injury is to induce
stromelysin-1 production by mucosal mesenchymal cells. It is also
worthy of comment that although interstitial collagenase was markedly
increased in PWM-stimulated explants, it was only minimally decreased
when TNFR-IgG was added. This also suggests that stromelysin-1 is more
important than interstitial collagenase in this model, but it also
suggests that, whatever is causing the elevated collagenase, it is a
factor other than TNF-, such as IL-1ß. Alternatively, the cell
source in the tissue may be different. Perhaps the collagenase is made
predominantly by macrophages whereas the stromelysin is made by
TNF--activated mesenchymal cells.
One of the puzzles in interpreting the therapeutic effects of
anti-TNF- therapy in Crohn’s disease as specific inhibition of
TNF- is that the treatment should have no effect on other
proinflammatory cytokines such as IL-1ß, unless one evokes a complex
feedback loop whereby TNF- promotes the production of other
cytokines. IL-1ß concentrations are markedly elevated in IBD (39),
and in rabbit colitis, IL-1R antagonist (IL-1RA) is highly effective at
preventing gut injury (40). Studies in man have revealed an imbalance
of IL-1ß/IL-1RA ratios (41, 42) in the gut in IBD as well as elevated
concentrations of platelet-activating factor, IL-6, IL-8, IL-15, etc.
(43). There have, however, been no reported clinical studies
on the use of IL-1RA in IBD. Even if one neutralizes TNF-, however,
one would expect that local IL-1ß would also act on mucosal
mesenchymal cells to increase stromelysin-1 production (Fig. 5) and
maintain tissue injury. However, this does not appear to be the case,
either in fetal gut explants or in patients, so perhaps in the tissue
microenvironment there is sufficient IL-1RA to inhibit IL-1ß, despite
the altered ratios.
Finally, we also investigated TIMP-1 production following T cell
activation in explants and in mesenchymal cells activated with
cytokines. In the explants, there was a modest increase in TIMP-1 in
the PWM-stimulated explant supernatants, which was only slightly
decreased by TNFR-IgG. In vitro, mesenchymal cells secrete marginally
more TIMP-1 when stimulated with rTNF- and this was inhibited by the
TNFR-IgG. Nothing is known about the regulation of TIMP-1 production in
the gut, but these preliminary results suggest that it may be possible
to down-regulate MMPs without such a dramatic effect on TIMP-1, thereby
increasing the capacity of the gut’s endogenous inhibitors to prevent
MMP-mediated matrix degradation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to: Sylvia L. F. Pender, Department of Paediatric Gastroenterology, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, Suite 31, Dominion House, 59 Bartholomew’s Close, London, EC1A 7BE, U.K. E-mail address:
3 Abbreviations used in this paper: IBD, inflammatory bowel disease; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; IL-1RA, IL-1 receptor antagonist; H&E, hematoxylin and eosin.
4 This study received ethical approval from the Hackney and District Health Authority, London, U.K.
Received for publication September 23, 1997. Accepted for publication December 22, 1997.
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References |
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TopAbstractIntroductionMaterials and Methods4ResultsDiscussionReferences |
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for Crohn’s disease. N. Engl. J. Med. 337:1029. chimeric Ab (cA2) effectively maintains cA2-induced remission in Crohn’s disease. Gastroenterology 112:(Suppl.):A1078.
in Crohn’s disease. Lancet 349:521.[Medline]
, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. J. Immunol. 157:1261.[Abstract]
and TNF-ß and inhibition of TNF activity. J. Biol. Chem. 266:18324. in colonic Crohn’s disease. J. Leukocyte Biol. 58:284.[Abstract]
and ligation of Fas antigen. J. Immunol. 155:4147.[Abstract]
. Matrix Biol. 14:241.[Medline]
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S Daum, U Bauer, H-D Foss, D Schuppan, H Stein, E-O Riecken, and R Ullrich Increased expression of mRNA for matrix metalloproteinases-1 and -3 and tissue inhibitor of metalloproteinases-1 in intestinal biopsy specimens from patients with coeliac disease Gut, January 1, 1999; 44(1): 17 - 25. [Abstract] [Full Text] [PDF]
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M.F. Neurath, B. Weigmann, S. Finotto, J. Glickman, E. Nieuwenhuis, H. Iijima, A. Mizoguchi, E. Mizoguchi, J. Mudter, P.R. Galle, et al. The Transcription Factor T-bet Regulates Mucosal T Cell Activation in Experimental Colitis and Crohn's Disease J. Exp. Med., May 6, 2002; 195(9): 1129 - 1143. [Abstract] [Full Text] [PDF]
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