SMALL INTESTINE

Immunomodulatory effects of inhaled carbon monoxide on rat syngeneic small bowel graft motility

A Nakao¹, B A Moore², N Murase¹, F Liu³, B S Zuckerbraun¹, F H Bach⁴, A M K Choi³, M A Nalesnik⁵, L E Otterbein^3,*, A J Bauer^2,*

¹ Thomas E Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA
² Department of Medicine/Gastroenterology, University of Pittsburgh, Pittsburgh, PA 15261, USA
³ Department of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA
⁴ Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
⁵ Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261, USA

Correspondence to:
Correspondence to:
Dr A J Bauer, Department of Medicine/Gastroenterology, University of Pittsburgh, S-849 Scaife Hall, 3550 Terrace St, Pittsburgh, Pennsylvania 15261, USA;
tbauer{at}pitt.edu

Background: Intestinal transplantation provokes an intense inflammatory response within the graft muscularis that causes intestinal ileus. We hypothesised that endogenously produced anti-inflammatory substances could be utilised as novel therapeutics. Therefore, we tested the protective effects of inhaled carbon monoxide (CO) and an endogenous haeme oxygenase 1 (HO-1) anti-inflammatory mediator on transplant induced inflammatory responses and intestinal ileus in the rat.

Methods: Gastrointestinal transit of non-absorbable FITC labelled dextran and in vitro jejunal circular muscle contractions were measured in controls and syngeneic orthotopic transplanted animals with and without CO inhalation (250 ppm for 25 hours). Inflammatory mRNAs for interleukin (IL)-6, IL-1ß, tumour necrosis factor (TNF-), intercellular adhesion molecule 1 (ICAM-1), inducible nitric oxide (iNOS), cyclooxygenase 2 (COX-2), and IL-10 were quantified by real time reverse transcriptase-polymerase chain reaction and HO-1 by northern blot. Histochemical stains characterised neutrophil infiltration and enterocyte apoptosis.

Results: Transplantation delayed transit and suppressed jejunal circular muscle contractility. Transplantation induced dysmotility was significantly improved by CO inhalation. Transplantation initiated a significant upregulation in IL-6, IL-1ß, TNF-, ICAM-1, iNOS, COX-2, and HO-1 mRNAs with the graft muscularis. CO inhalation significantly decreased expression of IL-6, IL-1ß, iNOS, and COX-2 mRNAs. CO also significantly decreased serum nitrite levels (iNOS activity).

Conclusions: CO inhalation significantly improved post-transplant motility and attenuated the inflammatory cytokine milieu in the syngeneic rat transplant model. Thus clinically providing CO, the end product of the anti-inflammatory HO-1 pathway, may prove to be an effective therapeutic adjunct for clinical small bowel transplantation.

Keywords: transplantation; haeme oxygenase; carbon monoxide; cytokines; small bowel; rat

Abbreviations: BSA, bovine serum albumin; CO, carbon monoxide; SITx, small intestinal transplantation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO-1, haeme oxygenase 1; COX-2, cyclooxygenase 2; iNOS, inducible nitric oxide; NO, nitric oxide; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; TNF-, tumour necrosis factor ; MPO, myeloperoxidase; PBS, phosphate buffered saline; PMNs, polymorphonuclear neutrophils; RPA, RNase protection assay; RT-PCR, reverse transcriptase-polymerase chain reaction; SDS, sodium dodecyl sulphate

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