Cytokine-induced epithelial permeability changes are regulated by the activation of the p38 mitogen-activated protein kinase pathway in cultured Caco-2 cells.

Increased intestinal/epithelial permeability in sepsis and endotoxemia has been noted to be induced by proinflammatory cytokines such as interferon-gamma, TNF-alpha, and IL-1beta. The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in regulating the inflammatory response induced by these cytokines. We tested the hypothesis that epithelial permeability changes are regulated through the p38 MAPK signaling pathway. Caco-2 cells were cultured for 21 days and then stimulated with a cytokine mixture (CytoMix: TNF-alpha, interferon-gamma, and IL-1beta). Epithelial barrier function was evaluated by measuring permeability in an Ussing chamber. CytoMix-induced changes of MAPKs (p38, c-Jun amino-terminal kinase, and extracellular-regulated kinase), NO production, and inflammatory responses (IL-6 and IL-8 levels) were also assessed. The signaling pathways were further studied by pretreating cells with SB203580, a specific p38 MAPK inhibitor. CytoMix increased permeability at 24 and 48 h but not at 4 h. This was associated with increased IL-6 and IL-8 production, as well as increases in phosphorylation of all three MAPKs. Treatment with SB203580 completely blocked p38 activity with transient inhibition of p38 phosphorylation. SB203580 also prevented the CytoMix-induced permeability increase and reduced NO, IL-6, and IL-8 levels. The results suggest that p38 MAPK plays an important role in regulating epithelial barrier function during inflammation.

Thermal injury elevates the inflammatory monocyte subpopulation in multiple compartments.

Recent publications have demonstrated that human resident and inflammatory monocyte (IM) subpopulations have equivalents in rodents. The effect of thermal injury upon these subpopulations has not been studied. Mice were given a scald burn and killed on postburn days (PBDs) 2, 4, and 8. Bone marrow, blood, and spleen white cells were isolated, and the percentage of resident monocytes (CD11b LY6C), IMs (CD11b LY6C), and monocyte progenitors (macrophage-colony-forming unit [M-CFU]) were determined. The ability of each monocyte population to make TNF-alpha was determined by intracellular cytokine staining. Finally, the ability of sorted fractions from PBD 8 spleen to inhibit lymphocyte proliferation was performed. We noted that there was an increase in M-CFU in the blood and spleen at PBD 8, but the marrow only had a nonsignificant increase in M-CFU. All compartments showed a significant increase in the number of IMs by PBD 8, but no significant changes in resident monocytes were seen. In all compartments, IMs were a major source of TNF-alpha. The postburn increase in IMs and monocyte progenitors in the spleen was accompanied by an increase in the monocyte chemokine monocyte chemoattractant protein 1 and constitutively high levels of the progenitor chemokine stromal-derived factor 1alpha. After burn injury, mice deficient in the receptor for soluble TNF-alpha had equal levels of splenic M-CFU and monocytes, as did wild-type mice, suggesting that this cytokine is not essential for this effect. We conclude that in this model, IMs are a significant source of in vivo TNF-alpha.

Postburn monocytes are the major producers of TNF-alpha in the heterogeneous splenic macrophage population.

Increased tumor necrosis factor (TNF)-alpha production by postburn splenic macrophages is well documented. Splenic macrophages are a heterogeneous population, and the effect of thermal injury on these subpopulations has not been documented. We examined the effects of scald injury on myeloid cells with the phenotype of red pulp, white pulp, and marginal zone monocyte/macrophages. We found that thermal injury greatly increased the number of splenocytes with the phenotype of white pulp monocytes. These cells were the major producers of TNF-alpha in the postburn spleen. Cells with the red pulp macrophage phenotype had an increased ability to make TNF-alpha after burn injury, but had only half the capacity to make TNF-alpha as did postburn monocytes. The postburn changes in TNF-alpha production correlated with an increased in vivo susceptibility to endotoxin. The increase in monocytes in the spleen from postburn days 1 to 10 correlated with an increasing ability of splenocytes to produce granulocyte colony-stimulating factor, monocyte chemoattractant protein 1, macrophage inflammatory protein 2, and macrophage inflammatory protein 1-alpha. These data suggest that the monocyte is a major source of inflammatory cytokines in the postburn spleen.

Effect of thermal injury on splenic myelopoiesis.

Thermal injury increases the number of macrophage progenitors in the bone marrow but leads to a decrease in the number of granulocyte progenitors. In the spleen, thermal injury increases the numbers of myeloid progenitors, but the lineage commitment of these cells is unknown. In this study mice were given a scald burn, and the number of splenic myeloid progenitors as well as their progeny was determined. BrdU uptake was used to monitor the de novo production of splenocytes for 8 days after the burn. Burn injury increased the numbers of splenic granulocyte-macrophage (GM), granulocyte (G), and macrophage (M) progenitors at postburn day 8 by 12-, 11-, and 18-fold, respectively. Scald injury increased the number of mature PMN (CD11b GR1(bright)) in the spleen and increased the number of white pulp monocyte/macrophages. Increased numbers of BrdU-positive PMN and monocyte/macrophages were seen after injury. Burn macrophages produced increased levels of the anti-inflammatory hematopoietic cytokine G-CSF. Our work clearly shows that the increased myelopoiesis observed postinjury leads to the production of mature myeloid cells. However, the effects of thermal injury on progenitors in the spleen and marrow are not equivalent.

Influence of glutamine-supplemented Caco-2 cells on cytokine production of mononuclear cells.

BACKGROUND: Caco-2 cells, cultured with mononuclear cells, were used as an in vitro model of human intestinal cell function. This study shows the effect of glutamine (Gln) supplementation on the production of tumor necrosis factor alpha, interleukin-10 (IL-10), and interleukin-6 (IL-6). METHODS: Confluent Caco-2 cells were cultured in media with Gln at 0 mmol/L, 4 mmol/L, or 10 mmol/L +/- 1 microg/mL lipopolysaccharide (LPS), treated with fluorescein isothiocyanate- (FITC-) conjugated intercellular adhesion molecule-1 (ICAM-1) mononuclear antibody, and assessed for ICAM-1 expression levels via flow cytometry. Confluent Caco-2 cells alone in apical inserts, or mononuclear cells (MNCs) alone in basal chambers of transwells, were cultured in media with 0 mmol/L, 4 mmol/L, or 10 mmol/L Gln. Supernatants were taken to assess cytokine and endotoxin levels. Confluent Caco-2 cells in apical inserts of transwells were cultured in media containing Gln at 0 mmol/L, 4 mmol/L, or 10 mmol/L, whereas MNCs were cultured in the basal chamber in media containing Gln at 4 mmol/L +/- LPS. Supernatants were collected to determine cytokine levels in each chamber. RESULTS: With Gln supplementation of the media at 10 mmol/L, enterocytes displayed a decrease in ICAM-1 expression. MNCs showed a decrease in tumor necrosis factor alpha and IL-6 production and an increase in IL-10 production when incubated with Caco-2 cells in media supplemented with Gln at 10 mmol/L. CONCLUSIONS: Although cytokine production by Caco-2 or mononuclear cells incubated alone was not influenced by the Gln concentration of the media, cultured together, Gln levels affected cytokine production by mononuclear cells, which suggests that Caco-2 cells produce mediators in Gln-rich conditions that can influence mononuclear cell cytokine production.