In the present study we found that LPMCs from involved tissue of UC patients contain CD4+ cells bearing an NKT cell marker (CD161) that, upon polyclonal activation, produces increased amounts of IL-13 (and IL-5) as compared with equivalent cells from control or CD tissue; in contrast, similar cells from CD patients produced increased amounts of IFN-γ. These findings were corroborated by four-color flow cytometry in which we showed that CD4+ T cells from UC patients bearing an NKT cell marker (CD161) express intracellular IL-13, whereas similar cells from CD patients express intracellular IFN-γ. Finally, we showed that UC LPMCs produce IL-13 when cocultured with a B cell line expressing high levels of surface CD1d (in the presence of PMA and IL-2), indicating that the IL-13 was being secreted by a CD1d-restricted cell. Collectively, these data strongly suggest that IL-13–producing, CD1d-restricted, NKT cells are uniquely associated with the UC but not the CD form of IBD.
The observation that UC is characterized by an increased Th2-oriented immune response partially coincides with an earlier study in which it was shown that in intestinal biopsies IL-13 mRNA, as well as IL-4 and IL-10 mRNA, was increased significantly in a larger percentage of UC patients than in “noninflammatory” controls; however, these results were difficult to interpret because IL-13 and IL-10 mRNA was not increased compared with “inflammatory” controls (including CD patients) (29
). In addition, our findings differed in that, in concert with prior studies, we found a decrease in IL-4 protein secretion rather than an increase in IL-4 mRNA (8
). We postulate that this decrease is due to the fact that an initial IL-4 response in UC is superseded by an IL-13 response, as it is in oxazolone colitis (10
). It will be interesting in this regard to examine patients early after onset of UC to determine if the lesion is initially associated with IL-4.
The Th2 orientation of the UC lesion is also congruent with an earlier finding that mRNA encoding a recently discovered cytokine chain known as EBI3 was shown to be increased in UC tissues as compared with CD or control tissues (30
). Of interest, EB13-deficient mice manifest poor Th2 responses and are resistant to the development of oxazolone colitis (32
). In addition, while such mice exhibit normal numbers of both naive and mature CD4+
cells, they have markedly reduced numbers of NKT cells. These studies thus suggest that an EBI3-associated cytokine (such as IL-27) may be necessary for the development of the Th2-cytokine–producing NKT cells that characterize both UC and oxazolone colitis.
NKT cells are defined by the fact that they are cells expressing both a TCR and NK receptors such as NK1.1 (CD161) (reviewed in ref. 16
). In addition, NKT cells recognize antigens (usually glycolipid antigens) in association with CD1d (CD1 in mice), a MHC class I-like molecule present on the surface of professional APCs (dendritic cells) as well as on nonprofessional APCs such as intestinal epithelial cells (IECs) (33
). The TCRs used by classic NKT cells are invariant in that they use a particular Vα chain (in humans, Vα24/JαQ) usually associated with a particular Vβ chain (in humans, Vβ11) (19
). On this basis, the classical invariant NKT cells (iNKT cells) recognize a simple glycolipid, α-GalCer, and can be identified with α-GalCer-loaded tetramers (16
). Since glycolipids are also found among self-antigens or antigens in the mucosal microflora, NKT cells may be self-antigen–reactive cells. This probably explains the finding noted here that they react to cells expressing CD1d in the absence of exogenous antigen, presumably because under these circumstances a self-glycolipid is present within the CD1d groove.
Whereas invariant NK T cells comprise the majority of CD1d-restricted NKT cells, the latter also include nonclassical NKT cells that express noninvariant (diverse) or, alternatively, semi-invariant TCRs that nevertheless react with antigens presented by CD1d (as do iNKT cells) (16
). The cell population producing IL-13 and associated with the UC lesion appear to belong to this category of nonclassical NKT cells since they do not express an invariant TCR as indicated by their inability to bind α-GalCer tetramers and are stimulated by CD1d on the surface of CD1d-transfected EBV-transformed B cells (i.e., they are CD1d restricted) but not by α-GalCer. Such reactivity to CD1d in the absence of a specific antigen required partial activation by PMA as well as high-level expression of CD1d on the surface of the stimulating cell, probably reflecting the fact the cells are being stimulated by a relatively low-affinity self-antigen expressed by B cells in association with CD1d, as discussed above. It is interesting to postulate that these non- or semi-invariant CD1-restricted (NKT) cells in patients with UC recognize glycolipids in the bacterial microflora of the gut that cross-react with glycolipid self-antigens. Studies to establish the nature of this antigen or class of antigens will be among the next important steps in the elucidation of UC.
NKT cells secrete both Th1 and Th2 cytokines very soon after activation and thus can be considered a component of the innate immune system that “conditions” subsequent adaptive immune responses (15
). In addition, NKT cells may also function as regulatory cells since there is evidence that these cells protect mice against the development of autoimmune diabetes, experimental allergic encephalitis, and intestinal inflammation, possible through the production of Th2 cytokines (37
). More relevant to their role in UC, however, is evidence that NKT cells also act as effector cells that induce inflammatory disease. This is seen clearly not only in the oxazolone colitis model, but also in models of allergen-induced airway hyper-reactivity (41
) and in concanavalin A–induced (conA–induced) hepatitis, a model of autoimmune liver disease (42
). In addition, it has recently been shown that non-iNKT (nonclassical) cells contribute to liver injury in a murine model of hepatitis B virus infection (43
Another key finding in this study is that NKT cells, in general (in the form of iNKT cell lines), as well as the nonclassical NKT cells in UC tissues can act as cytotoxic cells for human epithelial target cells and that such cytotoxicity is enhanced by IL-13. This finding recalls the many studies of UC performed in the 1960s–1980s in which various investigators attempted to show that this disease is associated with (if not, in fact, due to) cells capable of causing epithelial cell cytotoxicity and thus the characteristic ulcerative lesions of UC (reviewed in ref. 44
). These studies were ultimately unable to prove the existence of bona fide cytotoxic cells with ability to lyse epithelial cells in UC, but, of course, they were performed before the present knowledge of NKT cells and their requirements for CD1d-expressing targets and IL-13 was available. It should be noted in this context that the target cells used in these studies were LPS-stimulated human epithelial target cells that expressed increased levels of CD1d as a result of LPS stimulation and, indeed, cytotoxicity was blocked by the addition of anti-CD1d. On the basis of these cytotoxicity findings it is reasonable to postulate that the immunopathology of UC involves first the stimulation of NKT cells through antigen presented to these cells by CD1d-bearing DCs or epithelial cells. The NKT cells so stimulated then begin to produce IL-13, at which point they become capable of lysing epithelial cells, and this leads to epithelial cell loss, ulceration, and breaches in the IEC barrier. Further intensification of the inflammation then ensues due to entry of organisms into the lamina propria proper as well as a result of IL-13–induced chemokine production (45
) that leads to the influx of acute inflammatory cells. Evidence for this postulated series of events as a cause of UC are the aforementioned studies of conA–induced hepatitis in which it has been shown that NKT cells cause hepatocellular cytolysis (26
) and the studies of oxazolone colitis in which it was shown that the inflammation can be aborted by agents that block either NKT cell formation or function or IL-13 (10
). Further work in which specific inhibitors of IL-13 and/or NKT cells are administered to patients will be necessary to prove this hypothesis, however.