A crosstalk between muscarinic and CRF2 receptors regulates cellular adhesion properties of human colon cancer cells.

Patients with inflammatory bowel disease often suffer from chronic and relapsing intestinal inflammation that favor the development of colitis associated cancer. An alteration of the epithelial intestinal barrier function observed in IBD is supposed to be a consequence of stress. It has been proposed that corticotrophin-releasing factor receptor (CRF2), one of the two receptors of CRF, the principal neuromediator of stress, acts on cholinergic nerves to induce stress-mediated epithelial barrier dysfunction. Non-neuronal acetylcholine (Ach) and muscarinic receptors (mAchR) also contribute to alterations of epithelial cell functions. In this study, we investigated the mechanisms through which stress and Ach modulate epithelial cell adhesive properties. We show that Ach-induced activation of mAchR in HT-29 cells results in cell dissociation together with changes in cell-matrix contacts, which correlates with the acquisition of invasive potential consistent with a matrix metalloproteinase (MMP) mode of invasion. These processes result from mAchR subsequent stimulation of the cascade of src/Erk and FAK activation. Ach-induced secretion of laminin 332 leads to α3ß1 integrin activation and RhoA-dependent reorganization of the actin cytoskeleton. We show that Ach-mediated effects on cell adhesion are blocked by astressin 2b, a CRF2 antagonist, suggesting that Ach action depends partly on CRF2 signaling. This is reinforced by the fact that Ach-mediated activation of mAchR stimulates both the synthesis and the release of CRF2 ligands in HT-29 cells (effects blocked by atropine). In summary, our data provides evidence for a novel intracellular circuit involving mAchR acting on CRF2-signaling that could mediate colonic mucosal barrier dysfunction and exacerbate mucosal inflammation.

RhoA-dependent switch between alpha2beta1 and alpha3beta1 integrins is induced by laminin-5 during early stage of HT-29 cell differentiation.

Integrin-mediated interactions between the basement membrane and epithelial cells control the differentiation of epithelia. We characterized the modulation of adhesive behaviors to basement membrane proteins and of integrin function in the human colon adenocarcinoma HT-29 cell line, which differentiates into enterocytes after the substitution of galactose for glucose in the medium. We demonstrate an increased capability of these cells to adhere to collagen type IV during the early stage of differentiation. This effect occurs without any changes in integrin cell surface expression but rather results from an alpha2beta1/alpha3beta1 integrin switch, alpha3beta1 integrin becoming the major collagen receptor. The increase in laminin-5 secretion and deposit on the matrix is a key factor in the mechanism regulating cell adhesion, because it is responsible for the activation of alpha3beta1 integrin. Furthermore, down-regulation of RhoA GTPase activity occurs during HT-29 cell differentiation and correlates with the activation of the integrin alpha3beta1. Indeed, C3 transferase, a RhoA GTPase inhibitor, induces a similar alpha2beta1/alpha3beta1 switch in undifferentiated HT-29 cells. These results indicate that the decrease in RhoA activation is the biochemical mechanism underlying this integrin switch observed during cell differentiation. The physiological relevance of such modulation of integrin activity in the functioning of the crypt-villus axis is discussed.

Polymorphism in the regulatory sequence of the human hsp70-1 gene does not affect heat shock factor binding or heat shock protein synthesis.

A bi-allelic polymorphism found in the regulatory region of the human heat shock (HS) protein (HSP) hsp70-1 gene, which comprises an A-->C transversion, 3 bp upstream of the HS element (HSE), has been associated with extended HLA haplotypes. In view of the chaperoning and protective functions of Hsp70, we investigated whether this hsp70-1 bi-allelic polymorphism could modulate the stress response, which may relate to enhanced resistance or susceptibility to certain diseases. We compared the basal and HS-induced HS factor (HSF)-binding activity of the two polymorphic HSEs, hsp70-1 mRNA accumulation and HSP expression in two human Epstein Barr virus (EBV)-transformed B cell lines typed for hsp70-1 promoter alleles. Our results suggest that hsp70-1 promoter polymorphism does not influence HSF-binding activity, hsp70 mRNA accumulation or synthesis in human EBV-transformed B cell lines.

Heat shock and proinflammatory stressors induce differential localization of heat shock proteins in human monocytes.

Heat shock (HS) proteins (HSP) are a family of molecular chaperones induced by environmental stresses such as oxidative injury, and contribute to protection from and adaptation to cellular stress. We investigated in human monocytes the expression and subcellular distribution of hsp70 and hsc70 after HS and inflammation-related stresses leading to generation of reactive oxygen species by these cells, such as the phorbol ester PMA and erythrophagocytosis (E phi). By combining immunofluorescent staining and Western blot on subcellular fractions, we found that all three stress factors resulted in an increased hsp70 expression, however the subcellular distribution pattern was different depending on the type of stress. While HS induced a rapid translocation of hsp70 into the nucleus, no nuclear translocation of hsp70 was observed after PMA or E phi. Neither of the examined stresses induced membrane expression of hsp70. The observed differences in subcellular distribution pattern might relate to distinct regulation and specific functions of hsp70 in inflammation.

Covalent binding of C3b to tetanus toxin: influence on uptake/internalization of antigen by antigen-specific and non-specific B cells.

Antigen opsonization by the C3b fragment of complement is a significant event in the modulation of cell-mediated immune response, but its mechanism is still largely unknown. The structural characteristics of C3b allow it to act as a bifunctional ligand between antigen and cells via their membrane C3b receptors. It was thus of interest to study the influence of the covalent link between C3b and antigen on the fixation and internalization of this antigen by antigen-presenting cells. Tetanus toxin (TT) was used as antigen, either free or covalently linked to C3b (TT-C3b). The antigen-presenting cells were TT-specific (4.2) or non-specific (BL15) Epstein-Barr virus (EBV)-transformed B cells. C3b was found to play an important role in antigen fixation and internalization by both antigen-specific and antigen non-specific cells. Covalent binding of C3b on TT (1) permitted fixation and internalization of this antigen by non-specific cells via their complement receptors; (2) enhanced antigen fixation and resulted in cross-linking between membrane immunoglobulins and complement receptors on antigen-specific cells. The consequences of covalent C3b binding to TT were analysed using antigen-specific and antigen-nonspecific cells. In both cases, a net increase in antigen fixation was observed. At the intracellular level, covalent C3b binding to TT resulted in a large TT incorporation in endosomes of nonspecific cells, similar to that observed in antigen-specific cells. Thus, C3b covalently linked to antigen enlarges the array of B-cell types capable of presenting antigen, including non-specific cells.

Cellular basis of ECD brain retention.

UNLABELLED: Clinical observations have shown discrepancies between ECD and HMPAO regional cerebral perfusion, particularly in brain tumors and during stroke recovery. We investigated the nature of the process(es) involved in ECD accumulation in vitro at the cellular level. METHODS: Time course incorporation of ECD was studied in a fast-growing human premonocytic line, U937, in a human astrocytic-derived cell line, U373, and a human hybridized endothelial cell line, EaHy926. Cells were further used in experiments aiming to correlate esterase activity and ECD retention. RESULTS: Significant differences in ECD retention between these cell lines were observed: %UECD (cpm cells/cpm standard of injected) plateaued within 2 hr in all cases but %UECD was significantly higher in U937 cells (25.1 +/- 3.9% at 120 min) than in the other cell lines (6.1 +/- 0.7% and 8.2 +/- 2.0% at 120 min for U373 and EaHy926, respectively). Contrary to what we expected, total cellular esterase activity (EATOT) was inversely correlated to %UECD.EATOT was 5-fold lower in U937 cells than in U373 and 20-fold lower than in EaHy926. Thus, we compared the membranar to the cytosolic esterase activity of U937 and analyzed the influence of temperature and diisopropylfluorophosphate (DFP, an inhibitor of cytosolic esterase activity) on both ECD retention and enzymatic activities. When cells were exposed to DFP, %UECD was reduced by 80%; while when cells were maintained at 4 degrees C, %UECD continuously increased, corresponding to a passive diffusion since both cytosolic and membranar esterase activities were inhibited. CONCLUSION: For optimal uptake of ECD, the membranar fraction of the esterase activity has to be low, while, in contrast, the cytosolic fraction of the esterase activity plays an important role in ECD cell retention. ECD-SPECT is likely able to reflect regional cerebral blood flow in normal and pathological states accurately, but in the event of unusual observations, the membranar esterase activity should be considered to explain reduced ECD retention.

Selective induction of the glucose-regulated protein grp78 in human monocytes by bacterial extracts (OM-85): a role for calcium as second messenger.

Heat shock/stress proteins (HSP) act as molecular chaperones, protect cells from injury, and are involved in the immune response. We investigated the effects of the immunomodulating bacterial extracts OM-85 on the stress response in normal human peripheral blood monocytes. While OM-85 did not induce the classical HSP, we show here, using 2D gel electrophoresis combined with immunoblotting, the induction of the glucose regulated protein grp78 (the immunoglobulin heavy chain binding protein BiP) along with the described accumulation of pro-interleukin-1 beta. The increased Ca2+ mobilization observed with OM-85 is the likely second messenger for grp78 induction. Recent studies are in favor of a protective role of grp78 against cytokine-mediated cytotoxicity and apoptosis. We suggest that grp78 induction following exposure to OM-85 explains, at least in part, the immunodulatory and protective effects of the bacterial extracts.

Dual regulation of heat-shock transcription factor (HSF) activation and DNA-binding activity by H2O2: role of thioredoxin.

The heat-shock (HS) response is a ubiquitous cellular response to stress, involving the transcriptional activation of HS genes. Reactive oxygen species (ROS) have been shown to regulate the activity of a number of transcription factors. We investigated the redox regulation of the stress response and report here that in the human pre-monocytic line U937 cells, H2O2 induced a concentration-dependent transactivation and DNA-binding activity of heat-shock factor-1 (HSF-1). DNA-binding activity was, however, lower with H2O2 than with HS. We thus hypothesized a dual regulation of HSF by oxidants. We found that oxidizing agents, such as H2O2 and diamide, as well as alkylating agents, such as iodoacetic acid, abolished, in vitro, the HSF-DNA-binding activity induced by HS in vivo. The effects of H2O2 in vitro were reversed by the sulphydryl reducing agent dithiothreitol and the endogenous reductor thioredoxin (TRX), while the effects of iodoacetic acid were irreversible. In addition, TRX also restored the DNA-binding activity of HSF oxidized in vivo, while it was found to be itself induced in vivo by both HS and H2O2. Thus, H2O2 exerts dual effects on the activation and the DNA-binding activity of HSF: on the one hand, H2O2 favours the nuclear translocation of HSF, while on the other, it alters HSF-DNA-binding activity, most likely by oxidizing critical cysteine residues within the DNA-binding domain. HSF thus belongs to the group of ROS-modulated transcription factors. We propose that the time required for TRX induction, which may restore the DNA-binding activity of oxidized HSF, provides an explanation for the delay in heat-shock protein synthesis upon exposure of cells to ROS.

TNF alpha alters mitochondrial membrane potential in L929 but not in TNF alpha-resistant L929.12 cells: relationship with the expression of stress proteins, annexin 1 and superoxide dismutase activity.

Tumour necrosis factor alpha (TNF alpha) cytotoxicity is mediated, at least in part, by oxidative stress and phospholipase A2 activation. The first post-receptor events to be observed in TNF alpha-sensitive lines are the generation of superoxide anion (O2-) within the mitochondria and the activation of phospholipase A2. Using the lipophilic dye JC-1 to determine mitochondrial membrane potential, we showed that TNF alpha induces time-dependent alterations in mitochondrial membrane potential in L929 cells but not in the TNF alpha-resistant L929. 12 subclone. Heat shock (HS) proteins (HSP) and superoxide dismutase (SOD) have been shown to protect cells from TNF alpha cytotoxicity, while glucose regulated proteins (GRP) and annexins might also be involved in cellular protection. We thus compared the expression of HSP, grp78 and annexin 1 as well as SOD activity in TNF alpha sensitive and resistant lines. We found no difference in the expression of HSP, grp78 or annexin 1, but an increase in the constitutive activity of SOD in the L929.12 cells as compared to L929. Furthermore, SOD was inducible by TNF alpha in L929 cells, but not in L929.12 cells. These data suggest that in TNF alpha-resistant lines, mitochondrial damage by TNF alpha is prevented by an increase in SOD rather than in overexpression of stress proteins or annexins.

Oxido-reductive state: the major determinant for cellular retention of technetium-99m-HMPAO.

UNLABELLED: Several clinical observations have suggested that HMPAO cerebral uptake might be related not only to regional cerebral perfusion but also to the nature of the lesion. Our aim was to investigate at the cellular level the nature of the process(es) involved in HMPAO accumulation in vitro. METHODS: Time-course incorporation of HM-PAO was studied in a fast-growing human premonocytic line, U937, in a human astrocytic-derived cell line, U373 and a human hybridized endothelial cell line, EaHy926. Minimal differences of HMPAO retention between these cell lines were observed and plateau of %U(HMPAO) (cpm cells/cpm standard of injected) were achieved within 2 hr. Because HMPAO cell retention was related to the intracellular content in glutathione, experiments studying effects of redox were conducted by preexposing U937 cells to D, L dithiothreitol or 2-Mercaptoethanol. RESULTS: Overnight incubation with NAC or BSO did not significantly modified the kinetic of 99mTc-HMPAO incorporation while overnight incubation with NAC resulted in a 2-fold increase in intracellular glutathione content and overnight incubation with BSO nearly abolished the intracellular glutathione content. At the opposite, presence of these reducing agents in the medium during the experiments completely abolished 99mTc-HMPAO retention. CONCLUSION: Our data thus provide in vitro evidence to support that overall intracellular retention of HMPAO is more dependent upon the redox activity of the tissue than the intracellular glutathione content. SPECT-HMPAO may accurately reflect regional cerebral blood flow in a normal state but possibly not in all pathological situations in which cell metabolism disturbances are characterized by alterations in the redox status.

Differential expression and regulation of hsp70 and hsp90 by phorbol esters and heat shock.

Human peripheral blood monocytes (PBM) produce superoxide anions (O2-.) by a process involving electron transfer from NADPH to O2, catalyzed by the respiratory burst enzyme NADPH oxidase. We have previously shown that phagocytosis, while activating NADPH oxidase, induced in PBM the synthesis of heat shock (HS) proteins (HSP). The present study was undertaken to establish whether this increase in HSP expression was related to O2-. and/or to classical second messengers such as protein kinase C (PKC). Thus, the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) were compared with those of heat shock on the expression, in PBM, of the major HSP, hsp70 and hsp90, using biometabolic labeling, Western and Northern blotting, and gel mobility shift assays. PMA induced the accumulation of mRNA and an increased expression of hsp90 and, to a lesser extent, hsp/hsc70 (hsc is the cognate, constitutive form). This induction was also observed in PBM from patients with chronic granulomatous disease, a genetic defect in NADPH oxidase, and was abolished by the PKC inhibitors staurosporine and H-7. PMA did not cause activation of the HS factor, and the PMA-induced overexpression expression of HSP was not blocked by the transcriptional inhibitor actinomycin D. HSP-specific mRNA stability was increased after PMA exposure as compared with heat shock. These results suggest that O2-. is not involved in the PMA-mediated induction of hsp70 and hsp90 and that, in contrast to HS, PMA increases the expression of HSP as a result of PKC-induced mRNA stabilization rather than of transcriptional activation of HS genes.

Differential induction of stress proteins and functional effects of heat shock in human phagocytes.

Induction of specific heat shock (HS) proteins (HSP) has been described as a response of human monocytes to phagocytosis, and HSP may play protective roles in infection and immunity. Here we compared the stress response in monocytes and polymorphonuclear neutrophils during exposure to the classical inducers of HSP, i.e., HS and cadmium. We also investigated the stress response in these two phagocytic cells after particulate (phagocytosis) and nonparticulate [f-Met-Leu-Phe (FMLP)] activation of the respiratory burst enzyme NADPH oxidase. HS and cadmium induced stress protein synthesis in both cell types. In contrast, phagocytosis induced HSP in monocytes only, while FMLP did so in neutrophils only. This differential regulation of stress proteins might relate to physiological and functional differences between monocytes and neutrophils. With respect to functional effects of HS, we examined, in human monocytes and in neutrophils, the effect of HS on NADPH oxidase-mediated O2- generation as well as on phagocytosis, bacterial killing, and superoxide dismutase (SOD) activity. In monocytes, as in neutrophils, NADPH oxidase activity was inhibited by HS, while thermotolerance prevented this inhibition. Phagocytosis and bacterial killing were unaltered by HS. SOD activity transiently increased in monocytes but decreased in neutrophils upon exposure to HS. These observations indicate differential induction of HSP in human phagocytes and differential regulation of phagocytes' functions by HS.

Modulation of antigen processing and presentation by covalently linked complement C3b fragment.

Ligands such as complement fragments (C3, C4), IgG or alpha 2-macroglobulin, which bind antigen (Ag) before their uptake by antigen-presenting cells (APC), are likely to modulate the different steps of Ag processing and presentation. These ligands contribute to internalization and endosomal targeting of Ag; they also influence its processing and, consequently, the binding of resulting peptides to major histocompatibility complex (MHC) class II molecules before presentation to T cells. Complement protein C3 contains, like other members of the alpha 2-macroglobulin family, an intrachain thiolester bond. Conformational alteration or limited proteolysis of C3 into C3b leads to breaking of the thiolester with transient capacity of the revealed carbonyl group to esterify hydroxyl groups of Ag. Ester-linked complexes including tetanus toxin (TT) and C3b were prepared to analyse the influence of bound C3b on TT processing and presentation by APC. Covalent binding of C3b to TT resulted in increased and prolonged stimulation of specific T-cell proliferation. This effect was observed with non-specific B cells, as well as with a TT-specific B-cell clone, as APC. On the other hand, SDS-PAGE analysis of proteolysates of TT or C3b-TT, obtained with endosome/lysosome-enriched subcellular fractions prepared from human Epstein-Barr virus (EBV)-transformed B cells, indicated a delay of TT proteolysis when TT was associated to C3b. Treatment of APC with protease inhibitors, before and during exposure of the cells to Ag, resulted in differences in the inhibition of TT and C3b-TT proteolysis. Using purified cathepsins B and D, we demonstrated that covalent binding of C3b to TT totally abolished TT proteolysis by cathepsin D, while proteolysis by cathepsin B was preserved. This finding and the absence of cathepsin B in endosomes may explain a delay in TT processing when it is associated to C3b. Confirming these data, presentation by formaldehyde-fixed cells of C3b-TT proteolysates showed higher stimulation of specific T-cell clones than formaldehyde-fixed TT proteolysates.

Protective effects of hsp70 in inflammation.

Inflammation results from the recruitment to a given tissue or organ and the activation of leucocytes, among which the monocytes-macrophages play a major role. These phagocytic cells produce high levels of reactive oxygen species (ROS) as well as cytokines. Whereas both ROS and cytokines have the potential to regulate the expression of heat shock (HS)/stress proteins (HSP), it appears that these proteins in turn have the ability to protect cells and tissues from the deleterious effects of inflammation. The mechanisms by which such protection occurs include prevention of ROS-induced DNA strand breaks and lipid peroxidation as well as protection from mitochondrial structure and function. In vivo, HS protects organs against a number of lesions associated with the increased production of ROS and/or cytokines. In an animal model for adult respiratory distress syndrome, an acute pulmonary inflammatory condition, HS completely prevented mortality. HSP (hsp70 in particular) may also exert protective effects in the immune system by contributing to the processing and presentation of bacterial and tumoral antigens. The analysis of the expression of hsp70 may prove of diagnostic and prognostic value in inflammatory conditions and therapeutical applications are being considered.