investigators have reported a high incidence of portal and systemic endotoxemia in .... ware (Apple Computers Inc., Abacus Concepts Inc.,. Berkeley, CA).
ANNALS OF SURGERY Vol. 227, No. 2, 205-212 © 1998 Lippincott-Raven Publishers
Increased Intestinal Permeability and Altered Mucosal Immunity in Cholestatic Jaundice Fenella K. S. Welsh, F.R.C.S,* Carol W. Ramsden, F.I.M.B.L.S.,* Kenneth MacLennan, F.R.C.Path.,* Maria B. Sheridan, F.R.C.R.,* G. Robin Barclay, Ph.D.,t Pierre J. Guillou, F.R.C.S.,* and John V. Reynolds, F.R.C.S.I. * From the Professorial Surgical Unit, Department of Pathology, and Department of Radiology, St. James's University Hospital, Leeds, England,* and the Regional Blood Donor Center, t Edinburgh, Scotland
Objective To examine the effects of cholestatic jaundice on gut barrier function.
Summary Background Data Gut barrier failure occurs in animal models of jaundice. In humans, the presence of endotoxemia indirectly implicates failure of this host defense, but this has not previously been investigated in jaundiced patients.
Methods Twenty-seven patients with extrahepatic obstructive jaundice and 27 nonicteric subjects were studied. Intestinal permeability was measured using the lactulose-mannitol test. Small intestinal morphology and the presence of mucosal immunologic activation were examined in endoscopic biopsies of the second part of the duodenum. Systemic antiendotoxin core IgG antibodies and serum interleukin-6 and C-reactive protein were also quantified. Intestinal permeability was remeasured in 9 patients 5 weeks after internal biliary drainage.
Results The median lactulose-mannitol ratio was significantly increased in the jaundiced patients. This was accompanied by upregulation of HLA-DR expression on enterocytes and gutassociated lymphoid tissue, suggesting immune activation. A significant increase in the acute phase response and circulating antiendotoxin core antibodies was also observed in the jaundiced patients. After internal biliary drainage, intestinal permeability returned toward normal levels.
Conclusions A reversible impairment in gut barrier function occurs in patients with cholestatic jaundice. Increased intestinal permeability is associated with local immune cell and enterocyte activation. In view of the role of gut defenses in the modern paradigm of sepsis, these data may directly identify an important underlying mechanism contributing to the high risk of sepsis in jaundiced patients.
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Sepsis and its sequelae are the leading cause of morbidity and mortality in jaundiced patients,' and this risk correlates with the severity of the jaundice. A potential relation with gut barrier function was suggested more than 30 years ago, when Fine et al.2 postulated that the intestine was a source of systemic endotoxemia. Many subsequent investigators have reported a high incidence of portal and systemic endotoxemia in jaundice, and endotoxin has been incriminated in the pathophysiology of postoperative complications in such patients.3 Bile salts and lactulose may neutralize gut endotoxin and alter enteric microflora, and their administration has been reported to result in clinical benefit in jaundiced patients.45 Although a number of related abnormalities result from jaundice, including impaired endotoxin clearance by Kupffer's cells6 and altered systemic immunity,7 these observations indirectly suggest that impairment of gut barrier function may be relevant in jaundiced patients. There is an emerging consensus that a failure of the gut barrier to exclude endogenous bacteria and toxins from the portal and systemic circulation contributes to the pathophysiology of the systemic inflammatory response syndrome, sepsis, and multiple organ failure.89 In humans, this hypothesis is supported by the frequent absence of an identifiable focus'0 in septic patients with microbiologic evidence of infection and by unexplained sepsis and multiple organ failure in patients with no microbiologic evidence of infection." This theory of gut-origin sepsis is supported by data from animal models.9 Although the integrity of intestinal defenses and barrier function is central to the thesis, there have been few studies of gut barrier function in relevant clinical situations in humans. The aims of this study were to determine whether gut barrier function was impaired in jaundiced patients and to investigate the effects of any altered barrier function on intestinal morphology or gastrointestinal immune function. In addition, we attempted to determine whether increased intestinal permeability was associated with an altered cytokine profile and acute phase response, or with evidence of endotoxin exposure. Intestinal permeability was also examined before and after internal biliary drainage.
METHODS Patients Ethical approval was obtained from the Ethics Committee of St. James's University Hospital, according to the Helsinki agreement. Informed consent was mandatory. Address reprint requests to John V. Reynolds, Academic Surgical Unit, St. James's University Hospital, Leeds LS9 7TF, United Kingdom. Accepted for publication January 1997.
Ann. Surg. * February 1998
Twenty-seven patients with extrahepatic obstructive jaundice and 27 nonicteric controls were investigated. The groups were matched for age, sex, and the presence of neoplastic disease. Postoperative patients, patients with evidence of infection or sepsis, and patients taking steroids or nonsteroidal anti-inflammatory drugs were excluded from the study. Each patient was defined as being well-nourished or mildly or moderately malnourished, using the method of subjective global assessment, as previously described.12 Severely malnourished patients were excluded from the study because of the impact nutritional depletion may have on mucosal permeability.
Intestinal Permeability Intestinal permeability was quantified using the lactulose-mannitol test.'3 The principle of this test is that lactulose and mannitol are absorbed from the intestine by different pathways. Mannitol, the smaller molecule, is absorbed via the transcellular route, lactulose by the paracellular route. Both probes are rapidly excreted in the urine. In conditions associated with increased permeability, the lactulose-mannitol excretion ratio is increased through relatively greater absorbence of lactulose compared to mannitol. After a 6-hour fast, a pretest urine specimen was collected from each patient, who then drank the test solution containing 5 g lactulose, 2 g mannitol, and 22.4 g glucose in 100 mL of water. Urine was then collected for the following 5 hours into 0.2 g chlorhexidine. Urinary mannitol was measured by spectrophotometry, using a color change reaction with potassium periodate; urinary lactulose was measured by paper chromatography.'4 The results are expressed as a ratio of the recovery of the two molecules. The normal range for the urinary lactulose-mannitol ratio is 0 to 0.07.
Intestinal Morphology and Immunohistochemistry All patients underwent endoscopy, at which time biopsies were taken from the second part of the duodenum, distal to the ampulla of Vater. One biopsy was fixed in 10% buffered formalin and embedded in paraffin, and 5/,m sections were cut and stained with hematoxylin and eosin. Intestinal villus height and crypt depth were measured using a microscope eyepiece graticule. At least 10 villi or crypts were measured for each section, and the mean value was determined. A second biopsy was snapfrozen in liquid nitrogen; 5-,um sections were cut in a cryostat and the sections were stained using a standard three-stage immunoperoxidase reaction as follows. After blocking nonspecific binding sites with 5% serum in phosphate buffered saline (PBS) and avidin and biotin blocking solutions (Vector AB Blocking kit; Vector, UK),
Jaundice Impairs Gut Barrier Function
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Table 1. PRIMARY ANTIBODIES USED Antibody
Cellular Distribution
Source
Description
Dilution
CD3 CD4 CD8 CD30 CD68 HLA-DR (MHC class 11)
T cells Helper T cells Cytotoxic T cells Late activated T cells Macrophages B cells, activated T cells, macrophages Secretory IgA
DAKO* PGt PG DAKO DAKO DAKO
Rabbit polyclonal Mouse monoclonal Mouse monoclonal Mouse monoclonal IgGl Mouse monoclonal IgGl Mouse monoclonal IgGl
1:100 1:40 1:50 1:100
DAKO
Rabbit polyclonal
1:500
IgA *
DAKO, High Wycombe, Buckinghamshire, United Kingdom. = personal gift, Dr. Ros Banks, Institute of Cancer Studies, University of Leeds, United Kingdom.
t PG
tissue sections were incubated at room temperature for 30 minutes with a primary antibody (Table 1) or serum (negative control). After washing in PBS, sections were incubated with a biotinylated second-layer antibody for 30 minutes at room temperature (swine antirabbit [DAKO, High Wycombe, Buckinghamshire, UK]; polyclonal primary antibodies) or rabbit antimouse (DAKO; monoclonal primary antibodies). After a further washing step, samples were incubated with StreptABComplex/ HRP (DAKO) and visualized with a diaminobenzidine chromogen (Sigma, Poole, Dorset, UK). Sections were counterstained with hematoxylin. The numbers of positive cells and the intensity of staining within cells were scored from + 1 to +4. Mucosal morphology and immunohistochemistry were scored by a consultant pathologist who was unaware of the source of the tissue.
signed ranks test. Analysis of categorical variables was performed using the chi square test with Yates' correction.
RESULTS Patient Demographics The characteristics of the control subjects and jaundiced patients are shown in Table 2. The groups were matched for age, sex, and the presence of neoplastic disease. Twenty of the 27 jaundiced patients had pancreatic cancer. By subjective global assessment, 5 subjects (19%) from the control group and 12 patients (37%) from the jaundiced group had evidence of malnutrition (chi square = 3.1, p = 0.08, Yates' correction).
Intestinal Permeability Serum Assays Evidence of chronic exposure to endotoxin was sought by measuring the IgG antibody response to endotoxin.'5 Serum concentrations of antiendotoxin core antibody (EndoCAb) of the IgG class were quantified using an enzymelinked immunosorbent assay (ELISA), as previously described."6 Serum interleukin-6 (IL-6) was measured using a commercially available ELISA (R&D Systems, Abingdon, Oxfordshire, UK) and serum C-reactive protein (CRP) was determined by routine autoanalysis (Beckman autoanalyzer, Beckman, Brear, CA).
Statistical Analysis Statistical analysis was performed using Statview soft(Apple Computers Inc., Abacus Concepts Inc., Berkeley, CA). Data were expressed either as individual data points or as median (with interquartile range in parenthesis). Nonparametric analysis was performed using the Mann-Whitney U test or the Wilcoxon matched pairs ware
Lactulose-mannitol ratios from control and jaundiced shown in Figure 1. The median lactulose-
persons are
Table 2. PATIENT DEMOGRAPHICS*
Number of patients Age (yr) Male:female Neoplastic disease (n) Gastroesophageal carcinoma Pancreatic carcinoma Cholangiocarcinoma Colorectal carcinoma Lymphoma Gall stones (n) Plasma bilirubin (rmolL-1)
Controls
Jaundiced
27 69 (62-78) 19:8 23 16 1 0 6 0 0 9 (7-11)
27 69 (60-81) 12:15 24 1 20 2 0 1 3 356 (261-386)t
Values are medians, with interquartile range in parentheses. t p < 0.01, Mann-Whitney U test.
*
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Ann. Surg. * February 1998
_._
'1.08
LactuloseMannitol Ratio
Table 3. IMMUNOHISTOCHEMISTRY RESULTS*
266 el.77
0.6
0.4
0:0. 0.2 00
u
*Control
*sx Jaundiced
Figure 1. Intestinal permeability in control and jaundiced patients. Individual data points are shown. Median values are represented by horizontal lines. p < 0.001.
mannitol ratio of 0.16 (0.03-0.5) in the jaundiced group was significantly greater than that in the controls (0.02 [0.01-0.05], p < 0.001). The control values were not
significantly different from nonpatient population controls (0-0.07).13 Sixteen of the 27 (59%) jaundiced patients had an increased lactulose-mannitol ratio versus only 4 of the 27 (26%) controls (chi square = 9.8, p < 0.01, Yates' correction).
Intestinal Morphology There was a significant reduction in villus height in the jaundiced patients (335 am [317-360]) versus 396 pm (368-410) in the controls (p < 0.02). However, there was no difference in crypt depth between the groups (64 Mm [60-83] in jaundiced patients vs. 75 ILm [60-90] in the controls, p not significant). There was also a significant increase in the numbers of lamina propria mononuclear cells in the jaundiced patients, with a median score of 3 (2-4) versus 2 (2-2) in the controls (p < 0.05).
Intestinal Immunohistochemistry The intensity of antibody staining of gut-associated lymphoid tissue and enterocytes is shown in Table 3. There was no difference in total mucosal T-cell numbers between groups. However, there was a significant increase in mucosal macrophages (CD68+ cells) infiltrating the lamina propria in the sections from the jaundiced patients.
Secretory IgA CD3, CD4, CD8 CD68 CD30 HLA-DR (lamina propria mononuclear cells) HLA-DR (epithelial cells) *
Controls
Jaundiced
2 2 2 1
2 2
4t 3t
1 1
3t 3t
Median scores.
tp
