Defective macrophage handling of Escherichia coli in Crohn's disease.

BACKGROUND AND AIM: Escherichia coli can be isolated from lamina propria macrophages in Crohn's disease (CD), and their intramacrophage persistence may provide a stimulus for inflammation. To further determine the contributions of macrophage dysfunction and E. coli pathogenicity to this, we aimed to compare in vitro functioning of macrophages from patients with CD and healthy controls (HC) in response to infection with CD-derived adherent-invasive E. coli (AIEC) and less pathogenic E. coli strains. METHODS: Monocyte-derived macrophages were cultured from patients with CD and HC. Intramacrophage survival of E. coli strains (CD-derived adherent-invasive [AI] and non-AI strains and laboratory strain K-12) was compared. Macrophage cytokine release (tumor necrosis factor alpha [TNFα], interleukin [IL]-23, IL-8 and IL-10) and monocyte phagoctyosis and respiratory burst function were measured after E. coli infection. For CD patients, laboratory data were correlated with clinical phenotype, use of immunomodulation, and CD risk alleles (NOD2, IL-23R, ATG16L1 and IRGM). RESULTS: Attenuated TNFα and IL-23 release from CD macrophages was found after infection with all E. coli strains. There was prolonged survival of CD-derived AIEC, CD-derived non-AIEC and E. coli K-12 in macrophages from CD patients compared to within those from HC. No abnormality of monocyte phagocytosis or respiratory burst function was detected in CD. Macrophage dysfunction in CD was not influenced by phenotype, use of immunomodulation or genotype. CONCLUSIONS: CD macrophage responses to infection with E. coli are deficient, regardless of clinical phenotype, CD genotype or E. coli pathogenicity. This suggests host immunodeficiency is an important contributor to intramacrophage E. coli persistence in CD.

What role do bacteria play in persisting fistula formation in idiopathic and Crohn's anal fistula?

AIM: The aetiology of Crohn's disease-related anal fistula remains obscure. Microbiological, genetic and immunological factors are thought to play a role but are not well understood. The microbiota within anal fistula tracts has never been examined using molecular techniques. The present study aimed to characterize the microbiota in the tracts of patients with Crohn's and idiopathic anal fistula. METHOD: Samples from the fistula tract and rectum of patients with Crohn's and idiopathic anal fistula were analysed using fluorescent in situ hybridization, Gram staining and scanning electron microscopy were performed to identify and quantify the bacteria present. RESULTS: Fifty-one patients, including 20 with Crohn's anal fistula, 18 with idiopathic anal fistula and 13 with luminal Crohn's disease and no anal fistula, were recruited. Bacteria were not found in close association with the luminal surface of any of the anal fistula tracts. CONCLUSION: Anal fistula tracts generally do not harbour high levels of mucosa-associated microbiota. Crohn's anal fistulas do not seem to harbour specific bacteria. Alternative explanations for the persistence of anal fistula are needed.

Apoptosis of endothelial cells precedes myocyte cell apoptosis in ischemia/reperfusion injury.

BACKGROUND: Apoptosis contributes to cell loss after ischemia/reperfusion injury in the heart. This study describes the time course and level of apoptosis in different cell types in the intact heart during ischemia/reperfusion injury. METHODS AND RESULTS: Isolated Langendorff-perfused rat hearts were subjected to perfusion alone (control) or to 35 minutes of regional ischemia, either alone or followed by 5, 60, or 120 minutes of reperfusion. Sections were stained by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and propidium iodide and with anti-von Willebrand factor, anti-desmin, or anti-active caspase 3 antibodies; they were then visualized by confocal microscopy. Sections were also examined by electron microscopy. No TUNEL-positive cells were seen in control hearts or hearts exposed to ischemia alone. Early in reperfusion, TUNEL staining was colocalized with endothelial cells from small coronary vessels. Endothelial apoptosis peaked at 1 hour of reperfusion and, at this time, there was clear perivascular localization of apoptotic cardiac myocytes, whose number was inversely proportional to their distance from a positive vessel. After 2 hours of reperfusion, apoptotic cardiac myocytes assumed a more homogeneous distribution. Active caspase 3 labeling was seen independent of DNA fragmentation during ischemia alone, but it colocalized with TUNEL staining over the 3 time points of reperfusion. Immunocytochemical findings were confirmed by electron microscopy and Western blotting. CONCLUSIONS: In the very early stages of reperfusion, apoptosis is first seen in the endothelial cells from small coronary vessels. The radial spread of apoptosis to surrounding cardiac myocytes suggests that reperfusion induces the release of soluble pro-apoptotic mediators from endothelial cells that promote myocyte apoptosis.

Synthesis of TNF alpha and TGF beta mRNA in the different micro-environments within atheromatous plaques.

OBJECTIVE: To examine localisation of tumour necrosis factor (TNF alpha) and transforming growth factor beta (TGF beta) mRNA synthesis in human coronary artery atheromatous plaques, to explore how synthesis of these cytokines relates to distribution of macrophages and smooth muscle cells, and to correlate this with plaque micro-environments. METHOD: In situ hybridisation with digoxigenin-labelled sense and anti-sense riboprobes was used, combined with immunohistochemistry to detect TNF alpha protein, macrophage, lymphocyte and smooth muscle cell markers. RESULTS: In the intimal plaque TNF alpha mRNA is synthesised by monocytes/macrophages as well as by smooth muscle cells. Both TNF alpha and TGF beta mRNAs were present at the margins of the lesions and in reactive areas, where there was little lipid and fibrosis. Focal aggregates of macrophages in the adventitia expressed both TNF alpha mRNA and protein and TGF beta mRNA. CONCLUSION: Synthesis of these two cytokines by macrophages as well as smooth muscle cells contributes to the pathobiology of the plaque and that this is part of the 'reaction to injury', rather than a feature of a specific cell, or a specific layer, within the vessel wall.

Quantitative assessment of cardiac myocyte apoptosis in tissue sections using the fluorescence-based tunel technique enhanced with counterstains.

Apoptosis is a distinct form of cell death, induced, for example, by ischaemia/reperfusion injury, that results in characteristic alterations in cell morphology and fate. In tissue sections, the most commonly used technique to detect apoptosis is terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL) staining which labels the ends of DNA strand breaks characteristic of the apoptotic process. However, without the employment of additional staining, TUNEL is only a qualitative procedure that gives no information about the proportion of negative cells nor the cell type undergoing apoptosis. We have utilised propidium iodide (PI) as a counterstain to visualise TUNEL negative nuclei together with anti-desmin antibody in order to assess quantitatively apoptosis in specific cell types. The procedure has been evaluated in tissue sections from isolated perfused rat hearts subjected to ischaemia and reperfusion. Hearts were cross-sectioned into four 2.5 mm thick slices which were fixed in 4% formaldehyde and embedded in paraffin. Serial sections (5 microns) were cut, dewaxed and pretreated by incubation with trypsin at 37 degrees C for 30 min. After the employment of the TUNEL assay, sections were labelled with anti-desmin antibody, counterstained with PI and finally examined by confocal fluorescent microscopy. Apoptosis was not seen in sections from hearts subjected to ischaemia alone nor in control hearts. After 35 min of ischaemia the percentages of TUNEL positive cells were very low both in myocytes (0.1%) and in non-myocytes (0.3%). In ischaemic-reperfused hearts, the number of TUNEL positive cells was only significantly higher in vascular cells (44+/-5%) and cardiac myocytes (6+/-2%). This simple method therefore allows quantification of apoptosis in myocytic and non-myocytic cells in tissue sections. Use of alternative immunohistochemical markers would permit adaptation of the method to the quantitative assessment of apoptosis in other tissues.

Distinct microbial populations exist in the mucosa-associated microbiota of sub-groups of irritable bowel syndrome.

BACKGROUND: There is increasing evidence to support a role for the gastrointestinal microbiota in the etiology of irritable bowel syndrome (IBS). Given the evidence of an inflammatory component to IBS, the mucosa-associated microbiota potentially play a key role in its pathogenesis. The objectives were to compare the mucosa-associated microbiota between patients with diarrhea predominant IBS (IBS-D), constipation predominant IBS (IBS-C) and controls using fluorescent in situ hybridization and to correlate specific bacteria groups with individual IBS symptoms. METHODS: Forty-seven patients with IBS (27 IBS-D and 20 IBS-C) and 26 healthy controls were recruited to the study. Snap-frozen rectal biopsies were taken at colonoscopy and bacterial quantification performed by hybridizing frozen sections with bacterial-group specific oligonucleotide probes. KEY RESULTS: Patients with IBS had significantly greater numbers of total mucosa-associated bacteria per mm of rectal epithelium than controls [median 218 (IQR - 209) vs 128 (121) P = 0.007], and this was chiefly comprised of bacteroides IBS [69 (67) vs 14 (41) P = 0.001] and Eubacterium rectale-Clostridium coccoides [52 (58) vs 25 (35) P = 0.03]. Analysis of IBS sub-groups demonstrated that bifidobacteria were lower in the IBS-D group than in the IBS-C group and controls [24 (32) vs 54 (88) vs 32 (35) P = 0.011]. Finally, amongst patients with IBS, the maximum number of stools per day negatively correlated with the number of mucosa-associated bifidobacteria (P < 0.001) and lactobacilli (P = 0.002). CONCLUSIONS & INFERENCES: The mucosa-associated microbiota in patients with IBS is significantly different from healthy controls with increases in bacteroides and clostridia and a reduction in bifidobacteria in patients with IBS-D.

Clinical, microbiological, and immunological effects of fructo-oligosaccharide in patients with Crohn's disease.

BACKGROUND AND AIMS: The intestinal microbiota play a pivotal role in the inflammation associated with Crohn's disease through their interaction with the mucosal immune system. Some bifidobacteria species are immunoregulatory and induce increased dendritic cell interleukin 10 (IL-10) release in vitro. Fructo-oligosaccharides (FOS) increase faecal and mucosal bifidobacteria in healthy volunteers. The aim of this study was to assess the effect of FOS administration on disease activity, bifidobacteria concentrations, and mucosal dendritic cell function in patients with moderately active Crohn's disease. PATIENTS AND METHODS: Ten patients with active ileocolonic Crohn's disease received 15 g of FOS for three weeks. Disease activity was measured using the Harvey Bradshaw index. Faecal and mucosal bifidobacteria were quantified by fluorescence in situ hybridisation, and mucosal dendritic cell IL-10 and Toll-like receptor (TLR) expression were assessed by flow cytometry of dissociated rectal biopsies. RESULTS: FOS induced a significant reduction in the Harvey Bradshaw index from 9.8 (SD 3.1) to 6.9 (3.4) (p<0.01). There was a significant increase in faecal bifidobacteria concentration from 8.8 (0.9) log(10) to 9.4 (0.9) log(10) cells/g dry faeces (p<0.001). The percentage of IL-10 positive dendritic cells increased from 30 (12)% to 53 (10)% (p=0.06). Finally, the percentage of dendritic cells expressing TLR2 and TLR4 increased from 1.7 (1.7)% to 36.8 (15.9)% (p=0.08) and from 3.6 (3.6)% to 75.4 (3.4)% (p<0.001), respectively. CONCLUSIONS: FOS supplementation increases faecal bifidobacteria concentrations and modifies mucosal dendritic cell function. This novel therapeutic strategy appears to decrease Crohn's disease activity in a small open label trial and therefore warrants further investigation.

Molecular characterization of the bacteria adherent to human colorectal mucosa.

AIMS: To study large intestinal mucosal bacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) profiling and sequencing of 16S rRNA gene polymerase chain reaction (PCR) products amplified from DNA extracted from colorectal biopsies taken from healthy individuals. The specific aims were to determine how similar the mucosa-associated bacterial communities are within and between individuals and also to characterize the phylogenetic origin of isolated DGGE bands. METHODS AND RESULTS: Human colorectal biopsies were taken at routine colonoscopy from 33 patients with normal looking mucosa. The DNA was extracted directly from single biopsies and the bacterial 16S rDNA PCR amplified. The PCR products were profiled using DGGE to generate a fingerprint of the dominant members of the bacterial community associated with the biopsy. The reproducibility of this method was high (>98%). Washed and unwashed biopsies gave similar DGGE banding patterns (Median Similarity Coefficient - MSC 96%, InterQuartile Range - IQR 3.0%, n = 5). Adjacent biopsies sampled from the same patient using different forceps gave similar DGGE profiles (MSC 94%, n = 2). Two colorectal biopsies sampled at locations 2-5 cm apart, from each of 18 patients, resulted in very similar profiles (MSC 100%, IQR 2.8%). Biopsies sampled from different locations within the large intestine of the same patient also gave similar DGGE profiles (MSC 98% IQR 3.3%n = 6). Although all patients (n = 33) gave different DGGE profiles, some similarity (c. 34%) was observed between profiles obtained from 15 patients arbitrarily selected. 35 DGGE bands were excised and sequenced. Many were found to be most closely related to uncultured bacterial sequence entries in the Genbank database. Others belonged to typical gut bacterial genera including Bacteroides, Ruminococcus, Faecalibacterium and Clostridium. CONCLUSIONS: Bacterial communities adherent to colorectal mucosa within a normal patient show little variation; in contrast, mucosal bacterial communities sampled from different patients with normal colorectal mucosa show a high degree of variation. SIGNIFICANCE AND IMPACT OF THE STUDY: This research demonstrates that DGGE profiling of 16S rRNA gene PCR products amplified from DNA extracted directly from mucosal samples offers fresh insight into the bacterial communities that are adherent to colorectal mucosa. These findings are important with respect to further studies on the gastrointestinal tract in health and disease.

IFN-gamma down-regulates MHC expression and antigen processing in a human B cell line.

IFN-gamma is a crucial mediator in the induction of cell-mediated Th1-type responses but is predominantly a negative regulator of B cell differentiation and proliferation. This cytokine is therefore a key factor in determining Th1 vs Th2 differentiation. This study investigates the action of IFN-gamma in modulation of HLA-DR expression and Ag presentation by EBV-transformed human B cell lines. In contrast to its action on the monocyte/macrophage, IFN-gamma down-regulates surface MHC expression on these B cells, and this regulation is posttranscriptional. In parallel with MHC down-regulation, there is a reduced capability to process and present exogenous protein and peptide Ag to T cell hybridomas. IFN-gamma does not change the rates of fluid phase endocytosis or exocytosis in this model system but correlates with an up-regulation of the lysosomal enzymes cathepsins B and D.

Differential regulation of HLA-DQ expression by keratinocytes and Langerhans cells in normal and premalignant cervical epithelium.

Keratinocytes in normal ectocervix did not express major histocompatibility complex class II molecules. In low-grade intraepithelial lesions expression was confined to HLA-DR, while in high-grade disease there was expression of HLA-DR and occasional expression of HLA-DQ. HLA-DR was expressed constitutively on the majority of Langerhans cells. In contrast, few Langerhans cells expressed HLA-DQ in normal cervix, but there was a steady upregulation of the proportion expressing HLA-DQ which paralleled the severity of disease. There was no direct correlation between human papillomavirus 16 and the expression of major histocompatibility complex class II by keratinocytes and Langerhans cells. Significant upregulation of HLA-DQ by Langerhans cells is observed in high-grade intraepithelial cervical lesions, suggesting antigen-presenting cell activation in papillomavirus-related premalignant disease.

Regulation of cathepsin E expression during human B cell differentiation in vitro.

Cathepsin E is an aspartic proteinase which has been implicated in antigen processing in the class II major histocompatibility complex pathway. In this study we show that cathepsin E, measured at both the protein and message level, is up-regulated late in human B cell activation. The implications of this observation in terms of cathepsin E function are discussed.

Synthesis of tumor necrosis factor-alpha mRNA in bronchoalveolar lavage cells from human immunodeficiency virus-infected persons with Pneumocystis carinii pneumonia.

Bronchoalveolar lavage fluid cells from a cohort of 34 human immunodeficiency virus-infected persons with established Pneumocystis carinii pneumonia were examined for expression of tumor necrosis factors (TNF-alpha) mRNA by fluorescence in situ hybridization with an antisense riboprobe. Video image analysis was used to develop a quantitative assay that evaluates relative single-cell levels of mRNA. The resulting data were analyzed as an antisense-to-sense ratio and examined for correlation between TNF-alpha mRNA expression and other measures of disease severity. Higher levels of TNF-alpha mRNA were seen in persons who had higher levels of arterial oxygen.

Cathepsin E expression by normal and premalignant cervical epithelium.

We have investigated the expression of the aspartic proteinase cathepsin E and HLA-DR and the presence of HPV16 in normal squamous epithelium (n = 8) and low-grade (n = 21) and high-grade (n = 14) intraepithelial squamous lesions of the uterine cervix. Immunohistochemistry of cervical biopsies revealed that up-regulation of cathepsin E expression was related to increasing severity of the cervical intraepithelial neoplasia (CIN). Up-regulation of protein was associated with increased message as assessed by in situ hybridization. Langerhans cells and the majority of koilocytes did not express detectable cathepsin E levels. Although there was also an up-regulation of HLA-DR expression by cervical keratinocytes in cervical intraepithelial neoplasia lesions, as determined by immunohistochemistry, no significant correlation was found between HLA-DR and cathepsin E expression in these lesions; neither was expression of cathepsin E correlated to the presence of HPV16, detected by polymerase chain reaction. The expression of cathepsin E, an aspartic proteinase that is reported to play a role in antigen processing for presentation by class II major histocompatibility complex molecules, is associated with cellular dedifferentiation in cervical intraepithelial neoplasia.

The antigen-presenting environment in normal and human papillomavirus (HPV)-related premalignant cervical epithelium.

The activation of HPV-specific T cells within the cervical microenvironment is likely to play an important part in the natural history of cervical intraepithelial neoplasia (CIN). The extent and the type of T cell activation will depend critically on the expression of MHC, costimulatory cell surface molecules and cytokines by keratinocytes and Langerhans cells within the cervical lesion. Expression of MHC class II (HLA-A-DR and -DQ), costimulatory/adhesion molecules (CD11a/18, CD50, CD54, CD58 and CD86) and cytokines (tumour necrosis factor-alpha (TNF-alpha) and IL-10) was therefore investigated by immunohistochemistry in normal squamous epithelium (n = 12), low-grade (n = 23) and high-grade (n = 18) squamous intraepithelial lesions of the cervix. CIN progression was associated with de novo expression of HLA-DR and CD54, and increased expression of CD58 by keratinocytes. However, significantly, there was no expression of any adhesion/costimulation molecule by epithelial Langerhans cells in any cervical biopsy studied. Furthermore, TNF-alpha, a potent activator of Langerhans cells, was expressed constitutively by basal keratinocytes in normal cervix (12+/12). but expression of this cytokine was absent in a number of CIN samples (20+/23 for low-grade, 12+/18 for high-grade CIN). Conversely, the suppressive cytokine IL-10 was absent in normal epithelium (0+/12), but was up-regulated in a number of CIN lesions (12+/23 for low-grade; 8+/18 for high-grade CIN). The restricted expression of costimulation/adhesion molecules and the nature of the cytokine microenvironment within the epithelium may act to limit effective immune responses in some CIN lesions.

Immunopathology of cerebral malaria: morphological evidence of parasite sequestration in murine brain microvasculature.

A murine model that closely resembles human cerebral malaria is presented, in which characteristic features of parasite sequestration and inflammation in the brain are clearly demonstrable. "Young" (BALB/c x C57BL/6)F(1) mice infected with Plasmodium berghei (ANKA) developed typical neurological symptoms 7 to 8 days later and then died, although their parasitemias were below 20%. Older animals were less susceptible. Immunohistopathology and ultrastructure demonstrated that neurological symptoms were associated with sequestration of both parasitized erythrocytes and leukocytes and with clogging and rupture of vessels in both cerebral and cerebellar regions. Increases in tumor necrosis factor alpha and CD54 expression were also present. Similar phenomena were absent or substantially reduced in older infected but asymptomatic animals. These findings suggest that this murine model is suitable both for determining precise pathogenetic features of the cerebral form of the disease and for evaluating circumventive interventions.

1,25(OH)2D3 regulates c-myc mRNA levels in tonsillar T lymphocytes.

The effects of 1,25(OH)2D3 on proliferation, c-myc mRNA levels and 1,25(OH)2D3 receptor expression in activated tonsillar T lymphocytes were studied. Activation of resting T cells with phytohaemagglutinin (PHA) for 72 hr led to an increase in proliferation, c-myc mRNA levels and to induction of 1,25(OH)2D3 receptor expression. However, when activation was carried out in the presence of 1,25(OH)2D3, there was inhibition of PHA-stimulated proliferation and c-myc mRNA levels. Increased cell proliferation, c-myc mRNA expression and 1,25(OH)2D3 receptor number were also observed, albeit to a lesser extent, when T cells were stimulated by phorbol myristate acetate (PMA), anti-CD3 antibody or A23187. However, in these cases 1,25(OH)2D3 was unable to prevent increased proliferation or c-myc mRNA expression. PMA and anti-CD3 used in combination produced similar or greater changes in proliferation, c-myc mRNA levels, 1,25(OH)2D3 receptor expression and responsiveness to the hormone when compared to PHA alone. Thus the inhibition of c-myc expression in activated T lymphocytes by 1,25(OH)2D3 can be related to its anti-proliferative effects. Moreover this inhibition seems to be dependent on the level of 1,25(OH)2D3 receptor expression, which in turn appears to be related to the degree of cell activation.

Tumour necrosis factor-alpha (TNF-alpha) synthesis is associated with the skin and peripheral nerve pathology of leprosy reversal reactions.

Leprosy may be complicated by episodes of increased cell-mediated immunity towards Mycobacterium leprae (reversal reactions) which result in severe local immunopathology in skin lesions and peripheral nerves. Using in situ hybridization and MoAb techniques we have demonstrated TNF-alpha mRNA and TNF-alpha protein in macrophages infiltrating leprosy skin and peripheral nerve. Levels of TNF-alpha mRNA are significantly increased in reactional skin and nerve, particularly in borderline tuberculoid patients. TNF-alpha mRNA and TNF-alpha protein levels are higher in reactional nerves then reactional skin. In both reactional skin and nerve TNF-alpha mRNA is more abundant than TNF-alpha protein; this may reflect the rapid turnover of TNF-alpha protein in an immunologically dynamic situation, such as is seen in reversal reaction. Our findings emphasize the importance of documenting both mRNA and protein production when assessing the role of cytokines in pathology. The leprosy reversal reaction may be regarded as a useful model of tissue immunopathology in which TNF-alpha is generated as part of the host response to infection, but also produces local tissue damage.

Myocyte loss in chronic heart failure.

This study examined whether or not there is progressive loss of individual myocytes in established heart failure, accounting for the progressive left ventricular dysfunction; whether such loss is by necrosis or apoptosis; and whether such loss is more pronounced in ischaemic heart disease or idiopathic dilated cardiomyopathy. Tissue for patients undergoing cardiac transplantation for clinical end-stage heart disease was used. The clinical diagnosis was not known to the observer at the time of analysis. Indices of potential myocyte loss were: detection of apoptotic nuclei in situ, using the TUNEL method, immunohistochemistry for CD120a, CD120b, CD95, perforin and granzyme B; binding of C9 complex; and lipofuscin deposition within macrophages. Interstitial macrophages and T cells and their relationship to myocyte loss were also examined. There is indeed low grade myocyte loss in chronic heart failure, but there was no difference between the disease groups; rather, there was marked patient-to-patient variation within each category. Thus in chronic heart failure myocyte loss does occur, and both necrosis and apoptosis contribute to this loss, irrespective of the underlying nature of the disease. Any mechanism which accounts for myocyte loss must be common to both conditions, rather than specific for a pre-operative diagnosis.

Placentally derived prostaglandin E2 acts via the EP4 receptor to inhibit IL-2-dependent proliferation of CTLL-2 T cells.

A number of immunomodulatory molecules are present in the placenta, including cytokines, prostaglandins, progesterone and indoleamine 2,3-dioxygenase. An undefined factor capable of down-regulating T-cell activity has recently been reported [1] as being produced by short-term cultures of placental fragments. By careful repetition of these studies we have confirmed that chorionic villi isolated from term placenta produce a low molecular weight, heat stable factor capable of inhibiting the IL-2-dependent proliferation of mouse CTLL-2 cells. This activity was not due, however, to a previously unknown immunosuppressive molecule, but rather to prostaglandin E2 (PGE2). Expression of cyclooxygenase (COX)-2 was detected in the syncytiotrophoblast of chorionic villi explants using immunohistochemistry. Culture of the explants in the presence of the COX-1/COX--2 inhibitors indomethacin and diclofenac, or with the COX-2-selective inhibitor DFP, blocked the production of the immunosuppressive factor. The immunosuppressive activity was restored by adding PGE2 to the supernatants obtained from diclofenac-inhibited explants. A number of different receptors are involved in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that the immunosuppressive effect of PGE2 on CTLL-2 cells is exerted via the EP4 receptor. Thus, addition of an EP4-selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL-2 cells. This may have implications for attempts to selectively manipulate T-cell responses.