Cytokines and chemokines are key mediators that orchestrate the host immunoinflammatory response to bacterial infections (49
). Those that activate leukocytes (particularly PMNs) to produce inflammation are essential for the early, nonspecific eradication of invading microbes, often at the price of tissue destruction, which may be quite deleterious and possibly lethal to the host. Shigellosis is a clear example of this situation, in which a limited number of microbes causes acute destructive inflammation of the colonic mucosa. Control of the proper balance between bacterial eradication and tissue destruction requires an understanding of the complex network of interactions mediated by pro- and anti-inflammatory cytokines at the initial stage of tissue invasion by Shigella
IL-8 is a CXC chemokine which is chemotactic for PMNs. In addition to being produced by macrophages, along with IL-1, IL-6, and TNF-α, in situations of infection or tissue injury, it is also produced by other cells such as PMNs themselves, T cells, endothelial cells, and epithelial cells (2
). LPS, viruses, cytokines (IL-1, TNF-α), yeasts, and bacterial invasion of tissues are among the infection-related factors that cause IL-8 expression. In addition to chemotaxis, it increases adherence of PMNs to endothelial cells, thereby promoting their transendothelial migration, IL-8 also induces degranulation, respiratory burst, and LTB4 release by PMNs.
We have addressed the role of IL-8 in experimental shigellosis, a disease characterized by a massive influx of PMNs in the intestinal mucosa. Several observations pointed to IL-8 as a major candidate. (i) In human diseases characterized by massive PMN infiltration, IL-8 is detected at high concentrations in the corresponding bodily fluids in infections such as acute peritonitis, bacterial meningitis, endotoxemia, acute respiratory distress syndrome, Helicobacter pylori
gastritis, and urinary tract infection (19
). (ii) The use of blocking antibodies against IL-8 in animal models has shown attenuation of both clinical and histopathological symptoms in several situations characterized by acute inflammation (44
); this includes rabbit models of LPS-induced dermatitis, arthritis, and immune complex glomerulonephritis (18
), lung reperfusion injury (41
), and endotoxin-induced pleurisy (5
). These experiments have confirmed that IL-8 is a key mediator of tissue damage caused by acute inflammation. (iii) Finally, IL-8 is likely to be a key mediator in the pathogenesis of IBDs, particularly ulcerative colitis (10
). The role of intestinal epithelial cells as a source of IL-8 in IBDs remains controversial. In Crohn’s disease and ulcerative colitis, colonic crypt cells produce elevated levels of IL-8, and cells isolated from inflamed areas express more IL-8 than do cells from normal areas (27
). Also, short-chain fatty acid butyrate reduces in vitro secretion of IL-8 from isolated crypt cells (15
). Other studies have shown, however, that infiltrating macrophages and PMNs are the major source of IL-8 (10
In situations of intestinal infection, intestinal epithelial cells, which are the first cellular barrier against the pathogen, act as sentinels (43
) by orchestrating early nonspecific immune responses by the secretion of cytokines and chemokines. This is particularly obvious for colonic epithelial cells (i.e., the T84, HT-29, and Caco-2 cell lines and freshly isolated colonic cells). Gram-negative invasive bacteria appear to stimulate the highest levels of chemokines such as IL-8, MCP-1, and granulocyte-macrophage colony-stimulating factor and of TNF-α (21
In patients, in the course of shigellosis, secretion of cytokines, particularly IL-8, in stool extracts is correlated with disease severity (35
). As expected from the previous data, experimental shigellosis in the rabbit ligated-loop model of infection was associated with increasing levels of tissue IL-8, which reached a concentration eightfold higher after 8 h of infection when the infection was carried out with a wild-type invasive microorganism than when it was done with a noninvasive control. Although as already discussed for IBD, recruited monocytes and the PMN themselves may account for the production of a significant part of IL-8, immunostaining experiments indicated that IL-8 expression was essentially associated with epithelial cells, regardless of their invasion by bacteria, with areas of expression extending far beyond these zones of bacterial invasion. Similarly, in a SCID mouse model of human embryonic xenotransplantation followed by infection with Entamoeba histolytica
trophozoites, IL-8 was essentially produced by epithelial cells, even at locations far from areas of mucosal damage (42
), thereby confirming previous in vitro data (13
). However, in in vitro assays, bacterial invasion of the cells is required to elicit significant basal secretion of IL-8 (11
). In addition, as shown experimentally with Salmonella typhimurium
, factors other than IL-8 are required in order to allow PMN transmigration across an in vitro-reconstituted epithelial monolayer. IL-8 may attract PMNs from a distance but may not be directly responsible for their transmigration (28
In the case of Shigella
infection in similar in vitro systems, PMN transmigration also occurs and facilitates bacterial invasion via the basolateral pole of epithelial cells (32
), and LPS accounts for about 50% of PMN transmigration (3
). LPS acts both by induction of IL-8 production by T84 cells but also directly via its ability to transcytose through the epithelial lining from its apical to its basolateral pole (4
). It was therefore essential to examine where, exactly, IL-8 is involved during the infectious process in an experimental model of infection that closely reflects tissue invasion.
Neutralization of IL-8 in rabbits during infection with invasive Shigella
had a significant effect, both in attenuating the severity of the lesions and in loosening the barrier effect of the intestinal epithelium against Shigella
translocation. This indicates that in spite of the redundancy of the chemokine system (9
), as observed in other models reported above, IL-8 is a major chemoattractant for PMNs in infected tissues. IL-8 neutralization has a pleiotropic effect on intestinal tissues infected with Shigella
which can be summarized in three parts.
(i) The global decrease in the severity of intestinal lesions can be directly assigned to poor recruitment of PMNs in the lamina propria and epithelial lining, which prevents abscess formation in the presence of invading bacteria but also prevents global attraction of PMNs, even to zones of the epithelium which are not affected by bacterial invasion. In addition, it is likely that PMNs that are still recruited when IL-8 is neutralized do not reach the activation level (i.e., in terms of adherence properties, induction of an oxidative burst, and release of bactericidal or cytotoxic granules) of the PMNs recruited in response to invasive Shigella in control rabbits.
(ii) A decrease in PMN influx caused by the neutralization of IL-8 limits epithelial destruction by lowering the number and size of abscesses, as well as extensive epithelial detachment; this decrease has revealed a subjacent wave of mononuclear cells, mostly lymphocytes, infiltrating the paracellular space of the epithelial lining. This wave precedes the PMN influx in response to the presence of luminal invasive bacteria, but its presence and effect on the epithelial barrier are normally quickly overwhelmed by the influx and cytotoxicity of PMNs. We are currently characterizing these cells which seem to exert a strong cytotoxic effect on the epithelial lining. They may also regulate the transmigration of PMNs through the epithelial lining by producing IFN-γ (1
). If this is the case, early infiltration of the epithelium by these mononuclear cells may promote extension of inflammation at a distance from the infection foci and facilitate bacterial crossing of the epithelial barrier, which is necessary for Shigella
to penetrate epithelial cells via the basolateral pole (30
). Extensive loosening of junctional structures may facilitate access of bacteria or bacterial products of the luminal flora to subepithelial tissues and trigger extensive and diffuse PMN influx beyond foci of Shigella
(iii) Neutralization of IL-8 also had a dramatic effect on the characteristics of mucosal invasion by Shigella
, both qualitatively and quantitatively. Qualitatively, the domain of bacterial infection expanded deeply and diffusely into the lamina propria, instead of remaining restricted to the epithelium. IL-8 produced by epithelial cells may therefore play a major role in limiting Shigella
infection to the villus surface at the initial site of invasion, the epithelium. This is achieved by recruitment of PMNs which have the ability to control and restrict the infectious focus at the cost of severe epithelial destruction. One can conclude from these experiments that IL-8 is a chemokine essential for maintenance of the antitranslocating potential of the intestinal epithelial barrier. Whether this notion can be generalized to the translocation of noninvasive bacteria which do not seem to elicit significant IL-8 expression by epithelial cells (21
) remains to be demonstrated. Quantitatively, the number of bacteria associated with tissues appeared to be threefold higher when IL-8 was neutralized. Three major factors may explain this difference: a higher rate of translocation of bacteria across the epithelial barrier, a diminished number of PMNs recruited to the lamina propria, and a decreased bactericidal function of these PMNs. As a consequence, the number of bacteria achieving complete translocation and appearing in the mesenteric venous blood doubled when IL-8 was neutralized, indicating relative but significant alteration of the intestinal barrier.
Not only do these results indicate the important function that IL-8 has in the pathogenesis of shigellosis, but they also suggest caution when considering the use of an anti-IL-8 strategy against acute inflammatory diseases, particularly IBD and severe cases of infectious enterocolitis.