IL-1 (IL-1α and IL-1β) is a multipotent, primarily inflammatory cytokine which affects almost any cell type and cooperates with other cytokines, chemokines, and a variety of other mediators. IL-1 is predominantly synthesized by activated macrophages but can also be produced by members of the mononuclear phagocyte family, including dendritic cells, and by other cell populations, such as B lymphocytes, NK cells, endothelial cells, and even epithelial cells under particular circumstances, such as bacterial invasion (17
). Production of IL-1 is tightly regulated at the level of gene expression, and its activity is controlled by a variety of molecules, including surface receptors, such as IL-1RII, a nonagonist, decoy receptor (57
); soluble receptors, such as IL-1sRI and IL-1sRII (6
); and IL-1ra, a specific receptor antagonist (5
). This 23- to 25-kDa protein, which, like IL-1, is produced by monocytes/macrophages but also by PMN, fibroblasts, and keratinocytes (4
), binds to IL-1RI with affinity almost equal to that of IL-1α and IL-1β (19
) but does not transmit a signal (18
). As a consequence, IL-1ra blocks IL-1 activity both in vitro and in vivo (4
There are two major forms of the protein, sIL-1ra, which likely corresponds to the above definition of a nonagonist competitor for IL-1RI, particularly as an antagonist of the secreted form of IL-1β, and icIL-1ra, which shows an altered signal peptide. The physiological function of icIL-1ra, however, is unclear (29
In an increasing number of infectious and noninfectious clinical and experimental situations, data indicate that IL-1ra may be essential for the host defense against excessive, deleterious inflammation (20
). In IBD, and particularly in ulcerative colitis, which shares several clinical and histopathological characteristics with shigellosis, a significant imbalance of the IL-1ra/IL-1 ratio, mostly caused by a decrease of IL-1ra concentrations in intestinal tissues, was found at the acute phase of the disease (11
). The lowest IL-1ra/IL-1 ratios were observed in the most severe cases. More recently, an increase in IL-1α and IL-1β was demonstrated in biopsy specimens from patients suffering acute cases of Crohn's disease, ulcerative colitis, and other inflammatory diseases of the colon. The IL-1ra/(IL-1α plus IL-1β) ratio was consistently decreased in these situations. In addition, a genetic influence on the intensity of inflammation based on dysregulation of the IL-1ra/IL-1 balance may account for an increase in disease severity (3
In patients at the acute and convalescent stages of S. flexneri
and Shigella dysenteriae
1 infection, immunohistochemistry of rectal samples shows a pattern of IL-1, IL-4, IL-6, IL-8, tumor necrosis factor alpha, and gamma interferon hyperproduction. Severe disease is associated with increased numbers of IL-1β-, IL-6–tumor necrosis factor alpha, and gamma interferon-producing cells; IL-1 is essentially produced by monocytes/macrophages, unlike IL-6 and IL-8, which are expressed by epithelial cells (51
). These data suggest that IL-1 is a key factor in Shigella
-induced intestinal inflammation. Our previous observation that IL-1ra reduces intestinal inflammation in experimental shigellosis in the rabbit ligated-loop model of Shigella
) represents the experimental complement to these clinical observations and confirms the key role of IL-1 in the initiation of inflammation. These data also suggest an imbalance in the IL-1ra/IL-1 ratio, as observed in IBD (11
). Moreover, in a model of rabbit immune complex colitis, IL-1 gene expression and synthesis occur early (i.e., at 4 h) in the course of experimental disease and levels of IL-1 in tissues, which are in the same order of magnitude as those observed here, correlate with the degree of tissue inflammation, which is reduced by administration of IL-1ra (15
The aim of this work was to follow the kinetics of IL-1α, IL-1β, and IL-1ra in rabbit ligated loops infected by S. flexneri
, both in infected tissues and in the mesenteric blood. We followed the balance between these proinflammatory cytokines and their major antagonist during the infection. Infections were carried out in loops containing a Peyer's patch because the FAE that covers Peyer's patches is the major site of Shigella
invasion of intestinal tissues at early stages of incubation (i.e., at 2 to 4 h); thus, these areas are the major foci of the initiation of inflammation (47
). In addition, rapid entry into these areas facilitates synchronization of the infectious process, whereas invasion of villous areas of the intestinal epithelium is delayed and more widespread in time (52a
). In addition to invasive M90T, we used a control mutant strain, BS15, a plasmidless, noninvasive mutant of S. flexneri
M90T which expresses the AFR1 rabbit-specific adherence pilus of the enteropathogenic E. coli
). This adhesin causes selective binding to the M-cell surface, thus mediating the translocation of bacteria. In consequence, M90T and BS15 reach similar numbers in infected Peyer's patches (56
The results presented in this paper show an increase in production of IL-1 during progression of the infection. At all time points, tissues infected by the invasive strain M90T contained more IL-1α and IL-1β than those infected with the adhesive-noninvasive strain BS15. Tissue concentrations of IL-1α in Peyer's patches infected by M90T were roughly twofold greater than in those infected by BS15 at all time points, and tissue concentrations of IL-1β were roughly two- to fourfold greater, depending on the time point. This demonstrates that invasive shigellae cause significantly greater release of IL-1 and correlates with the observation that, in spite of its causing significant influx of PMN in the domes of lymphoid follicles after crossing the FAE, BS15 does not induce the extensive inflammatory destruction observed with M90T (56
). These results are also in agreement with immunohistochemical observations of human rectal samples (51
). Higher production of IL-1α and IL-1β is likely to reflect both higher expression of IL-1 in individual cells and stronger recruitment of these producing cells to the infection site. Accordingly, the number of transcripts for IL-1α and IL-1β followed a trend similar to that measured by the cytokines, as measured by RT-PCR performed on tissue samples. Immunostaining experiments carried out on infected Peyer's patch tissues clearly confirmed the titration experiments; there were higher numbers of IL-1α- and IL-1β-producing cells, as well as a greater amount of these cytokines visible on the tissue sections of Peyer's patches infected with M90T than on those infected with BS15.
Titrations carried out on the plasma obtained from the efferent mesenteric blood samples taken from infected loops were in agreement with these data, showing increasing concentrations of the two cytokines during the infection. Interestingly, however, the ratio of plasma IL-1α to tissue IL-1α was roughly 1 at all time points, whereas the ratio of plasma IL-1β to tissue IL-1β varied between 0.1 and 0.2, thus appearing 10-fold lower. The latter observation was unexpected, because in inflammatory situations, particularly in acute infections, significant levels of circulating IL-1β, but rarely of circulating IL-1α, are observed. This is essentially due to the property of IL-1α of remaining primarily intracellular and being released mostly in situations in which cytosolic leakage can occur, such as in the event of cell lysis (58
). Conversely, mature IL-1β is normally secreted by activated macrophages following cleavage of the promolecule by caspase 1 (ICE) (9
Macrophages from ICE knockout mice do not release mature IL-1β in vitro (36
). The present observation is likely to reflect a situation of massive killing of preactivated IL-1-producing cells in areas where invading Shigella
interacts with macrophages, particularly in the domes of follicular structures. This is precisely what was observed here with the early appearance (i.e., at 4 h) of apoptotic cells, likely macrophages, in the apical domes of Peyer's patches infected by M90T. Invasive Shigella
causes macrophage apoptosis both in vitro (67
) and in vivo (66
). Apoptotic killing in vivo has been studied under experimental conditions that were similar to those followed in the course of this study. Under such conditions, after 8 h of infection, the number of apoptotic cells is more than 50-fold greater in follicles infected by M90T than in those infected by BS15 (66
). IpaB is the major effector of apoptotic death by activating ICE (12
). This accounts for early release of mature IL-1β by infected macrophages before they complete their cell death program (31
), thereby initiating early inflammation in follicular zones (62
). The important extracellular release of IL-1α, which is reflected by its high circulating titer in the mesenteric blood, may be explained by massive killing of resident macrophages and recruited monocytes in the dome area. This profound level of apoptosis overwhelms the phagocytic clearance of apoptotic cells, thus allowing free extracellular IL-1α to be present in tissues and to subsequently pass into the circulation. Based on the above discussion, a model emerges in which not only the difference in transcriptional and translational expression of IL-1α and IL-1β but also the complex intricacy of macrophage activation and macrophage death caused by the invasive phenotype of M90T that makes both forms of IL-1 more readily available to bind IL-1RI and activate the large array of reactive cells present within the invaded zone can account for the difference in induction capacity of tissue inflammation and destruction observed between M90T and BS15.
Surprisingly, in addition to the higher expression of IL-1, M90T also caused a decrease in the expression of IL-1ra in infected tissues at 4 h after infection in comparison with BS15. A twofold and a fivefold decrease were observed at 2 and 4 h, respectively. At 8 h after infection, IL-1ra concentrations caught up, probably due to massive recruitment of producing cells to infected zones. The plasma concentrations reflected the tissue data, with a striking difference observed at 4 h after infection; IL-1ra concentrations were 25-fold lower in mesenteric blood samples corresponding to Peyer's patches infected with M90T than in those infected by BS15. These differences were confirmed when the IL-1ra/IL-1 ratios were calculated. In tissues infected by M90T, IL-1ra/IL-1α and IL-1ra/IL-1β ratios were consistently less than 100, in contrast to tissues infected by BS15, in which those ratios appeared constantly greater and, at 2 h, largely greater than 100.
The major differences appeared at the early 2- and 4-h time points of infection. Ratios in plasma samples dramatically accentuated these differences again at early stages of infection. The levels of mRNA clearly reflected the tissue levels of IL-1ra, with significantly lower activities after 2 and 4 h of infection in tissues infected by M90T than in those infected by BS15. This was observed both for sIL-1ra and icIL-1ra, thereby suggesting a common down-regulatory mechanism. It is possible that killing of macrophages at the early time points of infection accounts for this decrease.
It has previously been shown in human monocytes that LPS has the capacity to induce IL-1 and IL-1ra in the same cell (2
). It is therefore possible that after releasing IL-1, the same macrophages and recruited monocytes are unable to compensate for their proinflammatory effect by producing enough IL-1ra because they are apoptotic. After 8 h of infection there is recruitment of PMN and circulating monocytes. Together, these cells control bacterial growth and increase the number of cells that produce IL-1ra, thereby restoring the IL-1ra–IL-1 balance.
It appears, therefore, that the early stage of Shigella infection is characterized by an imbalance between IL-1ra and IL-1 whose hypothetical mechanism is summarized in Fig. . In addition to the higher level of IL-1 production that characterizes the invasive phenotype, the lack of a proper balance between IL-1ra and IL-1 becomes another key feature of the development of severe inflammation. These studies certainly emphasize the need for experimental systems and analytical tools that would allow us to dissect the very early stages of infectious processes, particularly the early time points of the innate immune response, which are crucial for the development and subsequent healing of the disease lesions.
FIG. 6 Scheme summarizing the possible link between macrophage apoptosis in the dome areas of lymphoid follicles infected by a noninvasive (A) or an invasive (B) Shigella strain and the imbalance between IL-1 and IL-1ra accounting for severe inflammation observed (more ...)