Björn R. Lúdvíksson

Reciprocal IFN-γ and TGF-β responses regulate the occurrence of mucosal inflammation

Abstract Recent studies of oral tolerance and experimental colitis indicate that the occurrence of gastrointestinal inflammation is determined by a balance between proinflammatory interferon γ responses and anti-inflammatory transforming growth factor β responses. Here, Warren Strober and colleagues discuss the new findings.

The effect of TGF‐β1 on immune responses of naïve versus memory CD4+ Th1/Th2 T cells

The role of TGF‐β1 in the regulation of T cell responses has been perplexing, possibly because it is dependent on the type of T cell being regulated and its cytokine microenvironment. In the present study, we demonstrate that TGF‐β1 has a profound inhibitory effect on naive CD4+ T cell undergoing differentiation under defined neutral, Th1 and Th2 priming conditions. In addition, we show that if CD4+ T cells are primed in the presence of TGF‐β1, they exhibit reduced secondary anti‐CD3/anti‐CD28‐induced and antigen‐specific immune responses (even when TGF‐β is absent during the secondary response), which is not due to reduced expression of co‐stimulatory molecules or to inadequate IL‐2 production. Finally, with respect to the effect of TGF‐β on fully differentiated antigen‐specific memory CD4+ T cells, we demonstrate that while antigen‐specific activation and cytokine secretion by memory Th1 T cells is inhibited by TGF‐β1, such inhibition is associated with partial down‐regulation of IL‐12 receptor β2 chain expression. In contrast, memory Th2 T cells are not subject to TGF‐β1‐mediated suppression. In summary, these studies reveal that TGF‐β1 is a powerful negative regulator of the primary immune response of CD4+ T cells, but only Th1 T cells are subject to such regulation after the memory stage of T cell differentiation has been reached. Thus, these studies define the potential regulatory role of TGF‐β1 in Th1 and Th2 T cell‐mediated autoimmunity.

The Pathogenesis of Mucosal Inflammation in Murine Models of Inflammatory Bowel Disease and Crohn Disease

Dr. Warren Strober (Mucosal Immunity Section, National Institute of Allergy and Infectious Diseases [NIAID], National Institutes of Health [NIH], Bethesda, Maryland): Recent advances in knowledge of T-cell differentiation and cytokine production are providing new insights into the nature of autoimmune diseases and other diseases involving immune dysregulation [1]. These advances are often based on studies of various spontaneous or induced animal models of immune dysfunction, particularly models that resemble human disease. We focus on Crohn disease, a major form of inflammatory bowel disease in which murine models have been used to great advantage [2-4]. Studies of T-cell and macrophage function in patients with Crohn disease and parallel studies of various newly described murine models of inflammation resembling Crohn disease can be combined to provide important insights into the underlying mechanisms of disease [5-9]. Furthermore, murine models can be used to devise potential cytokine- or anti-cytokine-based treatments for disease. Crohn disease manifests primarily as a transmural inflammation involving the full thickness of the bowel wall that frequently leads to bowel obstruction, fistulas, and abscess formation. It thus differs from ulcerative colitis, which is a relatively superficial inflammation of the mucosa that leads to early ulcer formation. These two forms of inflammatory bowel disease also differ with regard to their distribution in the intestine. Crohn disease can occur anywhere in the gastrointestinal tract and has a predilection for the terminal ileum and ascending colon; it is discontinuous, with areas of inflammation alternating with normal areas. Ulcerative colitis, in contrast, is limited to the colon and usually begins in the rectal and sigmoid areas and progresses upward continuously [10]. Histologic studies of Crohn disease show a dense accumulation of activated T cells and macrophages, which in some cases are organized into typical granulomas. The earliest microscopic lesion in Crohn disease consists of a focal accumulation of lymphocytes and macrophages located next to an intestinal crypt. When this lesion is associated with epithelioid cells, it has the appearance of a protogranuloma; when it is associated with crypt epithelial-cell destruction, it leads to the formation of an aphthous ulcer [11]. Thus, it seems that interactions between lymphocytes and macrophages are at the core of the pathology of Crohn disease. In contrast, in ulcerative colitis, the cellular infiltrate is more variegated and acute inflammatory events, such as neutrophils forming crypt abscesses, are prominent. Lymphocytes and macrophages are present, but granulomas are not [12]. The pathologic findings in Crohn disease seem to result from the interplay of environmental and genetic factors. The former are evident from the observation that the incidence of Crohn disease is greatly increased in urbanized, economically advanced societies, whereas the latter are expressed by the fact that Crohn disease (and ulcerative colitis) manifests a strong familial pattern and can be linked to disease susceptibility loci on chromosomes 3, 6, 7, 12, and 16 [10, 13-16]. We believe that these factors may work through the formation of a dysregulated mucosal immune response that leads to chronic mucosal inflammation. The clinical course of Crohn disease is characterized by recurrent episodes of abdominal pain and other symptoms arising from bowel inflammation and the sequelae of inflammation (bowel fistulae and abscesses). In more than half of patients, surgery is required to remove diseased tissue but is rarely curative, and 60% of surgical patients have recurrence within 10 years [17, 18]. Medical therapy for Crohn disease consists of anti-inflammatory agents, such as aminosalicylates or steroids, and immunosuppressive agents, such as 6-mercaptopurine. These drugs, however, suppress rather than cure the inflammatory disease. Clearly, new treatment approaches are needed for patients with Crohn disease. Cytokine Production in Crohn Disease Dr. Ivan J. Fuss (Mucosal Immunity Section, NIAID, NIH): Intestinal lesions in Crohn disease are replete with activated T cells. Investigators in our laboratory and others have attempted to characterize the function of these T cells with regard to their most cogent immunologic function: their capacity to produce various cytokines. We knew of recent studies showing that if naive CD4+ T cells are cultured under conditions that favor interleukin-12 production, Th1 T cells that produce IFN- and tumor necrosis factor are induced. In contrast, if naive CD4+ T cells are cultured under conditions that favor interleukin-4 production, Th2 T cells that produce interleukin-4, interleukin-5, interleukin-6, and interleukin-13 emerge. Furthermore, this pattern of T-cell differentiation is associated with distinct functional activities: Th1 T cells are the key players in delayed-type hypersensitivity reactions, whereas Th2 T cells are potent inducers of antibody-mediated immunologic reactions [1]. We sought to determine whether cytokine secretion in Crohn disease (or ulcerative colitis) allows characterization of T cells in the lesions as Th1-type or Th2-type T cells [19]. In our studies [2], we extracted purified CD4+ T cells from lesional intestinal tissue obtained at surgery from patients with Crohn disease and ulcerative colitis or from controls who came to surgery for cancer and other noninflammatory diseases. We cultured the cells in a polyclonal T-cell stimulant, the anti-CD2-anti-CD28 antibody pair that has been shown in previous studies [20, 21] to be a particularly good stimulant of activated T-cell populations, such as those in the intestinal lamina propria. As shown in Figure 1, stimulated CD4+ T cells from patients with Crohn disease produced approximately two times more interferon- than did CD4+ T cells from controls or patients with ulcerative colitis. In parallel studies, we enumerated stimulated CD4+ T cells that produce interferon- by using an enzyme-linked immunospot assay technique [2] in which cells that produce a particular cytokine are identified by the footprint that they leave on a surface coated with anticytokine antibody. As in the case with interferon- secretion, the number of cells secreting interferon- was increased in the inflamed lamina propria of patients with Crohn disease but not patients with ulcerative colitis. Finally, we found that mature (CD45RO+) CD4+ T cells in the peripheral circulation of patients with Crohn disease, when cultured and stimulated as described above, secreted three times more interferon- than did mature CD4+ T cells from controls. This circulating T-cell subpopulation is likely to contain mucosal cells that have re-entered the circulation; thus, the increased interferon- production seen in cultures of this subpopulation is probably a peripheral reflection of secretion by cells in the inflamed mucosa. Figure 1. Top and bottom left. Top and Bottom right. To obtain estimates of CD4+ T-cell production of interleukin-4 and interleukin-5 in the lamina propria, we analyzed culture supernatants (obtained as described above) for their interleukin-4 and interleukin-5 content. We found that production of interleukin-4 by cells from tissues of Crohn disease and ulcerative colitis was greatly decreased compared with production of interleukin-4 by cells from control tissues. By contrast, production of interleukin-5 by CD4+ T cells in the lamina propria of patients with Crohn disease decreased, whereas production of interleukin-5 by CD4+ T cells in the lamina propria of patients with ulcerative colitis increased substantially (Figure 1). As with the interferon- studies, these results were corroborated by studies using the enzyme-linked immunospot technique, which showed that CD4+ T cells obtained from the lamina propria of patients with ulcerative colitis contain an increased number of cells secreting interleukin-5. These data, in conjunction with data from previous studies of inflammatory bowel disease, establish that the cytokine secretion profile of lesional Crohn CD4+ T cells (that is, production of large amounts of interferon- and small amounts of interleukin-4 and interleukin-5) is most consistent with the Th1 category of T cells [2, 3, 22, 23]. As studies of several human diseases and experimental models of inflammation show [24-29], this T-cell category is associated with granulomatous inflammatory reactions. Thus, its presence in Crohn disease is consonant with and probably responsible for granulomatous inflammation. The cytokine data also show that T cells extracted from lesional colonic tissue in ulcerative colitis are qualitatively different from those in Crohn disease in that their reduced production of interferon- and their elevated production of interleukin-5 makes them more like Th2-type T cells. In this regard, only their unexplained lack of high interleukin-4 production prevents the definite assignment of lesional CD4+ T cells to this category. Recent work has shown that Th1 T-cell differentiation occurs when T cells interact with antigen-presenting cells and the latter produce interleukin-12, the key inductive cytokine of Th1 T-cell differentiation and growth [30]. Thus, if T cells in the lesional tissues of Crohn disease are in fact Th1 T cells, production of interleukin-12 in these tissues should also be increased. To explore this possibility, Neurath and colleagues performed immunohistochemical studies of frozen tissues from patients with Crohn disease and ulcerative colitis and from controls. They found that inflamed Crohn disease tissue stained positive for interleukin-12, whereas tissue with ulcerative colitis and control tissue did not (Neurath M, Fuss I, Pettersson S, Schurmann G, Herfarth C, Meyer zum Buschenfelde KH, et al. Upregulation of the interleukin-12/stat-4 pathway distinguishes Crohn disease from ulcerative colitis. In preparation)

Mucosal immunoregulation and inflammatory bowel disease: new insights from murine models of inflammation.

Membranous glomerulonephritis (MGN) is said to be caused by circulating autoantibodies against antigen(s) located to the epithelial side of the glomerular capillaries. Membranous glomerulonephritis with severe renal damage can be produced experimentally by injections of heterologous antibodies, but this model is obviously of questionable relevance. It can also be produced by immunizing the animal with antigen mixed with Freund’s adjuvant, but this model does not prove that the damage is exerted by antibodies because Freund’s adjuvant is nephrotoxic and is itself able to produce MGN. It has not yet been demonstrated experimentally that autologous antibodies alone can produce more than trace or transient proteinuria; and kidney biopsies in unselected humans have shown that MGN is a frequent finding in individuals with normal urine and normal renal function, indicating that a subepithelial formation of immune complexes is also harmless to humans. Severe glomerulonephritis without immune complex formation can easily be produced experimentally with many nephrotoxic chemicals. It is therefore suggested that the primary event in MGN, and probably other subgroups, is a toxic or allergic tubulointerstitial reaction to chemicals or drugs and that the formation of glomerular immune complexes is a later and secondary phenomenon. In agreement, renal function and the course in MGN and other glomerulonephritides are strongly correlated with the degree of tubulointerstitial damage, but totally unrelated to the degree of glomerular damage. The hypothesis explains why a large number of patients with endstage renal failure owing to glomerulonephritis have been heavily exposed to environmental pollutants.

Murine Model of Oral Tolerance: Induction of Fas‐Mediated Apoptosis by Blockade of Interleukin‐12

We explored the immunological mechanisms underlying the development of oral tolerance with the use of ovalbumin (OVA) T-cell receptor (TCR)-transgenic mice. Feeding high doses of OVA induced tolerance in the peripheral lymphoid tissues, and the degree of peripheral tolerance was enhanced when antigen feeding was combined with systemic administration of antibodies to interleukin-12 (anti-IL-12). Using the TUNEL technique by which apoptotic cells can be specifically identified, we found evidence that peripheral clonal deletion occurs in OVA-TCR transgenic mice in vivo after oral antigen delivery and treatment with anti-IL-12, but only to a minor degree after antigen feeding alone. The mechanism that accounts for the dramatic loss of peripheral cells is Fas-mediated, since > 90% of antigen-specific Fas+ T cells were lost. In addition, antagonizing Fas but not TNF reversed the phenomenon when cells were stimulated in vitro. These findings suggest that IL-12 negatively regulates apoptosis, a major mechanism of peripheral tolerance. A combination of oral antigen feeding and administration of anti-IL-12 may thus be useful in the treatment of autoimmune diseases and may be a potent means to modulate peripheral tolerance.

When Immunization Leads to Autoimmunity: Chronic Inflammation as a Result of Thymic and Mucosal Dysregulation in IL-2 Knock-out Mice

In 1965 Wheelock identified IFN--/ as a new soluble substance produced by activated T cells that inhibited viral replication in fibroblasts [ I ] . This seminal observation led to the explosive research on lymphocyte mediated activators during various aspects of the cellular immune response [2-71, and was followed in 1976 by the discovery of IL-2 by Morgan et al. [S]. IL-2 was discovered as a secreted product of T cells activated by a polyclonal mitogen, phytohemagglutinin (PHA), that stimulated the proliferation of