Intestinal I/R injury is associated with increased microvascular permeability, interstitial edema, impaired vasoregulation, inflammatory cell infiltration, and mucosal ulceration.1
Neutrophils have been implicated as an important mediator in intestinal I/R injury.3
Previous studies found accumulated neutrophils in the gut after I/R injury.1, 27
Neutrophil depletion was found to decrease the incidence of gastritis in primates and gastric bleeding in rats after HS/R, 41, 42
and improved postischemic hypoperfusion of the intestines in rats.10
In the current study, we used the strategy of neutrophil depletion to determine whether the intestinal cytoprotective effects of HB-EGF were dependent upon the presence of neutrophils. HB-EGF treatment of mice subjected to HS/R led to decreased intestinal permeability. Neutropenia provided the same level of gut barrier protection as did HB-EGF. However, the protective effects of HB-EGF treatment on gut barrier function was not synergistic with neutropenia, since neutropenia combined with HB-EGF treatment did not confer further improvement in gut barrier function. This observation suggests that the ability of HB-EGF to protect gut barrier function is dependent on the presence of neutrophils.
PMN-EC interactions play vitals roles in the pathogenesis of intestinal I/R injury.10
To examine PMN-EC interactions, an in vitro
PMN-EC adhesion model was established.43
In this model, EC injured by A/R express various inflammatory mediators such as adhesion molecules, interleukins, growth factors, cytokines and chemokines,44
facilitating PMN-EC adherence. We found that treatment of PMN with HB-EGF significantly decreased PMN-EC adherence 4 h after A/R, and this effect was reversed with EGFR inhibition. Pretreatment of EC with HB-EGF significantly decreased PMN-EC adherence 12 h after A/R, and this effect was reversed in the presence of EGFR or PI3K inhibitors. These findings suggest that HB-EGF exerts its inhibitory effects on PMN-EC adherence via interaction with the EGFR and via the PI3K-Akt pathway.
PMN-EC adherence is mediated by a well orchestrated sequence of interactions between adhesion molecules on both EC and neutrophils.39
Some of these adhesion molecules including E-selectin, ICAM-1 and PECAM-1 are transcriptionally up-regulated once the PMN or EC are activated.39, 40
Others, including P-selectin, CD11b/CD18 and CD11c/CD18 are stored in intracellular granules that can be rapidly mobilized to the surface of EC or PMN by fusion of granule membranes with the cell membrane.39, 40
We found that HB-EGF treatment of EC led to inhibition of PMN-EC adherence at a late stage after A/R (12 h). However, HB-EGF treatment of PMN led to inhibition of PMN-EC adherence at an earlier stage after A/R (4 h in this study). In a previous study, we found that HB-EGF treatment of PMN began to inhibit PMN-EC adherence as early as 1 hour after A/R.32
These observations suggest that HB-EGF may regulate the expression of adhesion molecules on PMN and EC by different mechanisms.
Using similar PMN-EC adhesion assays, the transcription factor NF-κB was shown to be responsible for increased PMN-EC adherence 4 h after A/R by transcriptional upregulation of EC adhesion glycoproteins.43
The NF-κB/Rel family of transcription factors is composed of five distinct DNA-binding subunits - p50, p52, p65 (RelA), c-Rel, and Rel-B.43
Nuclear translocation of the activated p65 subunit was shown to increase significantly in EC within 30 min of A/R stimulation, and remained constant over 4 h.43
More importantly, the p65 subunit was shown to be responsible for the strong transcription activating potential of NF-κB, and binding sites for the p65 subunit was found in the promoter elements of E-selectin, ICAM-1 and PECAM-1. 35–38
In this study we found that treatment of EC with the NF-κB inhibitor MG132 led to decreased PMN-EC adherence 4 h and 12 h after A/R. HB-EGF treatment of EC decreased PMN-EC adherence, and down-regulated the mRNA expression and p;rotein production of E-selectin, ICAM-1 and PECAM-1 in EC 12 h after A/R. We found that p65 DNA binding was significantly increased 4 h and 12 h after exposure of EC to A/R, and that treatment of EC with HB-EGF inhibited p65 DNA at both of these time points. Furthermore, the inhibitory effects of HB-EGF on p65 DNA binding in EC, and the expression of the adhesion molecule PECAM 1 in EC, were reversed in the presence of inhibitors of EGFR inhibitor and PI3K. These findings suggest that the inhibition of NF-κB activation via the EGFR and PI3K pathway in EC is responsible for the inhibitory effects of HB-EGF on PMN-EC interactions.
Flow cytometric analysis of cell-surface membrane associated CD11b expression in PMN showed that CD11b was down-regulated by HB-EGF 30 min, 1 h and 4 h after fMLP addition to PMN. At the same time, Western blotting of cell extracts revealed that the total amount of CD11b in PMN remained unchanged. These findings suggest that HB-EGF inhibited the mobilization of CD11b to the cell surface rather than regulating the transcription of CD11b in PMN. ROS have been shown to be an important mediator of either CD11b/CD18 mobilization or transcription. Furthermore, PMN are the major source of ROS during inflammation. We showed that HB-EGF decreased ROS production in PMN in this study. These observations suggest that HB-EGF inhibits CD11b mobilization in PMN by decreasing ROS production in these cells. The signal pathways by which HB-EGF decreases ROS production in PMN remain unknown. We assessed possible signaling pathways by treating PMN with several signal pathway inhibitors, however the inhibitory effects of HB-EGF on PMN-EC adherence was only reversed by EGFR inhibition and not by inhibition of Erk1/2 or PI3K, suggesting that other signaling pathways must be involved in the anti-inflammatory effects of HB-EGF in PMN.
In conclusion, this study provides new insights into the anti-inflammatory role of HB-EGF in the preservation of gut barrier function after HS/R. This study demonstrates that the ability of HB-EGF to preserve gut barrier function is dependent upon the presence of neutrophils, that HB-EGF decreases PMN-EC adherence after A/R, that HB-EGF inhibits NF-κB activation via the PI3K pathway, that HB-EGF regulates the transcription of adhesion molecules in EC, and that HB-EGF inhibits adhesion molecule mobilization by decreasing ROS production in PMN. These findings further our knowledge regarding the mechanisms underlying the anti-inflammatory effects of HB-EGF, and support the clinical use of HB-EGF in the future to treat intestinal injuries associated with hypoperfusion/inflammatory states.