Induction of T lymphocyte apoptosis by sulphasalazine in patients with Crohn's disease.
BACKGROUND
Lamina propria T lymphocytes (LPL) of the intestinal mucosa are chronically activated in Crohn's disease (CD). Defective apoptosis of activated LPL was proposed as a key pathogenic mechanism. In fact, increased expression of antiapoptotic molecules was observed in CD LPL. In the present work, we aimed to analyse the effects and underlying molecular mechanisms of 5-amino salicylic acid (5-ASA) and derivatives on apoptosis of LPL and peripheral blood lymphocytes (PBL) in patients with CD compared with ulcerative colitis (UC) and in non-inflammatory controls.


METHODS
PBL and LPL were isolated by Ficoll-Hypopaque gradient centrifugation and the EGTA-collagenase method, respectively. PBL/LPL were stimulated with FasL, 5-ASA, sulphapyridine, and sulphasalazine for 24/48 hours and apoptosis was quantified by flow cytometry (annexin V- propidium iodide method) and immunofluorescence. The molecular mechanisms of drug induced apoptosis were analysed in wild-type and FADD-/- Jurkat T cells using western blots and caspase assays.


RESULTS
While PBL displayed a normal apoptosis pattern after Fas stimulation in patients with active CD, LPL from inflammatory areas were highly resistant. Comparable resistance to apoptosis was observed in LPL of UC patients. In contrast with 5-ASA, which did not induce apoptosis in lymphocytes, sulphasalazine proved to be a potent proapoptotic agent. Sulphasalazine induced T lymphocyte apoptosis was independent of the Fas pathway but associated with marked downregulation of antiapoptotic bcl-xl and bcl2, activation of the mitochondrial apoptosis signalling pathway, and subsequent activation of caspase-9 and caspase-3.


CONCLUSIONS
The beneficial effect of sulphasalazine in treating inflammatory bowel disease is at least in part attributable to its proapoptotic effects on LPL which allows potent downregulation of lymphocyte activation.
Hôpital Necker-Enfants Malades, Paediatric Gastroenterology, INSERM EMI 0212, University Paris V, 149, Rue de Sèvres, F-75743 Paris, Cedex 15, France.
J
J
Doering
B
B
Begue
M J
MJ
Lentze
F
F
Rieux-Laucat
O
O
Goulet
J
J
Schmitz
N
N
Cerf-Bensussan
F M
FM
Ruemmele
D000293:Adolescent; D000328:Adult; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D003093:Colitis, Ulcerative / Q000276:immunology / Q000473:pathology; D003424:Crohn Disease / Q000276:immunology* / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D018928:Immunity, Mucosal / Q000187:drug effects; D007413:Intestinal Mucosa / Q000276:immunology; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000187:drug effects; D019804:Mesalamine / Q000494:pharmacology; D013427:Sulfapyridine / Q000494:pharmacology; D012460:Sulfasalazine / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology
D000894:Anti-Inflammatory Agents, Non-Steroidal; D012460:Sulfasalazine; D019804:Mesalamine; D013427:Sulfapyridine