The present data demonstrate that rapid shedding of IR damaged intestinal epithelial cells attenuates the development of a classically observed vigorous inflammatory response in the reperfused jejunum. When analyzed over a prolonged reperfusion period, no increase in TNF-α, HO-1, IL-6, IL-8 gene expression, expression of adhesion molecules, PMN influx or activation and deposition of complement were observed. However, HIF1- α and C3 gene expression were up-regulated during reperfusion. This possibly illustrated an IR induced regenerative response, aimed at intestinal barrier restoration and prevention of bacterial translocation.
Development of IR induced organ damage is generally described to be characterized by an excessive and vigorous inflammatory response.
This inflammatory response is mainly triggered by apoptosis of cells that cannot be resolved in time by phagocytic cells and will become necrotic in time.
Such necrotic cells are a source of damage associated molecular patterns (DAMPS) or alarmins which recruit and activate innate immune cells, aimed at restoration of homeostasis and tissue repair.
However, in the context of ischemia and reperfusion such an inflammatory reaction often results in additional tissue damage.
It has been demonstrated in the kidney as well as other organs that IR induced inflammation and subsequent organ damage are dependent on the development of widespread apoptosis.
Rapid clearance of apoptotic cells as well as therapeutic strategies to reduce apoptosis have been shown to be critical in preventing the mostly harmful IR induced inflammatory response.
Conclusive data on intestinal ischemia and reperfusion pathophysiology have been obtained from experimental animal studies. Cytokine activity of TNFα, IL-6 and IL-8 have been associated with the inflammatory reaction following IR.
As a prototypic member of the TNF-family, TNF-α is a key mediator of acute inflammation of which expression is readily induced following ischemia reperfusion of the small intestine.
Interestingly, we did not detect an increase in TNFα message in our experiments. Similarly, IL-6 was demonstrated as an important component of the acute phase reaction, able to induce tissue injury and inflammation following mesenteric ischemia and reperfusion in IL-6 knockout (KO) mice.
IL-6 controls endothelial cell injury, mediating successive neutrophil influx. Similarly, the chemokine IL-8 is involved in intestinal ischemia reperfusion induced inflammation. Administration of an inhibitory anti IL-8 antibody over the course of mesenteric IR in rats prevented neutrophil infiltration and protected the small intestine from IR injury.
In keeping with these data, Il-6, IL-8 and TNFα gene expression did not increase in the absence of apoptotic cells, which were shed into the intestinal lumen immediately upon reperfusion.
E-Selectin (CD-62-E and formerly known as endothelial leukocyte adhesion molecule-1 (ELAM-1)) has an important role in the recruitment of leukocytes to sites of tissue injury. Its expression by vascular endothelial cells in response to injury is readily induced by cytokines IL-1 and TNFα. Interestingly, our data suggest that despite initial presence of damaged epithelial cells in response to an IR period, the rapid shedding of damaged epithelial debris largely averts endothelial cell activation. Consistent with this observation, PMN infiltration, assessed by total MPO tissue levels or the presence of HNP1-3 positive cells, in IR was not detected in our experiments. MPO, most abundantly released upon PMN activation, has been demonstrated to have a clear effect on the development of IR induced organ damage. Renal IR studies have demonstrated that MPO itself is able to contribute to the development of organ damage.
As a central innate immune regulator, the complement system is activated during intestinal IR and contributes substantially to IR induced inflammation, organ damage and failure.
Preventing complement activation has been proven to be beneficial to organ function after IR in general and intestinal IR more specifically.
Central in the activation of complement is the formation of C3a, an anaphylatoxin with the ability to attract inflammatory cells into the reperfused ischemic tissue. In our model no deposition of activated C3 in reperfused jejunum was detected, using immunohistochemical and Western blot analysis. Interestingly, our results do however suggest C3 gene expression in healthy jejunum tissue as well as an increased synthesis of C3 in response to IR. The lack of activated complement components as well as a clear increase in local C3 mRNA expression levels may illustrate the intestines response aimed at protection and preservation. The ability of small intestinal epithelial cells to express biological mediators such as C3 in response to IR was to be expected since previous work demonstrated C3 gene expression in inflamed small intestine.
To the best of our knowledge, however, these are the first results that demonstrate C3 mRNA expression in the healthy jejunum. Further analysis will have to elucidate whether increased expression of immune regulatory proteins is directed at preventing gut barrier bacterial translocation. However, when faced with massive intestinal barrier failure, increased expression of immune regulatory proteins able to prevent gut barrier bacterial translocation might be exceedingly useful.
The recently discovered molecular mechanism that recognizes and responds to excessive cell death consisting of SAP130 and the macrophage inducible C-type lectin (Mincle) fits in our observations.
The loss of excessive numbers of dead cells into the gut lumen prevents SAP130 released by the dead cells to reach tissue macrophages thus largely preventing the production of inflammatory cytokines driving rapid neutrophil infiltration. Similarly activation of complement by dead cells is prevented and maybe therefore the PMN chemotactic factors C3a en C5a will not be produced.
A mechanism of protection was suggested by the enhanced expression of HIF-1α in the surviving epithelium. Expression of HIF-1α in response to low oxygen tension during ischemia and early reperfusion triggers physiologic responses characterized by activation of functional proteins mucin, P-glycoprotein, intestinal trefoil factor and adenosine A2B receptor, aimed at preventing mucosal inflammation.
The role in preserving gut wall integrity in response to IR as well as the exciting role of HIF-1α in different cardiac and brain pre-conditioning IR models underlines a possibly protective influence of increased HIF-1α mRNA expression as detected in our model in response to IR of the human intestine.
Taken together, ischemia, sensed by the small intestine, induces normal physiological and IR induced responses. Our data provide new and compelling evidence of increased HIF-1α and also C3 gene expression during the reperfusion period. However, these responses are not paralleled by an IR induced inflammatory response, since important conditions to an inflammatory reaction have not been met by the absence of dead cells in the reperfused tissue. It is important to realize that these data indicate that the human intestine is more resistant to IR than initially thought. Its ability to shed damaged epithelial cells and repair its ever important barrier function without triggering massive inflammation can be seen as key features that prevent the gut from inflammation following splanchnic ischemia and reperfusion.