Inflammatory consequences of the translocation of bacteria and endotoxin to mesenteric lymph nodes

SCIENTIFIC PAPER

Inflammatory Consequences of the Translocation of Bacteria and Endotoxin to Mesenteric Lymph Nodes Ulrich Schoeffel, MD, Klaus Pelz, MD, Rudolf U. Ha¨ring, MD, Freiburg, Germany; Rainer Amberg, MD, Basel, Switzerland Rene Schandl, MD, Freiburg, Germany; Renate Urbaschek, MD, Heidelberg, Germany; Bernd-Ulrich von Specht, MD, PhD, Eduard H. Farthmann, MD Freiburg, Germany

BACKGROUND: Translocation of intestinal bacteria to mesenteric lymph nodes (MLNs) has been documented in humans under a variety of circumstances, yet its clinical significance remains to be established. The aim of this study was to correlate detectable translocation to MLNs of bacteria and endotoxin with local and systemic signs of inflammation. METHODS: From each of 10 patients with carcinoma of the cecal region two MLNs were harvested prior to resection. The presence of bacteria and endotoxin in the lymphatic tissue and blood was determined by culture methods and DNA preparation (PCR) and by a Limulus assay, respectively. Inflammatory mediators were determined in plasma and in MLN homogenates. RESULTS: Viable bacteria were detected in MLNs of 7 patients and in 9 of 20 lymph nodes. PCR revealed traces of bacteria in 4 patients and in 6 of their MLNs. Combining both modalities, the translocation rate was 80% and 55% for patients and MLNs, respectively. There was no detectable bacteremia. Endotoxin was found in the plasma of 7 patients and in 9 MLNs from 5 patients. There was no correlation between culture findings and endotoxin concentrations. Moreover, bacteriological data did not correspond to local or systemic inflammation. The group of MLN with detectable endotoxin differed significantly from LPS-negative nodes with respect to interleukin-6, interleukin-10, and sCD14. Systemic concentrations of endotoxin and inflammatory parameters did not correspond to levels within MLNs. CONCLUSION: Translocation to MLNs occurs in patients with cecal carcinoma. This, however,

From the Departments of Surgery (US, RUH, RS, BUVS, EHF) and Medical Microbiology (KP), University of Freiburg, Freiburg, Germany; the Department of Forensic Medicine (RA), University of Basel, Basel, Switzerland; and the Department of Medical Microbiology (RU), Klinikum Mannheim, University of Heidelberg, Heidelberg, Germany. Requests for reprints should be addressed to Ulrich Scho¨ffel, MD, Department of Surgery, Universita¨tsklinik, Hugstetterstr. 55, 79106 Freiburg, Germany. Manuscript submitted October 29, 1999, and accepted in revised form May 30, 2000.

© 2000 by Excerpta Medica, Inc. All rights reserved.

seems not to be of major clinical significance if no additional physiologic insults are encountered. Irrespective of the presence of bacteria, there are variations in inflammatory reactions between lymph nodes from one and the same patient, probably reflecting fluctuating response mechanisms to low-grade translocation. Am J Surg. 2000;180:65–72. © 2000 by Excerpta Medica, Inc.

I

ntestinal translocation is defined as the escape from the intestinal tract of bacteria and their toxins to extraluminal sites.1,2 A large body of evidence, derived from both experimental and clinical studies, suggests that it indeed occurs, yet its clinical significance is still a matter of controversy.3– 6 It has been suggested that some form of bacterial passage across the intestinal barrier even occurs under physiological conditions.7 Certain microorganisms reach the subepithelial space either within macrophages;8 or by active transcellular or intercellular penetration; or by endocytosis, absorption or diffusion. During this passage or on the further path toward the mesenteric lymph nodes (MLNs), intracellular or free bacteria may be killed by the host’s defense system (grade 0 translocation). Viable pathogens in the MLNs are a common finding in a huge variety of pathological and experimental conditions (grade I). The spread of bacteria beyond this level leads to their detection within the systemic circulation and in distant organs, such as the spleen, liver, or lung (grade II). Although the lymphatics are probably the most important route of dissemination, some bacteria may also enter small venules, thereby reaching the portal system, while others have been shown to pass across the muscularis propria into the peritoneal cavity.9 –11 Whether systemic spread has to be regarded as the decisive step in the development of a septic response (grade III) still remains unproven.3,4 Likewise, the role of endotoxin escaped from the intestinal tract or released from translocating bacteria remains to become established.11–15 Differences in reported translocation rates to specific extraluminal sites reflect differences in culture methods, of the species involved, and of the respective model. The magnitude of translocation, ie, the quantity of escaped bacteria as well as the degree of their spread, basically depends on three different factors: the bacterial factor, which is related to a disturbed colonization resistance, bacterial overgrowth, and an increased mucosal adherence;16 –18 0002-9610/00/$–see front matter PII S0002-9610(00)00410-4

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the bowel wall factor, combining the physical components of the intestinal barrier; and the defense factor, which encompasses all antibacterial effects both locally and systemically.19 –23 Under most pathological conditions at least one of these factors is involved. In patients with adenocarcinoma of the large bowel, disruption of the intestinal barrier is assumed. Bacterial translocation rates to MLNs ranging from 17% to 70% have been reported.24 –28 Common experience, however, does not suggest any major clinical consequences attributable to the presence of variable bacteria in MLNs in such patients. This might be explained by low-grade translocation leading to adaptive responses within the MLNs without being reflected systemically. To substantiate this hypothesis, the aim of the present study was to correlate detectable bacterial translocation with endotoxin levels and concentrations of inflammatory parameters in both, MLNs and the systemic circulation.

METHODS Patients A selected series of 10 patients with carcinoma of the cecal region (cecum and proximal ascending colon) without clinical and radiological signs of stenosis was investigated. Histologically, all tumors had penetrated the mucosal layer but were confined to the muscularis. Preexisting inflammatory disease was excluded by history, clinical examination, routine laboratory data, intraoperative findings, and the postoperative course. Patients were included only if no abnormalities were found concerning nutritional status, liver function, metabolism, and cardiovascular performance. Informed consent was obtained at least one day prior to the operation. All procedures were conducted in accord with the ethical standards of the Helsinki Declaration of 1975. Sampling of Blood and MLNs Thirty milliliters of central venous blood was collected at the start of the laparotomy for bacteriological assessments and the determination of systemic endotoxin and inflammatory parameters. Of that, 10 mL was used for aerobic and anaerobic blood cultures (Organon Teknika, Eppelheim, Germany), 10 mL for a lysis-centrifugation blood culture system (Isolator 10; Wampole Laboratories, Unpath GmbH, Wesel, Germany), 3 mL for the detection of endotoxin (Endo Tube ET, Chromogenix, Stockholm, Sweden), and 5 mL EDTA-blood for the determination of inflammatory parameters. Endotoxin vials and EDTAblood were centrifuged (2,000 g/10 minutes) and stored at ⫺70°C. After entering the abdominal cavity the ileocecal region was inspected, and two intermediate lymph nodes were removed from the adjacent mesentery before resecting the right colon. Under sterile conditions, the MLNs were weighed and 125 mg tissue from each lymph node was homogenized in 12 mL of 0.9% NaCl. A second small piece was collected for the detection of bacterial DNA (50 to 100 mg). The residual parts of the lymph nodes were divided and shock-frozen in liquid nitrogen or fixed in 4% formalin, respectively. The homogenate was aliquoted as follows: 3 mL in endotoxin vials (Endo Tube ET; Chro66

mogenix, Stockholm, Sweden), 5 mL in a blood culture bottle under anaerobic conditions (Organon Teknika, Eppelheim, Germany), 1 mL for direct plating, and 3 mL centrifuged (2,000 g/10 minutes) and stored at ⫺70°C for the determination of inflammatory parameters. At the final dilution, 1 mL of homogenate corresponds approximately to 10 mg of MLN tissue. Only after sample collections were completed was routine perioperative antibiotic prophylaxis administered. The postoperative courses were recorded on a daily basis and were uneventful in all cases. Microbiological Investigations For the detection of viable bacteria media bottles were incubated under aerobic or anaerobic conditions at 36°C for 7 days. The Isolator 10 system for semiquantitative analysis was further processed by conventional plating techniques according to standard microbiological practice. Aliquots of MLN homogenates also were plated on Columbia sheep’s blood (Oxoid CM 331; Oxoid, Wesel, Germany), Hematin (Columbia-agar heated to 85°C), heartbrain broth (Oxoid CM 225), and yeast-cysteine-blood (HCB, for the detection of anaerobes) agar culture plates. Culture plates were incubated at 36°C and organisms were identified by standard bacteriological techniques (BioMerieux, Marcy-lE´toile, France). Colony counts were expressed as colony-forming units per gram of MLN (CFU/g). Based on the dilutions used, the detection limit of viable bacteria in the MLN was 20 CFU/g. If bacteria grew only in media bottles but not on culture plates, their quantity may be considered to be less than 104 CFU/g MLN. The polymerase chain reaction (PCR) technique was used for the detection of bacterial DNA within MLNs as described previously.29,30 DNA extraction was performed using a commercially available kit (QIAamp tissue kit; QIAGEN Inc., Hilden, Germany). According to the manufacturer’s protocol, proteinase K digestion of approximately 25 mg tissue was followed by several column purification steps. Oligonucleotide primers 91E (GGA ATT CAA A(T/G)G ATT TGA CGG GGG C) and 13B (CGG GAT CCC AGG CCC GGG AAC GTA TTC AC), purchased from TIB Molbiol, Berlin, Germany, were used in a Robocycler (Stratagene, Amsterdam, Netherlands) to amplify an Escherichia coli 16S rRNA sequence (91E: positions 911–930; 13B: positions 1390 –1371, antisense) which is a common region in a wide range of bacteria. PCR products were finally detected by agarose gel electrophoresis with ethidium bromide. The sensitivity of the PCR method in vitro (without additional DNA) was less than 10 bacteria. An automated kinetic turbidimetric Limulus amebocyte lysate (LAL) microtiter test with individual internal standardization was used to measure endotoxin, as previously described.31 This assay takes into account plasma-related factors that interfere with the LAL-endotoxin reaction as well as the fact that the slope of the reaction curve for each plasma sample varies if spiked with defined concentrations of LPS. In each individual sample, an endotoxin reference curve was established by spiking 50 ␮L aliquots of the sample (diluted 1:5 and heated to 80°C for 15 minutes) with different amounts of LPS in 25 ␮L to obtain final concentrations of 2500, 500, 50, 5, 0.5, 0.05, and 0 pg/mL. As LPS, NP3 (Novo Pyrexal; Weidner, Waldorf, Germany;

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100 pg NP3 ⫽ 1 EU EC-5, FDA) was used. After the addition of 25 ␮L of lysate (Associates of Cape Cod, purchased from Weidner, Waldorf) the increase in optical density in the test sample was measured at 37°C and 405 nm at intervals of 30 seconds for 100 minutes (Thermomax Photometer, Molecular Devices, Ismaning, Germany). The deviation from the linear slope in the lower concentration range represents the unknown endotoxin content of the sample, which is calculated as follows: log(tonset) ⫽ A ⫹ B ⫻ log(LPS ⫹ C), where tonset ⫽ time at which the reaction reaches the threshold at 40 mAbs, A ⫽ intersection with y-axis; B ⫽ slope; LPS ⫽ concentration of LPS spike, and C ⫽ unknown endotoxin concentration in the native sample. The sensitivity of this assay is 0.1 pg/mL. MLN homogenates were tested undiluted and in a 100fold dilution with and without a LPS spike of 50 pg/mL. Results were obtained from a water standard curve (concentrations as above). Sample-related inhibition or augmentation of the reaction could not be observed. Inflammatory Parameters Measurements of the soluble form of the endotoxin receptor CD 14 (sCD14) and of the cytokines interleukin-6 (Il-6), Il-8, Il-10, and tumor necrosis factor alpha (TNF␣) in plasma and MLN homogenates were performed by commercially available assay systems (interleukins: Quantikine immunoassay, R&D Systems Inc., Minneapolis, Minnesota; sCD14: IBL, Hamburg, Germany). Detection limits were as follows: Il-6 ⬍0.7 pg/mL, Il-8 ⬍10 pg/mL; Il-10 ⬍2 pg/mL, TNFa ⬍4.4 pg/mL, and sCD14 ⬍1 ng/mL. Immunohistology CD14, Il-8, and TNFa were determined on frozen sections, Defensin 3 (DEF 3), macrophage-inhibiting factorrelated proteins 8 and 14 (MRP 8, MRP 14), Il-2, and Il-6 after paraffin embedment and subsequent deparaffinization. For the immunohistological staining of 2 ␮m serial MLN sections, an avidin-biotin-complex (ABC) method was applied. Endogenous peroxidase activity was quenched by 1% H2O2-methanol for 30 minutes. Unspecific binding was prevented by incubation with horse serum (Vector Laboratories, Burlingame, CA) at room temperature for 20 minutes. Following overnight incubation with the specific antibodies DEF 3, mouse [m] versus human [h], Dianova, Hamburg, Germany; MRP 8 [8-5C2] m versus h, Dianova; MRP 14 [S36.48] m versus h, Dianova; CD 14, m versus h, Dako Diagnostik, Hamburg; Il-2 [Il-2-66], m versus h, Immunotech, Marseille, France; Il-6 [AH 65], m versus h, Immunotech; Il-8, rabbit [r] versus h, Endogen, Woburn, Massachusetts; TNFa, r versus h, Genzyme, Ru¨sselsheim, Germany) the biotinylated link antibody (BA-2000, horse versus mouse, Vector Laboratories, or horse versus r) was applied for 30 minutes at room temperature. After incubation for another 30 minutes with the ABC-complex (Vectastain ABC Elite Standard, Vector Laboratories), the reaction was visualized by amino-ethyl carbazol (AEC, Sigma A 5754) for 40 minutes, counterstained with hematoxylin, and mounted in Crystal Mount (Biomedia Corp., Foster City, CA). Between the different incubation steps, slides were rinsed with a mixture of 0.5M TRIS/0.5M PBS at a 10-fold dilution.

TABLE I Comparison of Bacteriological Findings in Mesenteric Lymph Nodes Culture MLN 1

Culture MLN 2

PCR MLN 1

PCR MLN 2

0 0 Gram-positive 0 Gram-pos/neg 0 Gram-positive 0 0 0

Gram-positive 0 Gram-positive Gram-positive Gram-pos/neg 0 0 0 Gram-negative Gram-positive

0 positive positive 0 positive 0 0 0 0 0

positive positive positive 0 0 0 0 0 0 0

MLN ⫽ mesenteric lymph node; PCR ⫽ polymerase chain reaction.

Double evaluation of the slides was performed at a 40-fold magnification by two blinded observers. A semiquantitative grading (0 –⫹⫹⫹) was applied according to staining intensity or the relative number of stained cells, respectively. Statistical Analysis Data were analyzed by the Mann-Whitney nonparametric test if data measured on a continuous scale were compared between two groups. Differences among mean values of variables in more than two groups were calculated by one-way analysis of variance (ANOVA) and posttests with Bonferroni corrections. Correlations were tested by linear regression analysis. Statistical significance was accepted when probability values (two-tailed, if not otherwise indicated) were less than 0.05.

RESULTS Viable bacteria were found in MLNs from 7 patients (70%) and in 9 of 20 lymph nodes (45%). Using the PCR methodology, bacterial DNA was detected in 6 lymph nodes from 4 patients (Table I). However, only in 1 patient with a positive PCR result, and in both of his lymph nodes, were the respective culture findings negative. Thus, combining both methods, bacterial translocation could be observed in 80% of patients and in 55% of the MLNs, respectively. Species differentiation showed the translocation of E coli in 3 lymph nodes (2 patients); of coagulase-negative Staphylococci in 3 MLNs (2 patients); of Staphylococcus aureus in 2 MLN from 1 patient; of Bacteroides spp. in 2 (1), and of Corynebacteria, Enterococcus faecalis, and Streptococcus mitis in 1 MLN each. Only in 3 MLNs from 2 patients were two different species detected: Bacteroides vulgatus and S mitis in 1 node and Bacteroides fragilis and E coli in the other from 1 patient and S aureus and Corynebacterium jeikeium in 1 MLN of the second patient. Coagulase-negative Staphylococci, Streptococci and Bacteroides could only be found by direct plating, while S aureus only grew after enrichment in culture bottles. Blood cultures were consistently negative. Systemic endotoxin concentrations and the bacteriological results from the respective MLNs did not correspond (Figure 1). However, the highest endotoxin concentration

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TABLE II Bacteriological Results* and Inflammatory Parameters in Mesenteric Lymph Nodes Positive (n ⴝ 11)

Negative (n ⴝ 9)

Interleukin-6 5.87 ⫹ 11.4 3.5 ⫹ 4.8 Interleukin-8 77.67 ⫹ 104 71.6 ⫹ 100 Interleukin-10 2.63 ⫹ 2.82 7.8 ⫹ 18.6 TNF alpha 6.95 ⫹ 12.9 4.9 ⫹ 4.13 sCD14 9.9 ⫹ 19.56 4.7 ⫹ 6.15

Figure 1. Comparison between endotoxin concentrations in plasma and bacteriological findings in patients. One patient with Gram-negative isolates showed also a polymerase chain reaction (PCR)-positive node. In mesenteric lymph nodes of 2 patients in the PCR-positive group, Gram-positive bacteria were found. In the Gram-positive group, PCR results were negative. LPS ⫽ lipopolysaccharide (endotoxin).

P Value

MDC

0.21 0.45 0.484 0.224 0.17

0.7 pg/mL 18 pg/mL ⬍2 pg/mL 4.4 pg/mL ⬍1 ng/mL

Values are mean ⫾ SD. * Including polymerase chain reaction results. MDC ⫽ minimal detectable concentration; TNF ⫽ tumor necrosis factor.

TABLE III Endotoxin Concentrations and Inflammatory Parameters in Mesenteric Lymph Nodes

Interleukin-6 Interleukin-8 Interleukin-10 TNF-alpha sCD14

LPS Positive* (n ⴝ 9)

LPS Negative (n ⴝ 11)

P Value (OneTailed)

8.5 ⫹ 11.2 89 ⫹ 99.5 1.06 ⫹ 1.5 4.0 ⫹ 1.3 13.4 ⫹ 18.23

1.34 ⫹ 1.8 62.2 ⫹ 102 9.14 ⫹ 18.3 7.3 ⫹ 12.0 1.78 ⫹ 4.49

0.015 0.135 0.031 0.366 0.008

Values are mean ⫾ SD. * ⱖ10 pg/g mesenteric lymph node. LPS ⫽ lipopolysaccharide; TNF ⫽ tumor necrosis factor.

Figure 2. Comparison between endotoxin concentrations in mesenteric lymph nodes (MLNs) and bacteriological findings. Nodes with Gram-negative isolates include one which was also polymerase chain reaction (PCR)-positive. From three nodes in the PCRpositive group Gram-positive bacteria were also cultured. In MLNs with Gram-positive bacteria, PCR results were negative. The horizontal line indicates the detection limit for endotoxin (10 pg/g MLN).

was found in the plasma from a patient with several organisms, including E coli in both of his MLNs. The comparison between endotoxin concentrations in MLN homogenates and the respective bacteriology also did not reveal any significant correlations. Again, the highest value was derived from a lymph node in which the growth of E coli was observed. In 3 out of 9 bacteriologically negative MLNs, endotoxin concentrations from 30 up to 80 pg/g tissue were detected (Figure 2). If one lymph node of an individual patient contained more than 20 pg/g, endotoxin was also found in the second MLN. There was no correlation between systemic and local LPS concentrations. Only in 4 patients was endotoxin detected simultaneously in plasma and MLNs. The tissue concentrations of inflammatory cytokines and of the soluble endotoxin receptor CD14 did not differ significantly between the bacteriologically positive and negative MLNs (Table II). However, the comparison of MLNs containing detectable amounts of endotoxin with LPS-free MLNs revealed significant differences for Il-6, Il-10, and sCD14 (Table III). The correlation of actual MLN endotoxin values with the tissue concentrations of 68

Il-6 (r ⫽ 0.3348, P ⫽ 0.075), Il-10 (r ⫽ 0.2435, P ⫽ 0.15), and sCD14 (r ⫽ 0.3635, P ⫽ 0.055) failed to reach statistical significance (one tailed P values). Tissue concentrations of Il-6 correlated with the respective values of Il-8 (r ⫽ 0.7242, P ⫽ 0.0003) and sCD14 (r ⫽ 0.8871, P ⫽ 0.0001), but not with concentrations of Il-10 (r ⫽ ⫺0.2435, P ⫽ 0.3) or TNFa (r ⫽ ⫺0.137, P ⫽ 0.56). The comparison of findings in both MLNs reveals differences in concentrations, but some similarities in activation pattern as well (Figure 3A, B). With regard to the dilutions used in this study, the tissue concentrations of cytokines exceed the systemic levels by a factor of 100 on a per-mL basis. On the other hand, the levels of sCD14 per mL tissue are lower by a factor of 10 than those in the systemic circulation. By semiquantitative analysis of immunohistochemistry, approximately corresponding results were detected for almost all pairs of MLNs. Differences in the number of stained cells were found for MRP-8, -14, CD14, and DEF3, and in the staining intensity for Il-6 and Il-8. A relatively weak staining for Il-2 and a moderate staining intensity for TNF␣ were more equally distributed among the MLNs. The overall assessment of the activation state compared well to the local concentration of cytokines. However, a close correlation between cytokines detected immunohistochemically and those measured in the homogenates could not be found. Again, immunohistological staining intensity did not depend upon the presence of bacteria or endotoxin in the MLNs. Concerning the detectable systemic responses, only a minority of values lay beyond the normal ranges (Il-6:

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Figure 3. Cytokine levels in mesenteric lymph nodes. A. Values obtained from the homogenates of lymph nodes no. 1. B. Values from lymph nodes no. 2. Values represent pg/mL homogenate.

median 10.9, range 3.85 to 87.64, normal range 12.5 pg/ mL; Il-8: 34.96, 18.24 to 124.63, ⫺94 pg/mL; Il-10: 9.04, 0 to 14.51, ⫺7.8 pg/mL; TNF␣: 5.6, 3.97 to 12.64, ⫺15.6; sCD14: 5.65, 3.73 to 6.26, ⫺5.5 pg/mL). Most pronounced were the levels of Il-6 (87.64 pg/mL) in a patient with Staphylococci found in both lymph nodes and of Il-8 (142.63 pg/mL) in a bacteriologically negative patient. In general, plasma concentrations of inflammatory parameters corresponded neither to the bacteriological status of the patients’ lymph nodes, nor to local or systemic endotoxin levels. In addition, there was no correlation between the local and the systemic inflammatory response.

COMMENTS Ample evidence suggests that translocation occurs in a variety of pathological conditions. However, a causal relationship between microbial translocation and a subsequent systemic inflammatory response still remains to become established.3,4,6 Viable bacteria in the mesenteric lymph nodes generally seem to reflect a loss of gut barrier function. On the other hand, even under physiological conditions, some bacteria cross the epithelial border and reach the lamina propria and the mesenteric lymphatics where they are killed by local defense mechanisms.7 Thus, translocation is certainly not an all-or-nothing phenomenon

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and it may be the quality and the quantity of translocation in combination with the host’s defense capacities that determine its consequences. In addition, it is most difficult to differentiate whether an inflammatory response within the bowel wall promotes translocation or whether translocating microorganisms may elicit inflammatory responses in this area.16 In patients with colonic cancer, increased gut permeability can be explained by disruption of the bowel architecture by malignant tissue. An additional influence of altered gut flora or of a suppressed defense cannot be excluded since increased detection rates of viable bacteria have also been reported at sites distant from the tumor.25 In the present study, by adding PCR results to the results obtained by conventional culture methods, we found bacterial translocation rates which were higher than those reported earlier.24 –28 It has been reckoned that only 1 out of 1,000 translocating bacteria can be detected in MLNs by culture methods.10 PCR seems to be a promising methodology with a high sensitivity, especially if the species to detect are known.32 By the broad-range amplification of part of the 16S rRNA gene used in our study, a vast variety of different species could be detected theoretically.29 However, only in 1 single patient with culture-negative findings were PCR results positive. This suggests that dead bacteria are not present in large quantities in MLNs when viable bacteria are not detectable. The relatively broad range of species found makes a broad-range translocation of several species, with numbers often below the detection limit, more probable than different individual conditions allowing for restricted passage. Dilution of the homogenates led to a relatively high detection limit for endotoxin in MLN tissue. The fact that there was no overall correlation between bacteriological data and endotoxin results can be explained either (1) by independently translocating endotoxin, (2) by intermittent bacterial translocation with variable endotoxin release, or (3) by continuous translocation with variable amounts of Gram-negatives, or by respective combinations. Even more difficult to explain is the missing correlation between LPS findings in MLNs and in the systemic circulation, from where endotoxins are rapidly cleared. Major variations in the amount of endotoxin in different lymph nodes or individual differences in detoxifying capacities may account for this finding. We did not find sampleinternal inhibitory or augmentatory activities. However, the levels of LPS indeed showed wide variations between the individual nodes. Measurements of inflammatory responses by combinations of several inflammatory parameters in plasma or serum are well established.33,34 Out of the vast array of measurable cytokines, we used representatives from the group of proinflammatory mediators (Il-8 and TNFa), the inflammation-modulating Il-6, and a rather antiinflammatory cytokine (Il-10). Immunohistochemical staining of inflammatory proteins and surface antigens may correctly reflect the activation state of a defined anatomical area but has to rely on subjective evaluation criteria.21,34 In addition to the difficult assessment of focal variations within the lymph nodes, an enhanced protein release during homogenization must be taken into account. 70

With regard to the dilutions applied in our study, the concentration of inflammatory parameters in the MLN was high. This may be related to a state of “physiological inflammation”16 or to an acute response. Moreover, it is difficult to determine whether some activation occurred in the lamina propria of the affected bowel with following spillover, rather than in the examined lymph nodes. However, increased signs of local activation were not associated with the detection of bacteria in MLNs. Since microbial factors such as endotoxin stimulate the production of proinflammatory cytokines in activated cells, which in turn is followed by the appearance of counterregulatory factors, this finding seems surprising. If, however, a low-grade translocation up to the level of the MLN is considered normal, at least in states with an impaired intestinal barrier, the inability to detect bacteria in all MLNs must reflect short-term local variations in the amount of translocation. Fluctuating levels of cytokines thus can be explained as well as the missing correlation between bacteriological data and local endotoxin concentrations. To a certain extent, the local concentrations of Il-6, Il-10, and sCD14 corresponded to respective endotoxin findings. Missing correlations with actual LPS values, and between the concentrations of Il-6, Il-10, and TNF␣, may partly be due to a statistical type Il error. On the other hand, a low level of chronic stimulation cannot be expected to increase the concentration of acute inflammatory cytokines, such as TNF␣. Additionally, it has been reported that certain enteric bacteria inhibit, instead of stimulate, the production of some lymphokines by gastrointestinal mononuclear cells.35 There were only moderate signs of systemic responses detectable in our patients. None of the values of TNF␣ and only one of Il-8 lay beyond the normal range in plasma. Half of the patients showed elevated levels for Il-6, Il-10, and sCD14. Soluble CD14 has been shown to be associated with Gram-negative sepsis; however, the functional consequences are not yet clear.36 The missing correlations of the systemic response with the bacteriological findings in MLNs, with local and systemic endotoxin concentrations, and with the local inflammatory response are one of the key findings of this study. Obviously, the inflammatory response is confined in some way to the mesenteric compartment. The unrelated escape of endotoxin and inflammatory parameters into the thoracic duct lymph and thus into the systemic circulation is limited or inhibited by as yet unknown mechanisms. In 4 patients with multiple organ dysfunction syndrome and thoracic duct cannulation, the concentrations of Il-6 and TNF␣ in lymph and serum were shown to be extremely low and did not correlate to LPS findings.14 Higher concentrations in lymph versus serum were found for Il-1b in 2, for LPS in 2, and for lymphocytic activation markers in 3 patients. The authors concluded that T-cell activation and cytokine production occur at the gut level. The role of the gut in the production of cytokines has been shown previously.37 In a porcine model of endotoxicosis, bowel ischemia was not associated with gut release of endotoxin, Il-6, or TNF into the portal vein.38 The hypothesis has been confirmed recently,39 that the lymphatics rather than mesenteric veins are the primary route for delivery of inflammatory products into the

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circulation. On the other hand, the concept that the thoracic duct is a major route of endotoxin translocation could not be confirmed by a recent study in 8 patients with multiple organ failure.40 This again supports our hypothesis that continuous or intermittent low-grade translocation leads to variably detectable amounts of bacteria and bacterial products in the MLNs. Local inflammatory responses may occur along the way of translocation and can be shown in the sinus of mesenteric lymph nodes. Whether counterregulatory or degrading mechanisms are able to prevent a washout or escape of inflammatory mediators must remain open. Alternatively, the effect of a low lymphatic flow and systemic detoxifying capacities have to be discussed.

CONCLUSION From our results we can conclude that bacterial translocation up to the level of MLNs (grade I translocation) occurs almost regularly, at least in patients with a disrupted mucosal barrier. Regarding locally detectable inflammatory reactions, the MLNs seem well adapted to a continuous low-grade encounter with bacteria and their toxins. Obviously, our patients did not suffer from major inflammation. If, however, an additional physiologic insult comes into play, an exaggerated or uncontrolled response and a spillover into the systemic circulation may result, eventually leading to clinical sepsis.

REFERENCES 1. Berg RD, Garlington AW. Translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in a gnotobiotic mouse model. Infect Immun. 1979;23:403– 411. 2. Alexander JW, Boyce ST, Babcock GF, et al. The process of microbial translocation. Ann Surg. 1990;212:496 –512. 3. Nieuwenhuijzen GAP, Deitch EA, Goris RJA. Infection, the gut and the development of the multiple organ dysfunction syndrome. Eur J Surg. 1996;162:259 –273. 4. Lemaire LCJM, van Lanschot JJB, Stoutenbeek CP, et al. Bacterial translocation in multiple organ failure: cause or epiphenomenon still unproven. Br J Surg. 1997;84:1340 –1350. 5. Ferri M, Gabriel S, Gavelli A, et al. Bacterial translocation during portal clamping for liver resection. Arch Surg. 1997;132: 162–165. 6. MacFie J, O’Boyle C, Mitchell CJ, et al. Gut origin of sepsis: a prospective study investigating associations between bacterial translocation, gastric microflora, and septic morbidity. Gut. 1999; 45:223–228. 7. Wells CL, Maddaus MA, Simmons RL. Proposed mechanisms for the translocation of intestinal bacteria. Rev Infect Dis. 1998;10: 958 –979. 8. Wells CL, Maddaus MA, Simmons RL. The role of the macrophage in the translocation of intestinal bacteria. Arch Surg. 1987; 122:48 –53. 9. Wang X, Andersson R, Soltesz V, et al. Gut origin sepsis, macrophage function, and oxygen extraction associated with acute pancreatitis in the rat. World J Surg. 1996;20:299 –308. 10. Mainous M, Tso P, Berg RD, Deitch EA. Studies of the route, magnitude, and time course of bacterial translocation in a model of systemic inflammation. Arch Surg. 1991;126:33–37. 11. Alexander JW, Gianotti L, Pyles T, et al. Distribution and survival of Escherichia coli translocating from the intestine after thermal injury. Ann Surg. 1991;213:558 –567. 12. Scho¨ffel U, Lausen M, Ruf G, et al. The overwhelming inflammatory response and the role of endotoxin in early sepsis. Prog Clin Biol Res. 1989;308:371–376.

13. Danner RL, Elin RJ, Hosseini JM, et al. Endotoxemia in human septic shock. Chest. 1991;99:169 –175. 14. Sa´nchez-Garcı´a M, Prieto A, Tejedor A, et al. Characteristics of thoracic duct lymph in multiple organ dysfunction syndrome. Arch Surg. 1997;132:13–18. 15. Scho¨ffel U, Baumgartner U, Imdahl A, et al. The influence of ischemic bowel wall damage on translocation, inflammatory response, and clinical course. Am J Surg. 1997;174:39 – 44. 16. Boedecker EC. Adherent bacteria: breaching the mucosal barrier? Gastroenterology. 1994;106:255–257. 17. Katayama M, Xu D, Specian RD, Deitch EA. Role of bacterial adherence and the mucus barrier on bacterial translocation. Ann Surg. 1997;225:317–326. 18. Nieuwenhuijs VB, Verheem A, van Duijvenbode-Beumer H, et al. The role of interdigestive small bowel motility in the regulation of gut microflora, bacterial overgrowth, and bacterial translocation in rats. Ann Surg. 1998;228:188 –193. 19. Maddaus MA, Wells CL, Platt JL, et al. Effect of T-cell modulation on the translocation of bacteria from the gut and mesenteric lymph node. Ann Surg. 1988;207:387–398. 20. Brandtzaeg P, Halstensen TS, Kett K, et al. Immunobiology and immunopathology of human gut mucosa: humoral immunity and intraepithelial lymphocytes. Gastroenterology. 1989;97:1562– 1584. 21. Nakasaki H, Mitomi T, Tajima T, et al. Gut bacterial translocation during total parental nutrition in experimental rats and its countermeasure. Am J Surg. 1998;175:38 – 43. 22. Welsh FKS, Ramsden CW, MacLennan K, et al. Increased intestinal permeability and altered mucosal immunity in cholestatic jaundice. Ann Surg. 1998;227:205–212. 23. Dickinson EC, Gorga JG, Garretti M, et al. Immunoglobulin A supplementation abrogates bacterial translocation and preserves the architecture of the intestinal epithelium. Surgery. 1998;124: 284 –291. 24. Vincent P, Colombel JF, Lescut D, et al. Bacterial translocation in patients with colorectal cancer. J Infect Dis. 1988;1395– 1396. 25. Lescut D, Colombel JF, Vincent P, et al. Bacterial translocation in colorectal cancers. Gastroenterol Clin Biol. 1990;14:811– 814. 26. Koha M, Brisbar B, Wikstrom B, et al. Bacterial colonization and translocation in colorectal carcinoma. Mel Microbiol Lett. 1992; 1:168 –176. 27. Sedman PC, Macfie J, Sagar P, et al. The prevalence of gut translocation in humans. Gastroenterology. 1994;107:643– 649. 28. Scho¨ffel U, Ha¨ring R, Jacobs E, et al. Translocation of bacteria and endotoxin from the intestinal tract. Shock. 1995;3:15. 29. Relman DA, Schmidt TM, MacDermott RP, Falkow S. Identification of the uncultured bacillus of Whipple’s Disease. NEJM. 1992;327:293–301. 30. Kane TD, Johnson SR, Alexander JW, et al. Detection of intestinal bacterial translocation using PCR. J Surg Res. 1996;63: 59 – 63. 31. Ditter B, Becker KP, Urbaschek R, Urbaschek B. Quantitativer Endotoxinnachweis. Automatisierter, kinetischer Limulus-Amoebozyten-Lysat-Mikrotiter-Test mit Messung probenabha¨ngier Interferenzen. Arzneimittel Forsch/Drug Res. 1983;33:681– 687. 32. Kane T, Alexander JW, Johannigman JA. The detection of microbial DNA in the blood: a sensitive method for diagnosing bacteremia and/or bacterial translocation in surgical patients. Ann Surg. 1998;227:1–9. 33. Wheeler AP, Bernard GR. Treating patients with severe sepsis. NEJM. 1999;340:207–214. 34. Sendt W, Amberg R, Scho¨ffel U, Hassan A. The local inflammatory peritoneal response to operative trauma: studies on cell activity, cytokine expression and adhesion molecules. Eur J Surg. 1999;165:1024 –1030. 35. Klapproth JM, Donnenberg MS, Abraham JM, James SP. Prod-

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71

TRANSLOCATION OF BACTERIA TO MESSENTERIC LYMPH NODES/SCHOEFFEL ET AL

ucts of enteropathogenic E. coli inhibit lymphokine production by gastrointestinal lymphocytes. Am J Physiol. 1996;271:G841– 848. 36. Landmann R, Zimmerli W, Sansano S, et al. Increased circulating soluble CD14 is associated with high mortality in gramnegative septic shock. J Infect Dis. 1995;171:639 – 644. 37. Deitch EA, Xu DZ, Franko L, et al. Evidence favoring the role of the gut as a cytokine-generating organ in rats subjected to hemorrhagic shock. Shock. 1994;1:141–146. 38. Bathe OF, Rudstom-Brown B, Chow WC, Phang PT. Gut is

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not a source of cytokines in a porcine model of endotoxicosis. Surgery. 1996;120:522–533. 39. Magnotti LJ, Upperman JS, Xu DZ, et al. Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock. Ann Surg. 1998;228:518 –527. 40. Lemaire LCJM, VanLanschot JB, Stoutenbeek CP, et al. Thoracic duct in patients with multiple organ failure: no major route of bacterial translocation. Ann Surg. 1999;229:128 –136.

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