Crohn disease is a disorder characterized by inflammation of any portion of the gastrointestinal tract, from mouth to anus. Extraintestinal manifestations of the disease include arthralgias, ankylosing spondylitis, myalgias/myositis, uveitis, vasculitis, and sclerosing cholangitis. Skin manifestations include erythema nodosum and pyoderma gangrenosum. Granulomatous dermatitis or metastatic Crohn disease is rare in childhood. Although extensive published data exist on genetic, infectious, immunologic, and psychologic aspects of Crohn disease, the role of mitochondrial dysfunction as a primary or contributory cause has not been studied.
We report a pediatric patient with muscle weakness, seizures, and respiratory insufficiency beginning at age 1 year, who experienced severe metastatic Crohn disease at 8 years of age. She experienced no response to antibiotics, steroids, or bowel rest but showed dramatic improvement with anti–tumor necrosis factor-alpha (TNF-α) antibody (infliximab). The response to infliximab suggested that she might have some mitochondrial dysfunction (1,2). Her normal mononuclear cell thymidine phosphorylase activity ruled out the possibility of mitochondrial neurogastrointestinal encephalomyelopathy syndrome (MNGIE). Studies of mitochondrial oxidative phosphorylation revealed functional defects at Complex III and IV in isolated muscle mitochondria which suggest that mitochondrial dysfunction might play a role in the pathogenesis of her condition.
This 8-year-old Hispanic girl was born at term via spontaneous vaginal delivery without complications and was discharged on day 2 of life. At age 10 days she experienced seizures. She was treated unsuccessfully with phenobarbital. Seizures were controlled with carbamazepine. No etiology for her seizures was determined. She had normal development until age 11 months, when she was admitted to the pediatric intensive care unit (PICU) with obtundation, muscle rigidity, and hyperpyrexia, which was attributed to a neuroleptic malignant syndrome secondary to the combined use of carbinoxamine (Rondec) and carbamazepine. Her hospital stay was complicated by gastrointestinal hemorrhage from a large gastric antral ulcer. She was treated with amantadine and dantrolene for the neuroleptic malignant syndrome. Thereafter she experienced a gradual loss of developmental milestones, with resulting severe developmental delay.
An evaluation of her mental retardation and muscle weakness revealed normal serum concentrations of lactate, pyruvate, amino acids, and copper. Urine organic acids, thyroid function studies, biotinidase activity, and lactate-to-pyruvate ratios were normal. Computed tomography and magnetic resonance imaging of the head revealed thinning of the posterior body of the corpus collosum. Electromyographic study results were consistent with a myopathy. Mitochondrial point mutation studies for the syndromes of mitochondrial encephalomyelopathy, lactic acidosis, and strokes; neurogenic weakness, ataxia, and retinitis pigmentosa; and myoclonic epilepsy with ragged-red fibers were normal. No specific testing for MNGIE was performed during the initial evaluation. The patient continued to have recurrent pneumonia, pulmonary aspiration and hypoventilation. Nissen fundoplication with gastrostomy tube insertion was performed at 1 year. Esophagogastroduodenoscopy (EGD) at the time of surgery revealed hiatal hernia and esophagitis. At age 7 years, she was treated with a bilevel positive airway pressure device (BiPAP®; Respironics, Pittsburgh, PA, U.S.A.) at night because of persistent hypercarbia and hypoventilation.
At age 6 years, the patient began having repeated episodes of diarrhea and perirectal abscess. Her erythrocyte sedimentation rate was persistently high. During her third hospital stay for treatment of perirectal abscess and vulvar cellulitis, EGD, colonoscopy, skin biopsy and pelvic examination were performed under anesthesia. She had severe perianal inflammation with multiple anal fissures, anal skin tags, edema and erythema of the vulva, and inflammation of the rectum, colon, stomach, and esophagus. Biopsies of the labia majora, colon, and esophagus revealed granulomatous inflammation. Acid fast stains and fungal stains were negative (Fig. 1A and B). Biopsy specimens were consistent with the diagnosis of Crohn disease with metastasis to the vulvar skin. Her immediate treatment included ampicillin, gentamicin, Flagyl, intravenous steroids, sulfasalazine, bowel rest and total parenteral nutrition. After approximately 2 weeks of treatment without improvement, intravenous infliximab (5 mg/kg) was given. There was rapid improvement of the perianal disease and the metastatic disease of the vulva. Total parenteral nutrition was continued, and infliximab was given every 7 to 8 weeks. After approximately 3 months of treatment, feedings were initiated through her gastrostomy and were tolerated well. The patient regained the ability to stand and walk. Her intestinal symptoms and neuromuscular abnormalities suggested the possibility of MNGIE. Therefore, a more detailed search for mitochondrial disorders was initiated.
MATERIALS AND METHODS
Quantitation of Serum Cytokines
Serum obtained at the time of initial evaluation of her apparent myopathy that had been stored at −70°C and serum obtained at the time of the diagnosis of Crohn disease were used for acute-phase cytokine quantification. Commercially available enzyme-linked immunosorbent assay kits for TNF-α, interleukin-6 (IL-6), and IL-1β, were used according to manufacturer's instructions (R&D, Minneapolis, MN, U.S.A.).
Thymidine Phosphorylase Activity
Excess blood drawn for routine studies was used for this assay, performed as described previously (3). Peripheral blood mononuclear cells were separated using Ficoll Hypaque centrifugation. Cells were washed three times with phosphate buffered saline, and cell pellet was placed in cold (0°C) extraction buffer containing 10 mM KPO4 pH 7.4 and 1 mM dithiothreitol. Cells in buffer were ground in a glass homogenizer. The homogenate was centrifuged at 20,000 ×g for 10 to 15 minutes. The supernatant was dialyzed with the same extraction buffer, but containing 5% glycerol, and used in the enzyme assay containing 0.1 mL of the dialyzed extract, 0.1 mL substrate (100 mM KPO4 pH 7.4, 1 mM dithiothreitol, and 8 mg/mL thymidine), and 0.1 mL of extraction buffer (total, 0.3 mL). This was incubated 30 minutes at 37°C. Then 0.5 mL cold 3% HClO4 was added and centrifuged. Next, 0.4 mL of the resulting supernatant was mixed with 1 mL of 0.3 N NaOH. This was read in a spectrophotometer at 300 nm against 0 time blank. The protein concentration of the dialyzed enzyme preparation was determined using Folin reagent. The thymidine phosphorylase activity was expressed in units. One unit equaled 1 nanomole of thymine formed per milligram protein per hour.
Muscle mitochondria were isolated as previously described (4). Mitochondrial respiration [O2 consumption] was measured with a Clark-type oxygen electrode. Incubation mixtures contained 0.5 mg/mL of mitochondrial protein. Both coupled and uncoupled respirations were measured using standard substrates (5). Mitochondrial electron transport chain activity was determined as reported (4,6). Complex III was measured using a diode assay spectrophotometer by following the increase in reduced cytochrome C absorbance (7). Assays for electron transport in Complexes II, III, and IV were also measured in cultured dermal fibroblasts (4).
A muscle biopsy revealed atrophic type 2 fibers and no ragged red fibers (Fig. 2). Electron microscopic study did not reveal any significant mitochondrial abnormalities. Studies of the electron transport chain in isolated mitochondria of skeletal muscle, not usually performed during the evaluation of Crohn disease, showed normal activity for Complexes I, II, III, and IV (Table 1). These results were confirmed in studies of cultured dermal fibroblasts for Complexes II, III, and IV. In isolated mitochondria from skeletal muscle, oxidative phosphorylation was impaired in Complexes III and IV. ADP-stimulated (State 3) rates of substrate oxidation were below the control rates for glutamate, glutamate + malate, palmitoylcarnitine + malate, and duroquinol (Table 2). The respiratory control ratios (State 3/State 4; State 4; ADP limited rate), which reflect the integrity of the mitochondria and the coupling of oxidative phosphorylation, were below the control range for these substrates because of the low rates of State 3 oxidation. The ADP/O ratio (ADP added/oxygen consumed in State 4), which indicates the efficiency of oxidative phosphorylation, was normal for each substrate tested. Maximum rates of substrate oxidation were measured in the presence of a high concentration of ADP (2 mM). These ratios were below the control range for glutamate, glutamate + malate, duroquinol, and TMPD/ascorbate (Table 2). The rates of oxidation for these substrates was not increased by the addition of an uncoupler. The rates of substrate oxidation were normal for other substrates tested. The data indicate functional abnormalities in Complexes III and IV and no apparent defects in Complexes I, II, and V. These data suggest a disruption of normal cellular respiration at the mitochondrial level. Thymidine phosphorylase activity in blood mononuclear cells was normal (469 units), excluding the diagnosis of MNGIE.
The abnormalities detected are not diagnostic of a specific mitochondrial disorder (10–12) but resembled the changes mediated by TNF-α and ceramide on mitochondrial respiration (1,2). Systemic levels of TNF-α at the time of initial presentation were normal (3.2 pg/mL), as assessed by enzyme-linked immunosorbent assay on banked serum (R&D). Mucosal TNF-α was not measured.
A number of reports have suggested that mitochondrial pathology may be associated with Crohn disease. The brown bowel syndrome, described in a patient with Crohn disease of 5 years duration, was shown to be attributable to lipofuscin pigment within degenerating mitochondria. The investigators concluded that the brown bowel syndrome was a smooth muscle mitochondrial myopathy (11). Others have noted changes in the mitochondrial histology of patients with Crohn disease, as well as functional defects in organelles isolated from rectal biopsy specimens (12). Our patient had defective oxidation of Complex III and IV substrates in the mitochondria isolated from muscle, a site distant from her primary disease. Although defects were observed in isolated, intact skeletal muscle mitochondria during coupled oxidative phosphorylation, there were no defects observed in the enzyme complexes of skin fibroblast or skeletal muscle mitochondria. The apparent discrepancy between the normal enzyme complexes and the abnormal oxidative phosphorylation suggests that the defect is not in the subunit composition but is involving the integrated organization at the mitochondrial inner membrane.
The key questions raised by these observations are whether mitochondrial pathology played a primary role in the pathogenesis of Crohn disease in our patient and whether the functional defects were produced by circulating cytokines that inhibited mitochondrial respiration. We do not have data to answer these questions. However, we speculate that mitochondrial dysfunction may play a more important role in the development of Crohn disease than is currently recognized. If confirmed in larger studies, this would be of potential clinical and therapeutic significance.
One clinical observation deserves special comment. Our patient did not respond to standard therapies until anti–TNF-α antibodies were given. TNF-α is a pro-inflammatory cytokine that is a member of a family of cell death inducing ligands acting through the mitochondrial pathway (13,14). Its effect on mitochondrial respiration can be modulated through several molecules. Cell-permeable ceramide is a lipid second messenger that mediates the effects of TNF-α by directly inhibiting Complex III of the mitochondrial respiratory chain (1). Could this mechanism account for the defects of Complexes III and IV found in our patient's mitochondria? The response of our patient to treatment with anti–TNF-α antibodies would support this speculation. Severe fatigue, not explained by anemia, cortisol insufficiency, or mucosal disease severity is common in patients with Crohn disease (15). This symptom could be a result of abnormal mitochondrial function. Anti–TNF-α antibodies may be therapeutically useful in other patients with dysfunctional mitochondria. That the defects in mitochondrial function may be mediated at least in part by TNF is supported by clinical observations (16): the marked and immediate improvement in energy and sense of well-being experienced by many patients with Crohn disease treated with infliximab (sometimes within hours), long before mucosal healing could have occurred. Studies of the direct effect of infliximab on mitochondrial function have not been reported. Pharmacologic inhibition of TNF by infliximab and other agents previously has been shown to affect leptin, high-density lipoprotein, and triglyceride levels (17), also suggesting potential effects on energy metabolism.
Recent advances in molecular genetics also suggest a role for mitochondrial dysfunction in Crohn disease. NOD-2, also known as CARD-15, is a susceptibility gene for familial Crohn disease identified in 2001 (18). Mutations in the sequences involved in the recognition of the bacterial product muramyl dipeptide have been demonstrated in some patients with Crohn disease. These mutations cause abnormal activation of the proinflammatory transcription factor NF-KB. Mutations in the nucleotide-binding domain of CARD-15 have been associated with the development of Blau syndrome (19,20). There are no studies of the effects of mutations in the caspase activation domain or of the effects of the fully folded abnormal protein with the common Crohn disease mutations on caspase activation and mitochondrial function of this triple-domain peptide. Our patient was not tested for the common genetic mutations associated with Crohn disease. However, in light of the death of a similarly affected sibling, it is not unlikely that she possessed an underlying genetic/metabolic defect predisposing her to the combined metabolic-inflammatory abnormalities.
Very recently, NF-KB and IKB-alpha have been localized to the mitochondria (21). Mitochondrial exposure to TNF has confirmed the loss of expression of cytochrome c oxidase 3 and cytochrome b mRNA, which downregulate mitochondrial mRNA expression (21) Thus, growing evidence from clinical, genetic, and molecular biologic studies suggest that there may be cytokine-mitochondrial interactions in the pathogenesis of Crohn disease. Studies of additional patients using mitochondria from tissue directly involved in the inflammatory process should provide additional insight into the role of mitochondria in the pathogenesis of symptoms in patients with inflammatory bowel disease. If a role for mitochondrial dysfunction is confirmed, treatments that improve mitochondrial function, such as coenzyme or vitamin supplementation, may be of therapeutic value.
1. Gudz TI, Tserng K-Y, Hoppel CL. Direct inhibition of mitochondrial respiratory chain complex III by cell-permeable ceramide. J Biol Chem
2. Schulze-Osthoff K, Bakker, AC, Vanhaesebroeck B, et al. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J Biol Chem
3. Srivastava BI, Minowada J. Terminal transferase immunofluorescence, enzyme markers and immunologic profile of human leukemia-lymphoma cell lines representing different levels of differentiation. Leuk Res
4. Hoppel CL, Kerr DS, Dahms B, et al. Deficiency of the reduced nicotinamide adenine dinucleotide dehydrogenase component of complex I of mitochondrial electron transport: fatal infantile lactic acidosis and hypermetabolism with skeletal-cardiac myopathy and encephalopathy. J Clin Invest
5. Hoppel CL, DiMarco J, Tandler B. Riboflavin and rat hepatic cell structure and function. Mitochondrial oxidative metabolism in deficiency states. J Biol Chem
6. Krahenbuhl S, Chang M, Brass EP, et al. Decreased activities of ubiquinol: ferricytochrome C oxidoreductase (Complex III) and ferrocytochrome C; oxygen oxidoreductase (Complex IV) in liver mitochondria from rats with hydroxycobalamin [c-lactam]-induced methylmalonic aciduria. J Biol Chem
7. Krahenbuhl S, Talso C, Wiesmann U, et al. Development and evaluation of a spectrophotometric assay for Complex III in isolated mitochondria, tissues and fibroblasts from rats and humans. Clin Chim Acta
8. Roe CR, Coates PM. Mitochondrial fatty acid oxidation disorders. In: Schriver CR, et al., eds. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill, Inc; 1995:1501–25.
9. Shoffner JM, Wallace DC. Oxidative phosphorylation diseases. In: Schriver CR, et al., eds. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill, Inc; 1995:1535–83.
10. Heales SJR, Bolanos JP, Stewart VC, et al. Nitric oxide, mitochondria, and neurological disease. Biochim Biophys Acta
11. Lambert JR, Luk SC, Pritzker KP. Brown bowel syndrome in Crohn's disease. Arch Pathol Lab Med
12. O'Morain C, Smethurst P, Levi J, et al. Subcellular fractionation of rectal biopsy homogenates from patients with inflammatory bowel disease. Scand J Gastroenterol
13. Sanchez-Alcazar JA, Hernandez I, De la Torre MP, et al. Down-regulation of tumor necrosis factor receptors by blockade of mitochondrial respiration. J Biol Chem
14. Suematsu N, Tsutsui H, Wen J, et al. Oxidative stress mediates tumor necrosis factor alpha induced mitochondrial DNA damage and dysfunction in cardiac myocytes. Circulation
15. Minderhoud IM, Oldenberg B, van Dam PS, et al. High prevalence of fatigue in quiescent inflammatory bowel disease is not related to adrenocortical insufficiency. Am J Gastroenterol
16. Lichtenstein GR, Bala M, Han C, et al. Infliximab improves quality of life in patients with Crohn's disease. Inflamm Bowel Dis
17. Cauza E, Cauza K, Hanusch-Enserer U, et al. Intravenous anti-TNF-alpha antibody therapy leads to elevated triglyceride and reduced HDL-cholesterol levels in patients with rheumatoid and psoriatic arthritis. Wien Klin Wochenschr
18. Hugot JP, Zouali H, Lesage S. Lessons to be learned from the NOD2 gene in Crohn's disease. Eur J Gastroenterol Hepatol
19. Miceli-Richard C, Lesage S, Rybojad M, et al. CARD 15 mutations in Blau syndrome. Nat Genet
20. Kurokawa T, Kikuchi T, Ohta K, et al. Ocular manifestations in Blau syndrome associated with a CARD15/Nod2 mutation. Ophthalmology
21. Cogswell PC, Kashatus DF, Kiefer JA, et al. NF-kappa B and I kappa B alpha are found in the mitochondria. Evidence for regulation of mitochondrial gene expression by NF-kappa B. J Biol Chem