Regulation of adolescent rat intestinal epithelial inducible nitric oxide synthase expression in endotoxin tolerance: modulation of signal transduction

Endotoxin/lipopolysaccharide (LPS) tolerance is a state of induced hyporesponsiveness to endotoxin or LPS characterized by alterations in the release of inflammatory mediators. As the gut is both a source of infection and target of injury, we tested the hypothesis that alterations in intestinal epithelial signal transduction would account for the acquisition of endotoxin tolerance as defined by decreased induction of a key mediator of gut injury, inducible nitric oxide synthase (iNOS). Rats (15 days of age) were injected with saline or LPS (1 microg/g i.p.). Tissue was harvested after 1, 4, or 6 h for assessment of signaling and iNOS expression. Other animals received a second dose of LPS 1 to 7 days after the initial dose. Selected animals received the p38 inhibitor, SB203580 (10 microg/g), which was co-administered with the first dose of LPS. Induction of iNOS mRNA and protein was significantly attenuated after repeated LPS administration. Epithelial cells from LPS-tolerant rats showed a minimal level of iNOS expression by immunohistochemistry. The down-regulation of intestinal iNOS was not gender dependent. p38 inhibition enhanced tolerance rather than blocking it. LPS-mediated activation of NF-kappaB was attenuated in a manner consistent with a primary role in the induction of tolerance. Endotoxin tolerance can be demonstrated in intestinal epithelial cells using an in vivo model. Modulation of NF-kappaB signaling may be key in the down-regulation of LPS effect seen in tolerance.     

Conjugated primary bile salts reduce permeability of endotoxin through intestinal epithelial cells and synergize with phosphatidylcholine in suppression of inflammatory cytokine production

OBJECTIVE: Endotoxemia was shown to be integral in the pathophysiology of obstructive jaundice. In the current study, the role of conjugated primary bile salts (CPBS) and phosphatidylcholine on the permeability of endotoxin through a layer of intestinal epithelial cells and the consequent activation of basolaterally cocultured human mononuclear leukocytes were measured. DESIGN: In a coculture model, a layer of differentiated, confluent Caco-2 cells was apically stimulated with growth-arrested, nonpathogenic Escherichia coli. SETTING: Basic human cell culture laboratory. INTERVENTIONS: The effect of CPBS (0.5 mM and 1.5 mM), phosphatidylcholine (0.38 mM), and human bile (0.5% vol/vol) on the barrier function was assessed by the measurement of transepithelial electrical resistance, by endotoxin permeability through the intestinal epithelial cell layer, and by basolateral cytokine enzyme-linked immunosorbent assay measurement (tumor necrosis factor-[alpha], interleukins-6, -8, and -10). Micelles formed by CPBS were detected by dynamic light scattering. The association of endotoxin with CPBS micelles was tested by fluorescence resonance energy transfer. MEASUREMENTS AND MAIN RESULTS: Apical addition of CPBS suppressed the permeability of endotoxins through the intestinal epithelial cell layer significantly. In parallel, apical supplementation of CPBS dose-dependently reduced the basolateral production of all cytokines measured. Apical phosphatidylcholine supplementation enhanced this effect significantly. CPBS formed micelles (diameter, 134 +/- 7 nm), which were able to bind endotoxin to their surface. CONCLUSIONS: CPBS can reduce the permeation of endotoxin through intestinal epithelial cell layers by binding it to micelles. Thereby, the inflammatory processes beyond the mucosal surface are suppressed, an effect that is enhanced by phosphatidylcholine.     

Constitutive IKK2 activation in intestinal epithelial cells induces intestinal tumors in mice

Many cancers display increased NF-κB activity, and NF-κB inhibition is known to diminish tumor development in multiple mouse models, supporting an important role of NF-κB in carcinogenesis. NF-κB activation in premalignant or cancer cells is believed to promote tumor development mainly by protecting these cells from apoptosis. However, it remains unclear to what extent NF-κB activation exhibits additional protumorigenic functions in premalignant cells that could be sufficient to induce spontaneous tumor development. Here we show that expression of constitutively active IκB kinase 2 (IKK2ca) in mouse intestinal epithelial cells (IECs) induced spontaneous tumors in aged mice and also strongly enhanced chemical- and Apc mutation-mediated carcinogenesis. IECs expressing IKK2ca displayed altered Wnt signaling and increased proliferation and elevated expression of genes encoding intestinal stem cell-associated factors including Ascl2, Olfm4, DLK1, and Bmi-1, indicating that increased IKK2/NF-κB activation synergized with Wnt signaling to drive intestinal tumorigenesis. Moreover, IECs expressing IKK2ca produced cytokines and chemokines that induced the recruitment of myeloid cells and activated stromal fibroblasts to become myofibroblasts, thus creating a tumor-promoting microenvironment. Taken together, our results show that constitutively increased activation of IKK2/NF-κB signaling in the intestinal epithelium is sufficient to induce the full spectrum of cell-intrinsic and stromal alterations required for intestinal tumorigenesis.     

Functional interleukin-2 receptors on intestinal epithelial cells.

The presence of receptors for the cytokine IL-2 was assessed in the IEC-6 cell line established from normal rat crypt epithelium and primary intestinal epithelial cells. 125I-IL-2 was found to specifically bind to subconfluent IEC-6 cells. Maximal binding was observed within 30 min after addition of the ligand; binding could be inhibited by excess unlabeled IL-2 or addition of antibody to the IL-2 receptor. Both intermediate and low affinity receptors with approximate Kd of 10 and 100 pM, respectively were present. Kinetic analysis were consistent with the results of Western blot analysis using an antisera to the 75-kD IL-2 receptor beta chain. IL-2 receptors appeared to be functional; addition of IL-2 led to modulation of proliferation with initial stimulation at 24 h followed by inhibition at 48 h. This effect could be blocked by addition of antibody to the IL-2 receptor beta chain. IL-2 treatment could be shown to enhance expression (range = 4- to 50-fold stimulation) of TGF-beta, as well as the lectin protein mac-2, in IEC-6 cells. The relevance of observations in the IEC-6 cell line to intestinal mucosa in vivo was supported by the demonstration of a gradient of expression of the IL-2 receptor in primary rat intestinal epithelial cells by Western blot analysis. In addition, mRNA for the IL-2 receptor-beta chain was demonstrated by Northern blot analysis using mRNA from primary rat intestinal epithelial cells depleted of detectable contaminating intraepithelial lymphocytes by two cycles of fractionation on Percoll gradients. Collectively, these observations suggest that the range of cellular targets of the putative lymphokine IL-2 is broader than appreciated, and IL-2 may serve to integrate epithelial and lymphocyte responses in the intestinal mucosa.     

Apoptosis of human intestinal epithelial cells after bacterial invasion.

Epithelial cells that line the human intestinal mucosa are the initial site of host invasion by bacterial pathogens. The studies herein define apoptosis as a new category of intestinal epithelial cell response to bacterial infection. Human colon epithelial cells are shown to undergo apoptosis following infection with invasive enteric pathogens, such as Salmonella or enteroinvasive Escherichia coli. In contrast to the rapid onset of apoptosis seen after bacterial infection of mouse monocyte-macrophage cell lines, the commitment of human intestinal epithelial cell lines to undergo apoptosis is delayed for at least 6 h after bacterial infection, requires bacterial entry and replication, and the ensuing phenotypic expression of apoptosis is delayed for 12-18 h after bacterial entry. TNF-alpha and nitric oxide, which are produced as components of the intestinal epithelial cell proinflammatory program in the early period after bacterial invasion, play an important role in the later induction and regulation of the epithelial cell apoptotic program. Apoptosis in response to bacterial infection may function to delete infected and damaged epithelial cells and restore epithelial cell growth regulation and epithelial integrity that are altered during the course of enteric infection. The delay in onset of epithelial cell apoptosis after bacterial infection may be important both to the host and the invading pathogen since it provides sufficient time for epithelial cells to generate signals important for the activation of mucosal inflammation and concurrently allows invading bacteria time to adapt to the intracellular environment before invading deeper mucosal layers.     

Studies on the ATPase enzyme system in human intestinal epithelial cells

Human intestinal epithelial cells were fractionated to yield a soluble and particulate cell fraction, and a brush border fraction. (Na⁺-K⁺)-stimulated, ouabain sensitive ATPase (EC 3.6.1.4) was located in the brush border and soluble cell fraction. Mg²⁺-ATPase (EC 3.6.1.3) occurred throughout the epithelial cell. This enzyme was inhibited by Ca²⁺ in the brush border fraction, but was activated by Ca²⁺ and Mg²⁺ in the particulate cell fraction. The ATPase systems were separated and partially purified using Sephadex G-100. The molecular weights and enzymatic properties were examined. The results indicated that human intestinal epithelial cells contain two distinct types of (Na⁺-K⁺)-ATPase.     

Thioredoxin is associated with endotoxin tolerance in mice

OBJECTIVE: Oxidative stress and subsequent lipid peroxidation appear to be central to the lethal effect of lipopolysaccharide. We hypothesized that induction of an antioxidant protein, thioredoxin, would play an important role in the development of endotoxin tolerance and reduce mortality rates in lipopolysaccharide-treated mice. DESIGN: Prospective, randomized, controlled study. SETTING: University research laboratory. SUBJECTS: Adult, male, ddy mice. INTERVENTIONS: In survival-curve experiments, mice were pre-treated intravenously with a low dose of Escherichia coli lipopolysaccharide (20 microg/mouse, pretreatment group) or saline (control group). A large dose of lipopolysaccharide (200 microg/mouse) subsequently was injected into the tail vein 16 hrs after pretreatment. In experiments to measure the expression of thioredoxin after lipopolysaccharide challenge, mice were injected intravenously with different concentrations of lipopolysaccharide (between 20 and 200 microg/mouse, designated the lipopolysaccharide group) or with saline (control group). MEASUREMENTS AND MAIN RESULTS: The survival rate during the 72-hr observation period was significantly higher in the pretreatment group (82%) than in the control group (30%; p = .025 by Mantel-Cox log rank analysis). After lipopolysaccharide challenge, thioredoxin concentrations in the lung, heart, and liver of mice in the pretreated group were about 1.5- to 2-fold higher than those in the control group. Enhancement in these organs became apparent about 6 hrs after lipopolysaccharide challenge and lasted until at least 24 hrs. The levels of accumulation of 4-hydroxy-2-nonenal modified proteins after a large dose of lipopolysaccharide challenge were lower in the pretreatment group than in the control group. CONCLUSIONS: These results demonstrate that mice with an increased concentration of thioredoxin, induced by pretreatment with a low dose of lipopolysaccharide, had increased survival when given a subsequent high-dose challenge of lipopolysaccharide. Thus, thioredoxin is associated with the development of endotoxin tolerance in mice.     

Intracellular recognition of lipopolysaccharide by toll-like receptor 4 in intestinal epithelial cells

Toll-like receptor (TLR)4 has recently been shown to reside in the Golgi apparatus of intestinal crypt epithelial m-ICcl2 cells, colocalizing with internalized lipopolysaccharide (LPS). Here we demonstrate that disruption of the integrity of the Golgi apparatus significantly reduced LPS-mediated nuclear factor kappaB activation. Also, the TLR4 adaptor protein MyD88 and the serine/threonine kinase IRAK-1 were rapidly recruited to the Golgi apparatus upon stimulation. LPS-mediated activation required lipid raft formation and intact clathrin-dependent internalization. In contrast to macrophages, prevention of ligand internalization by use of LPS-coated beads significantly impaired recognition by epithelial cells. The localization of TLR4 to the Golgi apparatus was abrogated by expression of a genetically modified form of the TLR4 binding chaperone gp96. Thus, our data provide evidence that in contrast to the situation in macrophages, LPS recognition in intestinal epithelial cells may occur in the Golgi apparatus and require LPS internalization.     

Isolation of high density lipoproteins from rat intestinal epithelial cells.

Previous studies have defined forms of high density lipoproteins (HDL) in rat mesenteric lymph, suggesting that they have a secretory origin. This study describes the isolation and characterization of intestinal intracellular HDL. Two preparations were made as follows: (a) Rat enterocytes were isolated and a Golgi organelle fraction was prepared. (b) Cell homogenates were subjected to nitrogen cavitation and a cytoplasmic fraction was prepared. Lipoproteins were isolated from both preparations by sequential ultracentrifugation. When the HDL fraction (1.07-1.21 g/ml) was subjected to isopyknic density gradient ultracentrifugation, a peak of apoproteins A-I and B (apoA-I and apoB, respectively) was found at a density of 1.11-1.14 g/ml. Electron microscopy of the fraction showed spherical particles ranging in size from 6 to 13 nm. Immunoelectrophoresis revealed a precipitin arc in the alpha region against apoA-I which extended into the pre-beta region where a precipitin arc against apoB was also seen. ApoB antisera depleted the pre-beta particles whereas the alpha migrating particles remained. Lipid analysis of the whole HDL fraction revealed phospholipid, cholesteryl ester, and triglyceride as the major lipids. [3H]leucine was then administered into the duodenum and a radiolabeled intracellular HDL fraction was isolated. The newly synthesized apoproteins of the HDL fraction, as determined by gel electrophoresis, were apoB, apoA-I, and apolipoprotein A-IV (ApoA-IV). Immunoprecipitation of the apoB particles revealed apoA-I and apoA-IV in the supernatant. These data demonstrate that there are at least two intracellular intestinal forms of HDL particles, one of which contains apoB. The other particle contains apoA-I and apoA-IV, has alpha mobility, is spherical, and resembles a particle found in the lymph.     

Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage

Disruption of the intestinal epithelial barrier allows bacterial translocation and predisposes to destructive inflammation. To ensure proper barrier composition, crypt-residing stem cells continuously proliferate and replenish all intestinal epithelial cells within days. As a consequence of this high mitotic activity, mucosal surfaces are frequently targeted by anticancer therapies, leading to dose-limiting side effects. The cellular mechanisms that control tissue protection and mucosal healing in response to intestinal damage remain poorly understood. Type 3 innate lymphoid cells (ILC3s) are regulators of homeostasis and tissue responses to infection at mucosal surfaces. We now demonstrate that ILC3s are required for epithelial activation and proliferation in response to small intestinal tissue damage induced by the chemotherapeutic agent methotrexate. Multiple subsets of ILC3s are activated after intestinal tissue damage, and in the absence of ILC3s, epithelial activation is lost, correlating with increased pathology and severe damage to the intestinal crypts. Using ILC3-deficient Lgr5 reporter mice, we show that maintenance of intestinal stem cells after damage is severely impaired in the absence of ILC3s or the ILC3 signature cytokine IL-22. These data unveil a novel function of ILC3s in limiting tissue damage by preserving tissue-specific stem cells.The intestinal epithelium combines efficient uptake of nutrients and water while providing a physical barrier between the intestinal microbiota and the body (Peterson and Artis, 2014). Damage sustained by intestinal epithelial cells (IECs) needs to be swiftly and efficiently repaired to prevent inappropriate immune responses to commensal bacteria. Intestinal damage is an early event in the development of both graft-versus-host disease (Reddy and Ferrara, 2003) and alimentary mucositis (Sonis, 2004) and a driver of bacterial translocation and T cell activation in inflammatory bowel disease (Salim and Söderholm, 2011).A major pathway involved in the intestinal epithelial response to damage is the activation of Stat3, which is expressed along the crypt–villus axis of the intestinal epithelium (Grivennikov et al., 2009; Heneghan et al., 2013). Phosphorylated Stat3 translocates to the nucleus and activates genes involved in proliferation, survival, and mucosal defense (Bollrath et al., 2009; Pickert et al., 2009; Ernst et al., 2014). Mutations in STAT3 have been identified as susceptibility factors for inflammatory bowel disease (Bollrath et al., 2009; Anderson et al., 2011; Demaria et al., 2012), and in mice, upon DSS-induced colitis, epithelial Stat3 is required for mucosal wound healing (Pickert et al., 2009).Intestinal regeneration depends on the continuous differentiation of epithelial cells from crypt-residing intestinal stem cells (ISCs; Potten et al., 1978; Günther et al., 2013; Ritsma et al., 2014). Even though multiple intestinal progenitor cells have been described, the best-characterized populations are the Lgr5-expressing cells that reside at the crypt bottom, interspersed with Paneth cells. These stem cells have the ability to give rise to all IECs ex vivo (Sato et al., 2009). Similar to its role in differentiated epithelial cells, Stat3 activation is also an important pathway for survival of intestinal epithelial stem cells (Matthews et al., 2011).Type 3 innate lymphoid cells (ILC3s) are innate immune cells that reside in the lamina propria of both the small and large intestines and are involved in tissue homeostasis, early defense against enteric pathogens, and containment of microbiota (Spits and Cupedo, 2012; Artis and Spits, 2015). In the intestines, multiple ILC3 subsets exist, two of which can be distinguished by mutual exclusive expression of the natural cytotoxicity receptor NKp46 and the chemokine receptor CCR6 (Sawa et al., 2010; Reynders et al., 2011). Most Nkp46⁺ ILC3s are found dispersed throughout the lamina propria, a localization that depends on the expression of CXCR6 (Satoh-Takayama et al., 2014). In contrast, the majority of CCR6⁺ ILC3s are located in close proximity to the intestinal crypts in anatomically defined sites known as cryptopatches (Kanamori et al., 1996). Recent findings indicated that under inflammatory conditions, such as experimental graft-versus-host disease, ILC3s can interact with the epithelial stem cells in the crypts, protecting them from T cell–mediated killing (Hanash et al., 2012).The well-known ability of ILC3s to condition the local microenvironment, the close proximity of ILC3s to intestinal crypts, and the ability of ILC3s to communicate with epithelial stem cells led us to hypothesize that ILC3s are involved in directing intestinal epithelial responses to tissue damage. Using the methotrexate (MTX) model of small intestinal damage, we now show that ILC3s are activated immediately after MTX administration, leading to a rapid activation of epithelial Stat3 and maintenance of ISCs. Our data reveal a novel function for ILC3s as organizers of the intestinal epithelial response to tissue damage through activation of epithelial cells and maintenance of ISCs and suggest that ILC3s might in future be therapeutically harnessed to prevent stem cell loss during chemotherapy.     

The yin and yang of intestinal epithelial cells in controlling dendritic cell function

Recent work suggests that dendritic cells (DCs) in mucosal tissues are "educated" by intestinal epithelial cells (IECs) to suppress inflammation and promote immunological tolerance. After attack by pathogenic microorganisms, however, "non-educated" DCs are recruited from nearby areas, such as the dome of Peyer's patches (PPs) and the blood, to initiate inflammation and the ensuing immune response to the invader. Differential epithelial cell (EC) responses to commensals and pathogens may control these two tolorogenic and immunogenic functions of DCs.     

Microcirculatory alterations of hepatic and mesenteric microcirculation in endotoxin tolerance

LPSs getting access to the circulation of mammalian organisms cause typical systemic inflammatory reactions with symptoms characteristic for acute sepsis. One possibility to attenuate LPS effects is to expose a host to a challenge with low LPS doses, which results in the establishment of "endotoxin tolerance" (ET). Because the microcirculation is of particular importance in LPS action, it seemed of interest to analyze leukocyte-endothelial interactions in the mesentery and liver once endotoxin tolerance has been established and are challenged with LPS. The mesenteric and hepatic microcirculation was investigated by intravital microscopy. After induction of ET LPS, shock was induced by i.v. injection of LPS, and microcirculation of the mesentery and liver was examined. Endotoxin tolerance resulted in reduced ex vivo TNF-alpha synthesis of whole blood. In vivo LPS caused no increase of body temperature. In sinusoids, LPS challenge increased adherence of leukocytes in naive rats, which was almost completely prevented by ET induction. In contrast, in postsinusoidal venules, leukocyte adherence was more intense after ET induction and subsequent to LPS application. Similarly, in postcapillary mesenteric venules, increased adherence of leukocytes after LPS challenge in the ET group was observed. After LPS injection, the endothelial barrier was more disturbed in the nontolerant group when compared with the ET group. Soluble L-selectin and intercellular adhesion molecule were elevated in both ET and untreated rats. Endotoxin tolerance influences leukocyte-endothelial interaction differentially depending on organ and vessel area.     

应激状态下肠上皮细胞凋亡水平的变化及其机制

目的探讨在氧化应激状态下肠上皮细胞凋亡水平的变化以及凋亡异常发生的分子机制。方法使用过氧化氢(H2O2)处理培养的HT-29细胞模拟机体活性氧(ROS)损伤肠上皮细胞的体内状况,采用四甲基偶氮唑盐(MTT)微量酶反应比色法进行细胞生存力的检测;采用流式细胞术进行细胞凋亡的检测;采用蛋白质免疫印迹法(Westernblot)检测凋亡相关蛋白的表达。结果H2O2可降低HT-29细胞生存率,且呈现剂量依赖性和时间依赖性(P均<0.05);与空白对照组相比,随着H2O2浓度的增高,细胞凋亡率增加(P均<0.05),随着作用时间的延长,细胞凋亡率也增加(P<0.05);以不同浓度H2O2刺激HT-29细胞24h后发现,与空白对照组相比,Bax的表达随着H2O2浓度的增高而增加,Bcl-2的表达随着H2O2浓度的增高而降低。以500μmol/L,浓度的H2O2刺激HT-29细胞发现,Bax表达随着H2O2作用时间延长而增加,Bcl-2表达随着H2O2作用时间延长而降低。结论应激状态下,肠上皮细胞氧化应激水平与其凋亡程度相关,凋亡调控蛋白Bcl-2/Bax比值失调可能是肠上皮细胞凋亡过度的机制之一。     

Human intestinal Vdelta1+ lymphocytes recognize tumor cells of epithelial origin

gammadelta T cells can be grouped into discrete subsets based upon their expression of T cell receptor (TCR) variable (V) region families, their tissue distribution, and their specificity. Vdelta2+ T cells constitute the majority of gammadelta T cells in peripheral blood whereas Vdelta1+T cells reside preferentially in skin epithelium and in the intestine. gammadelta T cells are envisioned as first line host defense mechanisms capable of providing a source of immune effector T cells and immunomodulating cytokines such as interleukin (IL) 4 or interferon (IFN) gamma. We describe here the fine specificity of three distinct gammadelta+ tumor-infiltrating lymphocytes (TIL) obtained from patients with primary or metastatic colorectal cancer, that could be readily expanded in vitro in the presence of IL-1beta and IL-7. Irrespective of donor, these individual gammadelta T cells exhibited a similar pattern of reactivity defined by recognition of autologous and allogeneic colorectal cancer cells, renal cell cancer, pancreatic cancer, and a freshly isolated explant from human intestine as measured by cytolytic T cell responses and by IFN-gamma release. In contrast, tumors of alternate histologies were not lysed, including lung cancer, squamous cell cancer, as well as the natural/lymphocyte-activated killer cell-sensitive hematopoietic cell lines T2, C1R, or Daudi. The cell line K562 was only poorly lysed when compared with colorectal cancer targets. Target cell reactivity mediated by Vdelta1+ T cells was partially blocked with Abs directed against the TCR, the beta2 or beta7 integrin chains, or fibronectin receptor. Marker analysis using flow cytometry revealed that all three gammadelta T cell lines exhibit a similar phenotype. Analysis of the gammadelta TCR junctional suggested exclusive usage of the Vdelta1/Ddelta3/Jdelta1 TCR segments with extensive (< or = 29 bp) N/P region diversity. T cell recognition of target cells did not appear to be a major histocompatibility complex restricted or to be correlated with target cell expression of heat- shock proteins. Based on the ability of some epithelial tumors, including colorectal, pancreatic, and renal cell cancers to effectively cold target inhibit the lysis of colorectal cancer cell lines by these Vdelta1+ T cell lines, we suggest that intestinal Vdelta1+ T cell lines, we suggest that intestinal Vdelta1+ T cells are capable of recognizing cell surface Ag(s) shared by tumors of epithelial origin.     

Butyrate and the cytokine-induced alpha1-proteinase inhibitor release in intestinal epithelial cells

BACKGROUND: Alpha1-proteinase inhibitor (alpha1-PI), an anti-inflammatory protein thought to play a role in the intestinal inflammation, is synthesised by and released from the intestinal epithelial cells. IL-1beta is a key proinflammatory cytokine in the abnormal immune response that occurs in inflammatory bowel disease. Butyrate is a normal luminal constituent in the colon, known to be of benefit in preventing inflammatory bowel disease. Direct modes of action of butyrate in intestinal inflammation have been poorly studied so far. The aim of this study was to investigate the effects of butyrate on cytokine-mediated alpha1-PI release in intestinal epithelial cells. METHODS: Differentiated Caco-2 cells were incubated with IL-1beta in the presence or absence of 2 mM butyrate. Alpha1-PI expression in the cells was evaluated by Western blot analysis and alpha1-PI release by ELISA. RESULTS: Treatment with butyrate alone had no effect on alpha1-PI expression in differentiated Caco-2 cells. However, treatment of the cells with 2 mM butyrate significantly reduced the alpha1-PI level in IL-1beta-treated cells. In the cell culture medium, the presence of butyrate impaired the IL-1beta-induced alpha1-PI release to 17-35%. The treatment induced no change in the number of detached cells or the percentage of viable cells. CONCLUSION: Our data show that butyrate inhibits alpha1-PI release from Caco-2 colonocytes treated with IL-1beta. It is therefore likely that anti-inflammatory actions of butyrate occur via a mechanism that does not involve direct regulation of cytokine-induced anti-inflammatory protein expression in intestinal epithelial cells.