1. ALTERATIONS DE LA BARRIERE MUQUEUSE ET
Symposium Mayoly sur le Syndrome de l'Intestin Irritable.
Organisateur: Pr. Boucekkine
ALTERATIONS DE LA BARRIERE MUQUEUSE ET
SYNDROME DE L'INTESTIN IRRITABLE
L. Buéno
Unité de Neurogastroentérologie INRA
Toulouse, France
Alger, 25 Avril 2010

2. TRILOGY OF IBS PERIPHERAL PATHOPHYSIOLOGY
Altered gut sensitivity to Distension
Lower threshold of sensitivity (pain) to distension evidenced in 60-70% of IBS patients
Increased perception of pain for a given visceral stimulus (Whitehead et al.1998, ref.)
Colonic mucosal micro-inflammation
Increased number of mast cells, immune cells (Weston et al.1993 and ref.)
Presence of pro-inflammatory cytokines (Gwee et al. 2003)
Release of pro-inflammatory (eicosanoïds)(Jones et al.1982) and pronociceptive agents (Barbara et al. 2005)
Increased paracellular permeability
colonic or intestinal level in PI-IBS (Dunlop,2000),
intestinal in all Rome I (Marschall et al.2004)
colonic in IBS-D patients (Gesce et al. 2008)

3. Trimble et al.1995

4. 1995-2009: Evidence (25 articles) of allodynia in 60-70% of IBS patients
but not confirmed for all gut segments
Trimble et al.1995

5. Repetitive Stimulation Sensitizes the Spinal Cord
Dorsal root ganglion
Wind-up
Sensitized spinal circuits
Repeated balloon distention
Mechanosensitive afferent
Repeated inflation of a balloon to noxious levels in the recto-sigmoid region leads to lowered perception thresholds for discomfort and pain in IBS patients. This phenomenon is believed to be due to centrally mediated hyperalgesia. Centrally mediated hyperalgesia reflects hyperexcitability of second order sensory neurons in the dorsal horn of the spinal cord. Under conditions of severe and persistent nociceptive stimulation, small-diameter, nonmyelinated C fibers fire repetitively and their input progressively increases responses of second order dorsal horn neurons. “Wind-up” of the second order neurons is generated by the release of the neurotransmitter glutamate from the C fibers. Noxious stimulation of the type applied in balloon distention studies in IBS patients can produce long-term changes in the excitability of dorsal horn neurons. This phenomenon is called central sensitization to distinguish it from the sensitization that occurs at the endings of the sensory neurons in the intestinal wall during release of inflammatory mediators. Long-lasting changes in the excitability of dorsal horn neurons impart a memory for nociceptive input from C fibers. Upregulation in the expression of neuropeptides and neurotransmitters and their receptors presumably underlie the phenomenon of central sensitization. Elevated excitability in nociceptive dorsal horn neurons underlies spinally mediated hyperalgesia, which is termed central sensitization to distinguish it from sensitization that occurs at nociceptive terminals in the periphery. In conditions of severe tissue injury and persistent injury, nociceptive C fibers fire repetitively and the excitability of the second order neurons in the dorsal horn increases progressively in response to the elevated synaptic input. This effect is sometimes called wind-up and reflects the synaptic release of glutamate from the incoming C fibers and activation of N-methyl-D-aspartate (NMDA)-type glutamate receptors expressed by the second order neurons. One piece of suggestive evidence for wind-up hypersensitivity is the finding that second order neurons in the dorsal horn show induction of the early gene c-fos in response to noxious balloon distention of the colon in rats (96).
These long-lasting changes in the excitability of dorsal horn neurons are like a memory imprint of the nociceptive input. Accumulating evidence suggests that the spinal wind-up phenomenon may be operational and be an underlying factor in the intestinal hypersensitivity associated with IBS.
1. Basbaum AI, Jessell TM. The perception of pain. In: Principles of neuroscience 4th ed. Kandel ER, Schwartz JH, Jessell TM (eds.) New York: McGraw-Hill 2000:
2. Munakata J, Naliboff B, Harraf F, Kodner A, Lembo T, Chang L, Silverman DHS, Mayer EA. Repetitive sigmoid stimulation induces rectal hyperalgesia in patients with irritable bowel syndrome. Gastroenterology 1997;112:55-63.
3. Traub RJ, Pechman P, Iadarola MJ, Gebhart GF. Fos-like proteins in the lumbosacral spinal cord following noxious and non-noxious colorectal distention in the rat. Pain 1992;49:

6. HYPERALGESIA IN IRRITABLE BOWEL SYNDROME
(Use of “barostatic” distensions) 45
Baseline 40
Post sigmoid
stimulation 35
Rectal Pain
Threshold
(mm Hg)
These data reflect results for 14 patients with IBS and 11 healthy controls for which perception thresholds for discomfort and pain were determined for tonically-applied stimulus before and after repetitive high-pressure balloon distention of the sigmoid colon. Repetitive distentions consisted of phasic distentions of 15-seconds duration with a interstimulus intervals of 30 seconds for periods of 10 minutes. The tonic stimulus was a 3-minute duration distention. In patients with IBS, repetitive distention of the distal large intestine results in the development of central sensitization manifested as hyperalgesia and increased viscerosomatic referral during rectal distention and as spontaneous rectosigmoid hyperalgesia in the absence of applied stimuli. The results support a hypothesis that repetitive large-amplitude sigmoid contractions might induce rectosigmoid hyperalgesia in patients with IBS.
Munakata J, Naliboff B, Harraf F, Kodner A, Lembo T, Chang L, Silverman DH, Mayer EA. Repetitive sigmoid stimulation induces rectal hyperalgesia in patients with irritable bowel syndrome. Gastroenterology. 1997;112:55-63. 30 25 20
IBS
Controls
Munakata J, Gastroenterology 1997; 112:55

7. IBS MCC pACC Brain Activation with Noxious Visceral Stimulation
Prefrontal Cortex
Cing Cx included
MCC
pACC
Thalamus
Pf, Re, Cl, Li
Locus coeruleus
Subnucleus reticularis dorsalis
Spinal cord
Lamina 1
IBS

8. Evidence for colonic mucosal immune alterations and increased
density of mast cell and immunocytes in IBS
Mast cells
Lymphocytes T
Neutrophils
ECC
ICC
Ref.
Patients
Jejun/
ileum
Cecum/
colon
CD3+
CD4+
CD8+
CD25
“spastic colon” ++
Hiatt 1962
IBS- D/C ++
Weston et al. 1993
IBS- D/C/A +
O’sullivan et al.2000
IBS- D/C/A +PI ++ ++ ++
Thorbloom et al. 2002
IBS-D/C ++ ++ ++ +
Chadwick et al. 2002
IBS- D ++
Park et al. 2003
IBS-PI ++ ++
Dunlop et al. 2003
IBS-PI ++ ++
Spiller 2004
IBS- D+SII-C ++
Barbara et al. 2004
IBS- D/ PI ++
Chang et al. 2004
IBS- D ++ ++
Park et al. 2006
ECC: enteric chromaffin cells; ICC : interstitial cell of Cajal; IBS-C: constipated patients;
IBS-D: diarrheoic patients; IBC-A: alternated diarrhea-constipation; PI: post-infectious IBS.

9. Lymphocytose ganglionnaire dans le SII
Infiltration de lymphocytes dans les ganglions myentériques. La flèche noire indique un neurone et quelques lymphocytes à la base du neurone.Il ya plus de lymphocytes dans la zône délimitée par les les flêches bleues. Hématoxiline-éosine, X 380
(d’après Thorbloom et al. 2002)

10. Activation des lymphocytes T circulants CD4+ et CD8+
et expression de MAdCAM par l’endothélium colique dans l’IBS
Sang
Colon
IBS: D+C+A
UCr: RCH en rémission; UCa: RCH active; CTRL: Témoins
(Ohman et al.2005)

11. COLONIC MAST CELLS IN CLONIC BIOPSIES OF IBS PATIENTS
CTRL
IBS
Increased mast cell tryptase labeling in the sub-mucosa of IBS patients
Proximal colon biopsies in IBS = density of mast cells
amount of tryptase
colocalisation nerve-mast cells
( Barbara et al., 2004)

12. Facteurs principaux produisant la dégranulation ou l’activation
des mastocytes muqueux du tube digestif
Stress
CRF
Mastocyte
Degranulation
minutes
Allergie
Histamine
Leukotrienes
Cytokines*
proteases
NGF
IgE, SP,5-HT
PAF, ATP
Inflammation
heures
GM-CSF, IL-1
Secretion
Infection
NGF, NPY
Cytokines*
Chemokines
activation
Système S *
Cytokines: TNFa, IL-3, IL-5, IL-4, IL-13, IL-10, GM-CSF
(adapted from Shakoory et al,2004, Penicci et al.2003)

13. Distribution of nerve terminals close to mast cell
in IBS patients.
PI-IBS
CTRL
Mast cell number (mm2)
(ileal mucosa) MC
PI-IBS……… 11.2 ± 2.8*
(n=27) MC
Non PI-IBS… ±1.2*
(n=29)
Control………..6.1±0.5
(n=12)
*: from control at p<0.01
(X1000)
Red: nerve terminals (enolase labeling)
Blue: mast cells (alcian blue)
( Wang et al. Gut 2004 )

14. RELATIONSHIP BETWEEN MAST CELL-NERVES
CONNECTION AND PAIN IN IBS PATIENTS 4 4 3 3
Pain intensisity scoring
Pain frequency scoring 2 2 1 1 5 10 15 20 5 10 15 20
Number of mast cells at a distance < 5µm from nerves
(Barbara et al. Gastro. 2004)

15. Is increased gut permeability able to initiate mucosal
immune response and visceral hypersensitivity?
Increased paracellular permeability =
Entry of pathogens, toxins, antigens, bacteria
Epithelial cells
- activation of immunocytes
cytokines release
inflammatory mediators
Motility
disorders
ENS
disorders
Nociceptive
hypersensitivity
PAIN

16. Tight junction Pathogens Allergens Epithelial cells Mast cell
(LPS, DNA,peptidoglycans,…etc.)
Tight junction
Pathogens
Allergens
Epithelial
cells
Intestinal or colonic mucosa
Mast
cell
T-cell
B-cell
Sensory nerves
(nociceptive fibers)
Granulocyte
( from Perdue et al )

17. INFLUENCE OF FECAL SUPERNATANT FROM IBS PATIENTS INFUSED
ON PARACELLULAR PERMEABILITY OF MICE COLONIC STRIPS
Fecal supernatants (n=6)
IBS-D fecal supernatants (n=3)
300
200 *
100
IBS-D fecal supernatants (n=4) WT
PAR-2-/-
Increase in permeability triggered by apical application of IBS-D fecal supernatant is reduced by ser-protease inhibitor and is absent in PAR-2 -/- KO mice
(Gecse et al. 2008)

18. INFLUENCE OF FECAL SUPERNATANT FROM IBS-D PATIENTS INFUSED
INTRACOLONICALLY IN MICE ON COLONIC SENSITIVITY TO DISTENSION
(Gecse et al.
2008)

19. INFLUENCE OF FECAL SUPERNATANT FROM IBS-D PATIENTS ON
COLONIC EPITHELIAL TJ INTEGRITY IN MICE
P-MLC (green)
ZO-1 (green)
P-MLC in colonic mucosa
P-MLC is over expressed in colonic mucosa infused with IBS-D supernatant reflecting a EC cytoskeleton contractionC
Resulting opening of TJs is associated with a reduced apical expression of ZO-1
(Gecse et al. 2008)

20. Luminal ser- proteases
Mechanisms involved in long-term sensitization of mucosal sensory nerves in IBS-D patients
Luminal ser- proteases
PAR-2 activation
Increased
permeability
(mins to hours)
IFNg
tryptase
Mucosal
micro-inflammation
(hours to weeks)
T-cell
Mast cell
Inflammatory
mediators
tryptase SP
Nerve terminal
sensitization
(weeks to months)
Afferent neurons
Long-term
hypersensitivity
( Bueno et al. 2008)

21. Factors able to alter gut permeability/sensitivity in FGID
Inflammation
(gastroenteritis)
stress
sepsis
enzymes
Allergens, parasites
Biliary salts
bacteria (proteases?)
Bacterial secetion or lysis (acetaldehyde, LPS…etc)

22. IN VITRO MEASUREMENT OF PARACELLULAR PERMEABILITY OF COLONIC BIOPSIES (Ussing chambers)
The degree of porosity of biopsies from IBS patients is higher than that of healthy subjects independently of bowel habit alterations.
This altered permeability is associated with a decrease in the expression of ZO-1, a protein linking the actinomyosin apical ring to the proteins of the TJs
Piche et al. Gut 2009

23. EFFECTS OF COLONIC BIOPSY SUPERNATANT ON CaCo2 CELL PERMEABILITY AND CORRELATIONS WITH SYMPTOM SCORES
Increase of permeability of CaCo2 cells, 48h after mucosal exposure with supernatant of biopsies from IBS patients
Changes in permeability of CaCo2 cells is correlated with the pain score of explored IBS patients
Piche et al. Gut 2009

24. * RELATIONSHIPS BETWEEN GUT PERMEABILITY AND PAINFUL SENSATIONS
Zhou et al. Pain 2009
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Lactulose/ Mannitol
Normal
IBS
INTESTINAL PERMEABILITY
RELATIONSHIPS BETWEEN GUT PERMEABILITY AND PAINFUL SENSATIONS
TO DISTENSION MEASURED IN VIVO
Normal
IBS
10.0
7.5
5.0
2.5
0.0
VAS score (visceral)
VISCERAL PAIN* *
Pain measurement after repeated (2) 35mm Hg rectal distension performed during 30 sec. at 2 min. interval.

25. RELATIONSHIPS BETWEEN GUT PERMEABILITY AND SOMATIC* SENSITIVITY
IN IBS PATIENTS
120
100 80 60 40 20
FBDSI score
CONTROL
IBS
Patients with altered permeability *
skin thermal stimulus (Pelletier probe 3x3 cms) at 47°C applied to the left hand during 10 sec.
Zhou et al. Pain 2009

26. CONCLUSIONS
La douleur abdominale associée au SII à le plus souvent comme origine une hypersensibilité intestinale ou colique à la distension.
Cette hypersensibilité est associée à une micro-inflammation de la paroi pouvant être considérée comme résultant d'une augmentation de la “porosité“ de la muqueuse colique associée au passage de bactéries et toxines.
Cette augmentation de perméabilité à été montrée, in vivo et in vitro, être corrélée aux symptômes
Certains facteurs luminaux dont les protéases agissent sur les récepteurs des cellules épithéliales pour augmenter cette perméabilité