Mesenteric Th1 polarization and monocyte TNF-α production: First steps to systemic inflammation in rats with cirrhosis

Authors

  • Leticia Muñoz,

    1. Laboratorio de Enfermedades del Sistema Inmune, Unidad I+D Asociada al Centro Nacional de Biotecnología (CSIC), Departamento de Medicina, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
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    • L.M. and A.A. contributed equally to this article.

  • Agustín Albillos,

    Corresponding author
    1. Laboratorio de Enfermedades del Sistema Inmune, Unidad I+D Asociada al Centro Nacional de Biotecnología (CSIC), Departamento de Medicina, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
    2. Servicio de Gastroenterología, Hospital Universitario Ramón y Cajal, Madrid, Spain
    • Departamento de Medicina, Facultad de Medicina-Campus Universitario, Universidad de Alcalá, Carretera Madrid-Barcelona km. 33.600, 28871 Alcalá de Henares, Madrid, Spain
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    • L.M. and A.A. contributed equally to this article.

    • fax: (34) 91 8854526

  • Mónica Nieto,

    1. Laboratorio de Enfermedades del Sistema Inmune, Unidad I+D Asociada al Centro Nacional de Biotecnología (CSIC), Departamento de Medicina, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
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  • Eduardo Reyes,

    1. Laboratorio de Enfermedades del Sistema Inmune, Unidad I+D Asociada al Centro Nacional de Biotecnología (CSIC), Departamento de Medicina, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
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  • Lourdes Lledó,

    1. Departamento de Microbiología, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
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  • Jorge Monserrat,

    1. Laboratorio de Enfermedades del Sistema Inmune, Unidad I+D Asociada al Centro Nacional de Biotecnología (CSIC), Departamento de Medicina, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
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  • Eva Sanz,

    1. Laboratorio de Enfermedades del Sistema Inmune, Unidad I+D Asociada al Centro Nacional de Biotecnología (CSIC), Departamento de Medicina, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
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  • Antonio de la Hera,

    1. Laboratorio de Enfermedades del Sistema Inmune, Unidad I+D Asociada al Centro Nacional de Biotecnología (CSIC), Departamento de Medicina, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
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  • Melchor Alvarez-Mon

    1. Laboratorio de Enfermedades del Sistema Inmune, Unidad I+D Asociada al Centro Nacional de Biotecnología (CSIC), Departamento de Medicina, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain
    2. Servicio de Enfermedades del Sistema Inmune y Oncología, Hospital Universitario Príncipe de Asturias, Alcalá de Henares, Madrid, Spain
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  • Potential conflict of interest: Nothing to report.

Abstract

A systemic inflammatory state with increased circulating tumor necrosis factor alpha (TNF-α) has been related to the bacterial infection susceptibility and hemodynamic derangement of patients with cirrhosis. We compared the activation status of immune cell subpopulations defined by 4-color cytometry in mesenteric and peripheral lymph nodes and blood of rats with CCl4-cirrhosis to define the immune response initiation site, the T-cell and monocyte contribution to pro-inflammatory cytokine production, as well as the pathogenic role of enteric bacteria in the cirrhosis immune response. Th1 cells and monocytes were expanded in the mesenteric nodes (P < .001) and blood (P < .001) of rats with cirrhosis, and activated to produce interferon gamma (P < .0001) and TNF-α (P < .0001), respectively. The greater numbers of recently activated CD134+ Th cells in mesenteric nodes compared with blood, the correlation between their numbers in mesenteric nodes and blood (r = 0.66, P < .001), and the expansion of activated CD45RC Th cells, which are unable to re-enter lymph nodes, in mesenteric nodes but not in blood or axillary nodes points to mesenteric nodes as the origin site of activated Th cells. Abrogation of bacterial translocation by bowel decontamination reduced the number of activated Th cells and monocytes, and normalized interferon gamma production by Th cells and TNF-α production by monocytes in mesenteric nodes and blood, respectively. In conclusion, in cirrhosis, enteric bacteria start off an orchestrated immune response cascade in mesenteric nodes involving Th1 polarization and monocyte activation to TNF-α production. Later, the recirculation of these activated effector immune cells into blood promotes systemic inflammation. (HEPATOLOGY 2005;42:411–419.)

Patients with cirrhosis and ascites suffer a systemic inflammatory state that features blood lymphocyte and monocyte activation and an increased production of pro-inflammatory cytokines.1–3 Immune abnormalities in cirrhosis with ascites include diminished absolute numbers of naive and memory T cells, an increased percentage of activated T cells, and markedly increased numbers of activated monocytes in peripheral blood.1–4 Notably, monocytes spontaneously produce tumor necrosis factor alpha (TNFα),3 a pro-inflammatory cytokine that has been implicated in the hemodynamic derangement of cirrhosis.2, 5–7 The sites of activation of these circulating immune cells have not been established. However, immune system activation in cirrhosis might be promoted by enteric bacterial products, as suggested by its relationship with bacterial translocation to mesenteric lymph nodes (MLNs),7–9 its amelioration by intestinal decontamination with antibiotics,2, 10 and its association with an augmented risk of spontaneous bacterial infection in patients with high plasma levels of lipopolysaccharide-binding protein.11

The consensus model for the adaptive immune response against bacteria that enter the body is that T and B lymphocytes interact with antigen-presenting cells (APCs) to initiate responses in the draining regional lymph nodes,12 which for bacterial antigens of enteric origin are the MLNs.7, 13 After the initial challenge, the activated immune cells start to leave the draining lymph nodes and enter the circulation.14 Unlike naive cells, most effector memory T cells lack the ability to enter the lymph nodes from the blood. Effector memory T cells instead acquire the capacity to penetrate inflamed tissues, where they clear the pathogens and cause lesions, a task naive cells cannot perform.13–15

To investigate the initiation site of the immune response in cirrhosis, we used a CCl4 rat cirrhosis model. This model mimics some of the most relevant features of human cirrhosis, such as portal hypertension, a hyperdynamic circulatory state, ascites formation, and bacterial translocation to the MLN.7, 16, 17 Our rationale was that by using this experimental model we would be able to establish whether the MLN is the initiation site for the immune disturbance observed in cirrhosis, by comparing distributions of immune cell populations, their activation status, and the polarization patterns of the cytokines produced in MLNs with those in peripheral lymph nodes (PLNs), axillary lymph nodes, and blood. Finally, we suppressed the fecal load of aerobic bacteria by using non-absorbable antibiotics, to evaluate the impact of bacterial antigens of gut origin on the immune cell abnormalities observed in the lymph nodes and blood of rats with cirrhosis.

Abbreviations

TNFα, tumor necrosis factor alpha; MLN, mesenteric lymph node; APC, antigen-presenting cell; PLN, peripheral lymph node; FITC, fluorescein isothiocyanate; PE, phycoerythrin; IFNγ, interferon-gamma; IL, interleukin; Tc, T cytotoxic; MHC, major histocompatibility complex.

Materials and Methods

Animal Model.

Cirrhosis was induced in male pathogen-free Sprague-Dawley rats (120-160 g initial weight) by intragastric weekly CCl4 administration (Carbon Tetrachloride Carlo Erba, Farmitalia, Milan, Italy), along with phenobarbital (Química Farmacéutica Bayer, Barcelona, Spain) in the drinking water (35 mg/dL), as previously described.16, 17 The initial 20-μL dose of CCl4 was increased, depending on the animal's weekly change in body weight. CCl4 administration was continued for 2 weeks after ascites onset and then discontinued. Experiments were performed 7 days after the last CCl4 dose. On average, the animals were examined 18 weeks (range, 12-24 weeks) after the initial CCl4 dose. All tests were performed according to the Guide for the Care and Use of Laboratory Animals (NIH publication 86-23, revised 1985) and fulfilled local regulations.

Experimental Protocols.

The study was conducted as 2 experiments. The first was designed to establish whether systemic inflammation in cirrhosis initiates in the MLN by comparing the phenotype and activation status of lymphocyte and monocyte subpopulations as well as their activation state in the different immune compartments (MLN, peripheral blood, PLN). In this protocol, 28 rats with cirrhosis with ascites were compared with 20 healthy, phenobarbital-treated age- and sex-matched rats. In the second experiment, we investigated the impact of bowel bacterial decontamination on the immune cell effects of cirrhosis. To this end, 22 rats with cirrhosis and ascites were divided into 2 groups receiving either broad-spectrum, orally non-absorbable antibiotics (n = 12) or placebo (n = 10). Neomycin sulfate (180 mg/d; Sigma Chemical Co., St. Louis, MO), polymyxin B sulfate (150 mg/d; Sigma), and vancomycin (16 mg/d; Sigma) were dissolved in the drinking water, and administered for 3 weeks after ascites onset. A control group of 16 healthy phenobarbital-treated age- and sex-matched rats received a 3-week course of the same regimen of antibiotics (n = 8) or placebo (n = 8) as the animals with cirrhosis.

Study Design.

Before use, animals were fasted for 8 hours, but water was administered ad libitum. All experiments were performed under strictly sterile conditions. Anesthesia was induced with isoflurane (Forane; Abbott Laboratories, Madrid, Spain). Five to 15 mL peripheral blood was obtained by cardiac puncture. Subsequent to blood collection, MLN draining lymph from the terminal ileum, cecum, and ascending colon13 and the right axillary lymph nodes were dissected, removed, and weighed. Finally, a sample of cecal contents was taken.

Methods.

Peripheral blood mononuclear cells were separated by Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation. The cells were resuspended (1 × 106 cells/mL) in Dulbecco′s modified Eagle medium (DMEM; BioWhittaker, Verviers, Belgium) supplemented with glucose (4.5 g/L), L-glutamine, 10% heat-inactivated fetal calf serum (Gibco, Grand Island, NY) and 25 mmol/L HEPES buffer (BioWhittaker). Single-cell suspensions of the lymph nodes were obtained by pressing the nodes through a 150-μm pore mesh (Sefar Maissa SA, Madrid, Spain) and washing the cells by centrifugation in Hank's balanced salt solution.

Intracellular/Surface Immunofluorescence and Quantitative Flow Cytometry.

Proportions of monocyte, B-cell, and T-cell subpopulations were determined in cell suspensions from peripheral blood and lymph nodes by 4-color immunofluorescence and quantitative flow cytometry in a FACScalibur cytometer using Cell Quest software (Becton, Dickinson, San Jose, CA). Surface and intracellular antigen expression were quantified using a modification of the Dako QifiKit (Dako A/S, Glostrup, Denmark), as described elsewhere.3 Analyses were carried out using FlowJo software (Tree Star, San Carlos, CA). Cell suspensions were incubated with combinations of fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, peridinin chlorophyll protein–, and allophycocyanin (APC)-labeled monoclonal antibodies (Table 1). The rat monoclonal antibodies (BD Pharmingen, San Diego, CA) used were Biotin-CD3 (G 4.18), PE-CD4 (OX-38), peridinin chlorophyll protein–CD8a (OX-8), FITC-CD134 (OX-40), FITC-CD45RC (OX-22), FITC-CD11b (Mac-1, α-chain), PE-NKR-P1A (10/78), Cy-Chrome-CD45RA (OX-33), and Biotin-RT1B (OX-6). For the biotinylated monoclonal antibodies, APC-labeled streptavidin (Caltag Laboratories, San Francisco, CA) was used. After surface staining, cells were fixed and permeabilized, and the cytokine was stained with PE-labeled anti-TNFα or –interferon-γ (IFNγ) (BD Pharmingen), as previously described.18 Cytokine production was assessed in suspensions of mononuclear cells incubated 2.5 hours at 37°C in 5% CO2 in Dulbecco's modified Eagle medium, complete medium. To increase the sensitivity of cytokine detection, monensin (2 μmol/L; Sigma) was added to the culture medium. Because T cells arrest cytokine production quickly after the dissociation of their in vivo interaction with APCs,19 intracytoplasmic cytokine production was determined after culturing T cells 2.5 hours at 37°C in 5% CO2 in the presence of phorbol 12-myristate 13-acetate (0.05 μg/mL; Sigma) and ionomycin (1 μg/mL; Calbiochem-Novabiochem Corp., San Diego, CA).18 The number of immune cells in lymph nodes and blood was counted in a Neubauer chamber. Absolute cell counts of mononuclear cell subpopulations (cells/node × 10−3 and cells/μL blood) were calculated by multiplying the absolute number by the proportion of each subpopulation established by flow cytometry.

Table 1. Definition Criteria for the Immune Cell Populations Examined
Immune Cell SubsetsFunction Associated With the Indicated Surface Receptors
T Cells 
CD3Signal transduction module in the T-cell receptor for antigen (TCR/CD3 complex). Expressed in all T-cell subsets.
CD4TCR/CD3 co-receptor that recognizes major histocompatibility complex (MHC) class II molecules, which present antigens to Th cells. Th cell marker
CD8TCR/CD3 co-receptor that recognizes class I MHC molecules, which present antigens to Tc cells. Identifies Tc cell marker
CD45RCIsoform of the leukocyte common antigen that is lost in immunologically experienced cells. Positively identifies the subpopulation of naive Th cells that have not yet recognized the antigen
CD134Also termed OX-40 receptor. A co-stimulatory molecule that identifies a subpopulation of recently activated Th cells
NKR-P1AlowIts low expression is an activation marker on CD8+ Tc cells, whereas when highly expressed it is a recognition receptor of non-classic class I MHC molecules in NK cells
B Cells 
CD45RAIsoform of the leukocyte common antigen that identifies B cells
RT1BClass II MHC: responsible for antigen presentation to CD4+ Th cells. Expressed on antigen-presenting cell surfaces. Constitutively expressed in rat B cells, and further induced on B-cell activation.
Monocytes 
CD11bbrightIntegrin expressed on the surface of monocyte lineages
RT1BClass II MHC: responsible for antigen presentation to CD4+ Th cells. Expression is induced on activation in rat monocytes, negative on resting monocytes.
CD4NKR-P1AlowLow levels of NKR-P1A, as well as the loss of CD4, are monocyte activation markers.

Plasma Levels of Inflammatory Markers.

Blood samples were centrifuged and serum samples stored at −80°C until analysis. ELISA kits (Biosource International, Camarillo, CA) were used to determine TNFα, IFNγ, interleukin (IL)-12p70, and IL-18, according to the manufacturers' instructions. Sensitivity detection limits were 4, 13, 2.5, and 4 pg/mL, respectively. All determinations were performed in duplicate and the mean value used.

Bacteriological Study.

Samples of MLN and cecal contents were plated on McConkey and blood agar (Materlab, Madrid, Spain) and inoculated in thioglycollate (Scharlab, Barcelona, Spain). Thioglycollate culture bottles were incubated at 37°C for 48 hours and the presence of bacteria monitored by subculturing onto agar plates when turbidity was evident. Specific microorganisms were identified by a manual biochemical test or automated system (Microscan, Baxter, Irvine, CA) when necessary. Bacterial translocation from the intestinal lumen was defined as the presence of viable organisms in the MLN.7, 16, 17 The total intestinal aerobic count was defined as the sum of all the aerobic bacteria present in the cecal content sample, and expressed as colony-forming units per gram of stool.

Statistical Analysis.

Results are shown as means ± SD. Qualitative variables were analyzed using Fisher's exact test, and quantitative variables by the unpaired Student's t test. Correlations between selected variables were assessed by linear regression analysis. The level of statistical significance was set at P value of less than .05.

Results

Immune Cell Activation in the MLNs of Rats With Cirrhosis.

As shown in Table 2, there was a marked expansion of T cells (1.5-fold), B cells (3-fold), and monocytes (6-fold) in the MLNs of the rats with cirrhosis and ascites compared with age- and sex-matched controls. This simultaneous expansion of the 3 main lymph node immune cell populations was concurrent with a notable MLN enlargement in the rats with cirrhosis (53 ± 21 vs. 36 ± 12 mg, P < .01). Activated T-helper (Th) cells (CD3+CD4+CD8) accounted for most of the T-cell expansion in the MLNs of rats with cirrhosis. The Th-cell subset expressing the co-stimulatory receptor CD134, a marker of recent activation, showed an expansion that was greater than 7-fold. The latter was concurrent with the expansion of the total number of activated (CD45RC) Th cells15, 20, 21 (Table 2). Consistent with previous reports, CD134+ Th cells represent a small population in control rats20 (Table 2). Activation of MLN T cells also occurred in the T-cytotoxic (Tc) cells and monocytes of rats with cirrhosis. We found significantly increased numbers of Tc cells (CD3+CD4CD8+) that co-expressed NKR-P1Alow, an activation marker in non-NK cells22 (Table 2). Moreover, the CD11bbright monocyte population was markedly expanded and showed evident signs of activation. These signs included an increased number of class II major histocompatibility complex (MHC) receptors expressed on the surface of MLN monocytes (622 ± 484 vs 223 ± 91 molecules/cell, P < .0001), along with the increased number of CD4NKR-P1Alow monocytes (Table 2). As also shown in Table 2, changes in the distributions of immune cell subsets differed substantially between the peripheral blood and MLNs of rats with cirrhosis. There was a significant reduction in the numbers of B cells and Th and Tc cells in the blood, contrasting with their significant expansion in MLNs. Although numbers of recently activated CD134+ Th cells are significantly higher in the blood of rats with cirrhosis than in controls, total numbers of activated CD45RC Th cells and activated NKR-P1Alow Tc cells are not augmented in parallel, which contrasts with the marked expansion of the latter in the MLN. Hence, MLNs and blood share the expansion and activation of monocytes. Circulating monocytes showed an increased number of surface class II MHC receptors (194 ± 88 vs. 101 ± 28 RT1B molecules/cell, P < .01), as well as expansion of the activated CD4NKR-P1Alow subset (Table 2). Remarkably, there was direct correlation between the numbers of CD134+ Th cells (r = 0.66, P < .001) and of CD4NKR-P1Alow monocytes (r = 0.61, P < .01) found in the MLNs and blood of individual rats with cirrhosis (Fig. 1). To examine whether Th activation preferentially occurred in the MLNs of rats with cirrhosis, we quantified the number of total (CD45RC) or recently activated (CD134+) subsets present in the PLN as an extra-mesenteric location. This experimental design takes advantage of existing evidence that Th cells lose the CD45RC isoform of the leukocyte common antigen on activation in secondary lymphoid organs, such as the lymph nodes. A portion of the CD45RC Th population expands locally in the lymph nodes, whereas another fraction migrates through the lymph to the blood circulation. Notably, circulating CD45RC Th cells lose the capacity of naive (CD45RC+) Th cells to re-enter lymph nodes.15, 23 The expansion of total and recently activated Th cells in rats with cirrhosis is restricted to MLNs, because this did not occur in PLNs (Table 2, Fig. 2). Neither was evidence of monocyte expansion found in the PLN, further supporting the preferential nature of MLN activation. Moreover, the distribution and activation status of other immune cells of the PLN failed to differ significantly between rats with cirrhosis and control rats (Table 2). PLNs were also of a similar size in cirrhosis and control animals (58 ± 25 vs. 51 ± 23 mg).

Table 2. Immunophenotypic Profile of T and B Lymphocytes and Monocytes in the Mesenteric Lymph Nodes, Peripheral Blood, and Peripheral (Axillary) Lymph Nodes of Rats With Cirrhosis With Ascites and Control Rats
 Mesenteric Lymph Node (cells/node × 10−3)Peripheral Blood (cells/μL)Peripheral Lymph Node (cells/node × 10−3)
Rats with Cirrhosis (n = 28)Control Rats (n = 20)Rats with Cirrhosis (n = 28)Control Rats (n = 20)Rats with Cirrhosis (n = 28)Control Rats (n = 20)
  • *

    P < .001 vs. control rats

  • P < .01 vs. control rats

  • P < .05 vs. control rats.

Immune cell subsets      
T cells (CD3+CD11bbright−)7,754 ± 4,2175,173 ± 2,140447 ± 235685 ± 2512,965 ± 1,4703,139 ± 1,703
Total T helper (Th, CD4+CD8)6,048 ± 3,289*3,879 ± 1,604314 ± 146481 ± 2441,872 ± 9852,025 ± 1,699
Activated Th (CD45RC)3,392 ± 773*1,712 ± 344189 ± 65205 ± 71628 ± 361907 ± 476
Recently activated Th (CD134+)861 ± 414*122 ± 5522 ± 124.8 ± 1.140 ± 2638 ± 19
Total T cytotoxic (Tc, CD4CD8+)1,706 ± 8211,277 ± 495136 ± 58205 ± 1281,067 ± 6051,108 ± 684
Activated Tc (NKR-P1Alow)138 ± 59*39 ± 1433 ± 1841 ± 1970 ± 3662 ± 41
B cells (CD3CD45RA+RT1B+)9,231 ± 4,282*3,425 ± 1,34781 ± 42*208 ± 1241,240 ± 1,1581,683 ± 1,010
Monocytes (CD3CD11bbright+)466 ± 186*82 ± 25452 ± 263*69 ± 3823 ± 1825 ± 12
Activated monocytes (CD4NKR-P1Alow)83.9 ± 37*19.1 ± 923 ± 6*7.6 ± 2.17.5 ± 3.48.9 ± 4.7
Figure 1.

Direct correlation between the absolute numbers of recently activated (CD134+) Th (CD3+ CD11bbright− CD4+ CD8) cells (A) and activated monocytes (CD3CD11bbright+ CD4NKR-P1Alow) (B) present in mesenteric lymph nodes and in blood.

Figure 2.

Total activated (CD45RC) and recently activated (CD134+) CD4+ Th cells from a rat with cirrhosis and a control rat representative of each group. The dot plots depict the percentages of gated CD3+CD4+ Th cells from mesenteric lymph nodes, peripheral lymph nodes, and peripheral blood that express CD45RC and CD134. The negative limits were set using isotype-matched controls.

MLNs and Circulating Monocytes Are Activated to TNFα Production, and T Cells Polarized to IFNγ Production in Rats With Cirrhosis.

Given the monocyte and Th activation observed, we went on to examine their relative contributions to TNFα production. As shown in Table 3, monocytes are the main source of TNFα in both the MLNs and blood of rats with cirrhosis. The number of monocytes spontaneously producing TNFα in the MLNs of rats with cirrhosis was of 260 ± 104 cells/node × 10−3, compared with a mere 0.49 ± 0.12 cells/node × 10−3 in control rats (P < .0001). This marked difference resulted from an expansion in the total number of monocytes and an increase in the frequencies of monocytes producing TNFα in the MLN of rats with cirrhosis (56.2% ± 18% vs. 2.6% ± 0.9%, P < .001). TNFα production by monocytes in lymph nodes of rats with cirrhosis was selective of MLNs, because it was not observed in PLNs. In the peripheral blood of rats with cirrhosis, the expansion of the total number of monocytes and the increased proportion of monocytes spontaneously activated to TNFα production (75.1% ± 24% vs. 10.8% ± 3.8%, P < .001) resulted in a higher number of monocytes producing TNFα (Table 3). Unlike monocytes, T cells are not a relevant source of spontaneous TNFα production at any of the anatomical sites tested in this study. T cells polarized into Th1 produce IFNγ, which promotes pro-inflammatory immune responses that enhance TNFα production by monocytes.24, 25 We therefore addressed the question of whether the activated Th cells in MLNs and peripheral blood had the capacity to produce IFNγ in the rat with cirrhosis. Interestingly, the number of Th cells that were activated to produce IFNγ in the MLNs was much greater in rats with cirrhosis than in control rats (306 ± 159 vs. 17.9 ± 8.2 cells/node × 10−3, P < .0001), a difference that was the result of the expansion of Th cells and the increased proportion of Th cells producing IFNγ (5.01% ± 1.62% vs. 0.45% ± 0.1%, P < .001). In contrast, IFNγ production by T cells was similar in the PLNs of rats with cirrhosis and control rats (Table 3). The population of Th cells activated to IFNγ production was also expanded in the peripheral blood of rats with cirrhosis (Table 3), a difference that was entirely attributable to the increased frequencies of Th cells producing IFNγ (12.1% ± 3.6% vs. 1.4% ± 0.3%; P < .001). Notably, we observed a direct correlation between the number of Th cells activated to IFNγ production and the number of monocytes spontaneously activated to TNFα production both in the MLNs (r = 0.68, P < .001) and blood (r = 0.76, P < .001) of individual rats with cirrhosis (Fig. 3). Th1 polarization is promoted by the monokines IL-12p70 and IL-18, which are the major inducers of IFNγ production by T cells.26, 27 We therefore next considered whether IL-12p70 and IL-18 were augmented in the serum of rats with cirrhosis. As shown in Table 4, serum levels of both IL-12p70 and IL-18 were significantly increased in rats with cirrhosis compared with control rats. Interestingly, serum concentrations of IL-12p70 and IFNγ showed direct correlation (r = 0.76, P < .001) (Fig. 4). Table 4 also indicates that TNFα and IFNγ were also significantly increased in the circulation of rats with cirrhosis (Table 4), which is in accordance with the increased number of monocytes and Th cells producing those cytokines.

Table 3. Intracytoplasmic TNFα Expression by Monocytes and IFNγ Expression by Th Cells in the Mesenteric Lymph Nodes, Peripheral Blood, and Peripheral (Axillary) Lymph Nodes of Rats With Cirrhosis and Control Rats, and Rats With Cirrhosis Treated With Placebo or Antibiotics
 Rats With Cirrhosis (n = 28)Control Rats (n = 20)PRats With Cirrhosis Treated With Placebo (n = 10)Rats With Cirrhosis Treated With Antibiotics (n = 12)P
Spontaneous TNFα production by monocytes (CD3CD11bbright+)
 Mesenteric lymph node (cells/node × 10−3)260 ± 1040.49 ± 0.12<.0001226 ± 987.46 ± 3.6<.0001
 Peripheral blood cells (cells/μL)339 ± 1217.45 ± 3.1<.0001291 ± 11426.2 ± 17<.0001  
 Peripheral lymph node (cells/node × 10−3)0.46 ± 0.160.48 ± 0.14NS0.51 ± 0.190.54 ± 0.16NS
Recall IFN-γ production by Th-cells (CD3+CD11bbright−CD4+CD8)
 Mesenteric lymph node (cells/node × 10−3)306 ± 15917.9 ± 8.2<.0001321 ± 16737.1 ± 18<.0001
 Peripheral blood (cells/μL)38.1 ± 196.8 ± 2.9<.000131.5 ± 1414.1 ± 8.3<.0001
 Peripheral lymph node (cells/node × 10−3)6.71 ± 3.27.8 ± 5.1NS5.89 ± 3.16.1 ± 2.9NS
Figure 3.

Direct correlation between the absolute numbers of monocytes spontaneously expressing TNFα and Th cells showing recall expression of IFNγ in the mesenteric lymph nodes (A) and peripheral blood (B) of rats with cirrhosis.

Table 4. Serum Cytokines in Rats With Cirrhosis Treated With Placebo or Antibiotics
CytokineRats With Cirrhosis Treated With Placebo (n = 10)Rats With Cirrhosis Treated With Antibiotics (n = 12)P
  1. Serum cytokines in control rats (n = 20); TNFα, 4.2 ± 1.8 pg/mL; IFNγ, below detection limit (see Materials and Methods); IL-12p70, below detection limit; IL-18, below detection limit.

TNFα (pg/mL)45 ± 169.2 ± 4.1<.001
IFNγ (pg/mL)99 ± 34Below detection limit<.001
IL-12p70 (pg/mL)4.22 ± 1.61.85 ± 0.4<.001
IL-18 (pg/mL)5.68 ± 2.3Below detection limit<.001
Figure 4.

Direct correlation between serum IFNγ and IL-12p70 concentrations in rats with cirrhosis.

Bowel Decontamination With Antibiotics Normalizes Monocyte and Th Activation to TNFα and IFNγ Production, Respectively, in the MLNs of Rats With Cirrhosis.

We administered non-absorbable, broad-spectrum antibiotics to rats with cirrhosis to suppress enteric aerobic microflora, and we examined the effects of this action on the distributions and cytokine production profiles of the immune cells of the lymph nodes and blood of rats with cirrhosis. Bowel decontamination almost abolished the fecal load of aerobic bacteria (7.9 ± 0.9 vs. 1.2 ± 0.6 logCFU/g, P < .001). Bacterial translocation to the MLN was observed in 1 (8%) of the 12 rats receiving antibiotics (Pseudomonas aeruginosa), compared with 6 of 10 (60%, P < .05) of the placebo-treated rats (P < .05). Gut decontamination significantly reduced the signs of T-cell and monocyte activation in the MLNs of rats with cirrhosis, and also decreased the size of the mesenteric adenomegaly from 57 ± 22 to 38 ± 10 mg (P < .05). Antibiotic-treated rats with cirrhosis showed significantly diminished total numbers of Th cells (6,174 ± 2,476 vs. 4,475 ± 1,122 cells/node × 10−3, P < .05), activated (CD45RC) Th cells (3,371 ± 706 vs. 2,123 ± 735 cells/node × 10−3, P < .001), and recently activated (CD134+) Th cells (963 ± 485 vs. 546 ± 319 cells/node × 10−3, P < .001), as well as NKR-P1Alow Tc cells (146 ± 63 vs. 109 ± 56 cells/node × 10−3, P < .05). Antibiotics also reduced both the total number of monocytes (368 ± 174 vs. 136 ± 96 cells/node × 10−3, P < .01), and the number of activated monocytes (CD4NKR-P1Alow, 89 ± 44 vs. 25 ± 7 cells/node × 10−3, P < .01; intensity of class II MHC receptors on their surface, 637 ± 452 vs. 286 ± 79 molecules/cell, P < .01) in the MLN of rats with cirrhosis. Interestingly, the marked reduction in T-cell and monocyte activation in MLN after bowel decontamination was accompanied by a drastic reduction in the frequencies of monocytes spontaneously producing TNFα and of Th cells polarized to IFNγ production (TNFα, from 31.2% ± 12% to 5.49% ± 2.8%, P < .001; IFNγ, from 3.49% ± 1.9% to 0.83% ± 0.3%, P < .01) (Fig. 5) and their absolute numbers (Table 3). This effect of gut decontamination on proinflammatory cytokine production was extended to circulating immune cells with marked reductions in TNFα producing monocytes and Th cells producing IFNγ (Fig. 5, Table 3). Serum cytokine levels were also significantly reduced in antibiotic-treated rats (Table 4).

Figure 5.

Monocytes (CD11bbright+) spontaneously expressing TNFα and CD4+ Th cells showing recall expression of interferonγ in the mesenteric lymph nodes (A) and peripheral blood (B) from a placebo- and an antibiotic-treated rat with cirrhosis, and from a control rat representative of each group. The dot plots depict the percentages of gated monocytes (CD11bbright+) and CD3+CD4+ Th cells and interferon gamma, respectively. The negative limits were set using isotype-matched controls.

In control rats, antibiotics lacked any significant effects on the distribution, activation status, and cytokine production of immune cells in the MLN and peripheral blood (data not shown).

Discussion

In this experimental study, we demonstrated an innate and adaptive immune response occurring in the lymph nodes that drain the mesenteric region, as revealed by an intense expansion of activated immune cells, monocytes, and T and B cells. The systemic pro-inflammatory status in cirrhosis is initiated in the MLN, as indicated by the lack of immune activation in the extramesenteric lymph nodes and the distinct phenotypical profiles of MLNs and circulating T cells. Immune system activation in this model of cirrhosis features a pro-inflammatory profile involving Th1 polarization with increased IFNγ production, and monocyte activation to TNFα secretion. Our results indicate the pathogenic key role of enteric bacteria on the pro-inflammatory immune response of cirrhosis, because selective bacterial intestinal decontamination blunted the expansion of activated monocytes and Th cells in the MLN and normalized their polarization to IFNγ and TNFα production, respectively, in both the MLN and blood.

Whereas gut bacterial translocation in cirrhosis with ascites is a widely accepted phenomenon,7, 17 the cellular immune response elicited by these displaced enteric microorganisms has received little attention. Use of an established model of cirrhosis enabled us to demonstrate that the immune response promoted by translocated enteric bacteria commences in the MLN and thereafter expands to the systemic level. The finding of marked monocyte activation in the MLN along with intense T- and B-cell expansion indicates that immune cell activation in the MLNs of rats with cirrhosis involves not only phagocytic APCs but also an adaptive immune response implicating lymphocytes. Indeed, antigen sampling by APCs/monocytes may result in the presentation of bacterial products to naive cells, leading to the sequestration and activation of T cells in MLN. That activated Th cells are preferentially triggered in the MLNs of rats with cirrhosis was revealed by: (1) the lack of expansion of this population in the PLN, (2) the expansion in MLNs, but not in blood nor PLNs, of activated Th cells lacking the CD45RC receptor, (3) the greater number of recently activated CD134+ Th cells in the MLNs than in blood, and (4) a direct correlation between the numbers of recently activated CD134+ Th cells in MLNs and blood. The latter results are consistent with existing evidence that activated immune cells leave the lymph nodes, starting shortly after the onset of an immune response, and recirculating activated Th cells that lack CD45RC are unable to re-enter the lymph nodes.12, 15, 23 These activated immune cells that leave the MLN may thus account for the systemic activation observed in blood, in a manner that is non–mutually exclusive to the passage of enteric bacterial products into the circulation.

The expansion of monocytes and their activation to TNFα production in MLNs and blood is a hallmark of cirrhosis with ascites in the rat model. T cells do not contribute significantly to TNFα production, neither in MLNs nor in blood. Interestingly, Th-cell activation is accompanied by polarization toward a Th1 pro-inflammatory cytokine pattern, as indicated by the production of the prototype of Th1 cytokine IFNγ.24 Remarkably, our findings indicated direct correlation between the percentages of Th cells and monocytes activated to IFNγ and TNFα production, respectively, both in the MLN and blood. The latter result is consistent with evidence that IFNγ secretion by Th1 cells promotes TNFα production by activated macrophages.25 This regulatory interplay between monocytes and Th cells is bidirectional, because monocytes decisively affect the Th1/Th2 polarization balance.24–26 IL-12p70 is a key factor in the polarization of T cells to a Th-1 profile. A major effect of IL-18 is to increase the IFNγ-dominant T-cell response induced by IL-12.26, 27 Our analysis of circulating cytokines showed increased serum levels of the monokines IL-18 and IL-12p70, and direct correlation between serum concentrations of IL-12p70 and IFNγ. Taken together, these results suggest that, in cirrhosis, monocyte activation leads to increased IL-12p70 and IL-18 levels, triggering IFNγ production by T cells and, in turn, stimulating TNFα production by monocytes to complete the pro-inflammatory cascade.

These results in an experimental model of cirrhosis are coincident with prior studies in patients with cirrhosis with ascites that have indicated the systemic activation of T cells and monocytes in the blood, with reduced absolute numbers of both naïve and memory Th cells.3, 11 We show that the immune cell activation pattern in peripheral blood differs from that in the MLN. Total T- and B-cell numbers are significantly reduced in blood from rats with cirrhosis, despite signs of immune activation, whereas both are markedly expanded in MLNs. In contrast, monocytes are expanded and activated to TNFα production both in MLNs and blood. Recent research indicates that most effector memory T cells, unlike naive T cells, do not re-enter the lymph nodes but acquire the capacity to reach peripheral tissues.14, 28 On activation in the MLN, possibly immune cells return to the circulation and migrate to somatic tissues tracking signs of systemic inflammation, which might contribute to their relative depletion in blood. Similar to MLNs, blood-derived phagocytic/dendritic cells transport bacteria to the spleen, triggering lymphocyte responses.29 However, T cells activated in the spleen or other extra-MLN draining territories cannot account for the CD134+CD45RC pattern observed in MLNs. Further work is needed to address the role of spleen in the systemic inflammatory syndrome of cirrhosis.

Abrogation by antibiotics of both the enteric aerobic bacterial load and bacterial translocation to MLNs was found to attenuate the expansion of activated T cells and monocytes in the MLN and normalize TNFα and IFNγ production in the MLN and blood. These findings suggest a strong link between the ongoing pro-inflammatory immune response observed in the MLNs and blood of rats with cirrhosis and gut bacterial antigens. In effect, these results resemble the restored TNFα levels observed in patients with cirrhosis after selective bowel decontamination,2, 3 further reinforcing the similarity between cirrhosis in patients and this experimental model.

The immune cell abnormalities of the CCl4 rat-with-cirrhosis model were characterized by 4-color immunofluorescence. Our results indicate that in rats with cirrhosis and ascites, enteric bacteria set off an orchestrated immune response cascade in which MLN T cells are polarized to a Th1 profile, and monocytes activated to TNFα production. The recirculation of effector immune cells activated in the MLN later promotes systemic inflammation. Thus, enteric bacterial antigens seem to be at the root of the immune abnormalities observed at the systemic level in cirrhosis with ascites involving the innate and adaptive system. These results provide the basis for developing strategies able to modulate the systemic proinflammatory response in cirrhosis.

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