MAdCAM-1 is a 60 kDa endothelial cell surface molecule that is strongly expressed by mucosal endothelial cells, particularly following exposure of these cells to pro-inflammatory cytokines such as TNF-α. Expression of MAdCAM-1 has also been reported in the brain, and in the heart [44, 45]; based on these findings, it is now been suggested that MAdCAM-1 might play roles in chronic inflammation of these organs as well.
With respect to inflammatory bowel disease, MAdCAM-1 appears to be essential to the lymphocyte homing to the mucosa associated lymphoid tissue (MALT) [6, 37, 38, 46]. Since MAdCAM-1 is normally expressed mainly within the gut microvasculature, and is dramatically amplified during IBD, it has been suggested that increased MAdCAM-1 expression contributes to the etiology of IBD through its ability to direct homing of lymphocytes to the gut. This notion is well supported by several reports that show that antibodies directed against either MAdCAM-1, or its lymphocyte ligand, the α4β7 integrin will significantly attenuate several indices of gut damage in experimental models of colitis [9, 47].
Several studies have indicated that T helper (Th1) immune response has important roles in the development of IBD [48–50]. Moreover, dysregulation of cytokine networks is involved in Th1-dominant immune responses in IBD [48–50]. Among the Th1 cytokines, TNF-α is thought to be perhaps the most important cytokine responsible for driving the onset and evolution of IBD. Because of this prime role of TNF-α in IBD, anti-TNF-α antibody therapy has been very successfully used in IBD to reduce both colonic injury and expression of ECAMs in IBD .
IL-10, a cytokine produced by activated macrophages and Th2-type T cells, has crucial inhibitory effect against several Th-1 type immune responses, such as the antigen-presenting functions of monocytes and macrophages [52, 53]. IL-10 may play an important role in preventing the induction of IBD, since animals deficient in IL-10 will develop colitis spontaneously, and low levels of IL-10 are positively correlated with recurrences of Crohn's disease [25, 54]. However, unlike TNF-α based therapies, the administration of recombinant IL-10 (rIL-10) shows poor clinical efficacy. This may reflect the fact that TNF-α therapies for IBD are aimed at efficiently clearing TNF-α, while IL-10 therapies must increase IL-10 and recombinant IL-10 is likely too rapidly cleared from the circulation after in vivo administration to provide a uniform protection . On the other hand, IL-10 gene transfer technology has been used with some success in models of colitis, however its effectiveness is also variable. One reason for this variability may be that the final serum IL-10 concentration of gene-transfected mice might be below the critical threshold needed for gut protection [55, 56]. Therefore targeting of the IL-10 gene to the inflamed colon should ideally exploit tissue (i.e. gut) specific promoters to control selective organ gene transfer technology, endothelial specific promoters and also organ specific intra-arterial injection of vector to activate some genes in specific locations .
Administration of IL-10 in vitro prevents TNF-α stimulated expression of MAdCAM-1, and also blocks lymphocyte adhesion to endothelial cells to the same extent as dexamethasone treatment, currently a key therapy in IBD . Interestingly, the low basal levels of MAdCAM-1 expressed by control (cytokine unstimulated) cells were not affected by IL-10 transfection. This suggests that basal expression of MAdCAM-1, is IL-10 independent, while cytokine stimulated MAdCAM-1 is modulated by IL-10. While we previously showed that delivery of IL-10 to the endothelium in vitro is protective against TNF-α , in vivo IL-10 has not been as effective. There are at least two regulating pathways in ECAM expression, one is a cytokine-dependent pathway which is widely studied and the other is cytokine-independent . IL-10 apparently prevents cytokine-induced NF-kB binding activity to the ECAM promoter  and blocks the excessive expression of ECAMs in cytokine-mediated inflammatory conditions. In this regard, IL-10 has been used as a treatment in inflammatory diseases, like IBD for precisely this reason: because it has few effects at sites remote from inflammation. Similarly, we found that IL-10 transfection did not affect cell morphology, total protein level, or the expression of actin and vimentin in these cells (measured by Ponceau red S staining density on immunoblots). Therefore methods like endothelial gene transfection/transfer in vivo may effectively maintain adequate IL-10 concentrations near the endothelial cells that finally achieve the protection against cytokines that is not uniformly produced by systemic IL-10 administration.
The most important index for how well gene mediated recombinant IL-10 delivery might work in IBD appears to be measurement of the inhibition of lymphocyte-endothelium binding mediated by TNF-α induced MAdCAM-1 expression. In this experiment, IL-10 induction in the endothelium efficiently blocked TNF-α induced MAdCAM-1 expression and α4β7-dependent lymphocyte adhesion on SVEC endothelial cells. IL-10 deficiency in colitis increases expression of several ECAMs including ICAM-1, VCAM-1 and MAdCAM-1 . We performed an experiment, where under non-stimulated conditions only 55.7 ± 5.4% as many of the TK-1 lymphocytes bound to SVEC compared to TNF-α stimulation (100%, or 'maximal' binding). TK-1 were seen to interact with SVEC through ICAM-1 as well MAdCAM-1, but not VCAM-1, since an antibody against MAdCAM-1 reduced adhesion to 27 ± 4.6% of maximum and an anti-ICAM-1 reduced adhesion to 54.6 ± 8.3 % of maximum; anti-VCAM-1 reduced adhesion to 91% of maximum (blocks lymphocyte adhesion to endothelial cells (data not shown). Therefore we feel that adhesion in this system is mostly MAdCAM-1 dependent.
IL-10 transfection of endothelial cells reduced MAdCAM-1 expression by 42% (following TNF-α stimulation) and reduced TK-1 adhesion by 50%. Therefore, these data suggest that the reduction of TK-1 adhesion is approximately 84% (42%/50%) MAdCAM-1 dependent, and that the remaining 16% of adhesion depends on other ECAMs, (such as ICAM-1).
Although tissue specific promoters might further increase the organ specificity and transgene delivery of this approach, they have not been evaluated in this report. Our findings suggest that lymphatic or gut endothelial transfection with Th2 cytokines like IL-10 may be an effective method to reduce important clinical symptoms associated with IBD which are mediated by MAdCAM-1 dependent lymphocyte adhesion.