Differential expression of the IL-1 system components during in vitro myogenesis: implication of IL-1beta in induction of myogenic cell apoptosis.

We evaluated the expression of IL-1 system by normal human myogenic cells during in vitro myogenesis and the effect of exogenous IL-1beta. Expression of IL-1alpha and beta, IL-1 receptor antagonist (IL-1Ra), IL-1RI and II, IL-1R accessory protein (IL-1RAcP) and IL-1beta converting enzyme (ICE) was studied by immunocytochemistry, immunoblotting, ELISA and RT - PCR. Cell proliferation was evaluated by [3H]thymidine incorporation, cell fusion by flow cytometry and cell death by in situ end-labelling. Human normal myogenic cells constitutively produced IL-1beta and ICE, with a maximum expression at time of cell fusion. IL-1Rs and IL-1RAcP expression reached a peak at time of commitment to fusion. Myogenic cells produced small amounts of IL-1Ra at latest stages of culture, and only the intracellular isoform. Exposure of cultures to exogenous IL-1beta (1-5 ng/ml) induced myogenic cell apoptosis, without effect on cell proliferation or fusion. IL-1beta-induced cell death was associated with morphological changes including spreading appearance of cells and alteration of cell alignment. We conclude that (1) human myogenic cells constitutively produce IL-1beta; (2) IL-1 system components are differentially expressed during in vitro myogenesis; (3) IL-1 system participates to the coordinated regulation of cell density during normal myogenesis, which could serve to control the muscle mass in vivo.

Requirement of either the NH4Cl-sensitive or the cytochalasin D-sensitive pathway for ricin toxicity depends upon the enterocytic state of differentiation of HT-29 cells.

During the course of the present biochemical and ultrastructural studies, we found that the expression of either the undifferentiated or the differentiated HT-29 cell phenotype determined the intracellular fate of ricin. Although the recognition of ricin at the cell surface required interaction with the galactose-binding site on both cell populations, the lag time before ricin started to inhibit protein synthesis was longer in the differentiated than the undifferentiated cells. Dose-response studies and "time-addition" experiments performed with NH4Cl, which raises the pH of acidic vesicles and organelles, showed that ricin uptake as well as the movement of the toxin to the translocation site were affected in the differentiated cells. In contrast, NH4Cl acted on only post-internalization events in the undifferentiated cells. When the addition of cytochalasin D, an actin-depolymerizing drug, was staggered, the differentiated cells were found to be protected against ricin only during the very early stage of the internalization process. In contrast, the undifferentiated cells were protected during both the early and late stages of endocytosis. Moreover, electron microscopic examination showed that cytochalasin D altered the structure of the Golgi apparatus only in the undifferentiated cells. 3-Methyladenine, a specific inhibitor of the autophagic pathway, protected the undifferentiated and differentiated cells against ricin to about the same extent. We concluded that to enter the differentiated cells, ricin followed the classical endosome-Golgi pathway. In contrast, in the undifferentiated cells, ricin reaches the cytosol by two distinct routes: the minor one involves the endosome-Golgi pathway; the major one involves a cytochalasin D-sensitive pathway.

Interleukin (IL)-1 beta and IL-1 beta mRNA expression in normal and diseased skeletal muscle assessed by immunocytochemistry, immunoblotting and reverse transcriptase-nested polymerase chain reaction.

To confirm the production of IL-1 beta and to optimize detection and semiquantitation of IL-1 beta mRNA by polymerase chain reaction (PCR) techniques in skeletal muscle tissue, immunocytochemistry, immunoblotting and several procedures of RNA extraction and reverse transcription (RT)-PCR amplification were used on muscle samples from 12 patients with conditions associated with local production of IL-1 beta (AZT myopathy: 6 patients; sarcoid myopathy: 6 patients) and from 9 patients with normal muscle used as controls. Abundant IL-1 beta immunoreactivities, corresponding to both pro IL-1 beta and mature IL-1 beta as assessed by immunoblotting, were observed in all diseased muscles, either in inflammatory cells (sarcoid myopathy) or in atrophic muscle fibers (AZT myopathy). Acid guanidinium isothiocyanate phenol-chloroform extraction of RNA appeared less efficient for IL-1 beta mRNA detection by RT-PCR than proteinase K digestion followed by phenol-chloroform extraction. Even using the latter procedure, RT-single PCR for IL-1 beta mRNA was puzzlingly negative in all cases but one; in contrast, RT-nested PCR specified by DNA enzyme immunoassay yielded detection of IL-1 beta mRNA in all diseased muscles and in occasional controls, including the expected PCR product of 391 bp, but also another product of 935 bp, corresponding to IL-1 beta mRNA with unsplicing of the fourth intron. Semi-quantitative PCR showed that production of IL-1 beta mRNA was higher in sarcoid myopathy than in AZT myopathy, and in AZT myopathy than in controls. In conclusion, IL-1 beta expression can be reliably studied using immunocytochemistry, but assessment of IL-1 beta mRNA production in muscle tissue requires optimized extraction and RT-PCR procedures.

IFN-beta decreases adhesion and transmigration capacities of lymphocytes in Guillain-Barré syndrome.

The adhesion capacities, transmigration capacities, and integrin expression of lymphocytes from patients with Guillain-Barré syndrome incubated with interferon-beta were studied. Interferon-beta induced a dose-dependent inhibition of lymphocyte adhesion to recombinant vascular adhesion molecule-1 (p < 0.0001) and recombinant intercellular adhesion molecule-1 (rICAM-1) (p < 0.01) without modulation of very late activation molecule-4 and lymphocyte function-associated antigen-1 expressions and a dose-dependent decrease of lymphocyte transmigration across fibronectin (p < 0.0001). Inhibition of adhesion to rICAM-1 was similar after long (18 hours) or short (5 minutes) incubation time. These results support the potential therapeutic benefit of interferon-beta in Guillain-Barré syndrome.

Inhibition of the adhesion step of leukodiapedesis: a critical event in the recovery of Guillain-Barré syndrome associated with accumulation of proteolytically active lymphocytes in blood.

Intraneural inflammation, that reflects emigration of immune cells from blood to nerve tissue, is a critical event in Guillain-Barré syndrome pathogenesis. To investigate the adhesion and transmigration phases of leukodiapedesis, we determined in a series of patients with GBS: (1) circulating levels of soluble forms of adhesion molecules (sICAM-1 and sVCAM-1); (2) attachment capacities of circulating lymphocytes to rICAM-1 and rVCAM-1; (3) fibronectin-penetrating capacities of circulating lymphocytes; and (4) lymphocyte intracellular concentrations of MMP-9 at the different phases of GBS and in healthy controls. Circulating levels of sVCAM-1 and sICAM-1 were above normal values at the time of progression, markedly increased at the time of plateau (sVCAM-1: P<0.03; sICAM-1: P<0.02), and tended to normalize during recovery. The percentage of cells with attachment capacities to rVCAM-1 and to rICAM-1 decreased from progression to recovery by 30 and 31%, respectively (P<0.02). The number of circulating lymphocytes with fibronectin penetrating capacities was lower than controls at the time of progression (P<0.01), then progressively increased to reach values higher than controls at the time of late recovery (P<0.02). Cellular concentrations of MMP-9 in circulating lymphocytes paralleled their fibronectin penetrating capacities. These results suggest early emigration of lymphocytes into nerve, followed by shedding of adhesion molecules from endothelium, and late decrease of lymphocyte adhesion capacities. Plateau and recovery are associated with accumulation in the vascular compartment of still proteolytically active lymphocytes that can no longer adhere to endothelial cells. Modulation of the adhesion step of leukodiapedesis may be crucially involved in the switch from progression to plateau of GBS.

Monocyte chemoattractant protein 1 and chemokine receptor CCR2 productions in Guillain-Barré syndrome and experimental autoimmune neuritis.

Infiltration of activated lymphocytes and monocytes is a key phenomenon in the pathogenesis of Guillain-Barré syndrome (GBS) and experimental autoimmune neuritis (EAN). To investigate the role of chemokines, we determined the blood and nerve tissue expression of monocyte chemoattractant protein 1 (MCP-1), a major chemoattractant of monocytes and activated lymphocytes, and its receptor CCR2 in GBS and EAN. MCP-1 circulating levels (ng/ml) in GBS were increased at the time of progression, peaked at the time of plateau and normalized with recovery. MCP-1 circulating levels were the highest in the most disabled patients. The number of circulating CCR2 positive cells was lower in patients with GBS than in healthy subjects (p<0.004). In GBS, MCP-1 expression was observed in epineurial and endoneurial vessels, on infiltrating cells, Schwann cells and in the endoneurial extracellular matrix. Some CCR2 positive cells were observed in nerve biopsies of GBS patients. In EAN, a slight positivity for MCP-1 was observed in the sciatic nerve. There was no circulating CCR2 positive cells. However, at the time of plateau, a conspicuous infiltration of CCR2 positive cells was observed in the sciatic nerve that was no longer observed at the time of recovery. These results suggest that MCP-1 and CCR2 may participate to the recruitment of circulating mononuclear cells in nerve tissue in EAN and GBS.

Quality control of coated antibodies: new, rapid determination of binding affinity.

A procedure is described for the determination of the affinity constant between a fluid-phase biotinylated antigen and a solid-phase monoclonal antibody. This procedure allows evaluation of the efficiency of an antibody as a coated tool for an immunoassay. For this purpose, the biotinylation of the antigen and its further quantitative measurement by streptavidin-peroxidase led to a single reversible interaction, the binding affinity of which greatly determines the quality of the assay. The free and bound fractions of the biotinylated antigen were obtained in wells coated with a low level of immobilized antibodies. At the equilibrium state, the free antigen present in the supernatant of these wells was further transferred to high level antibody coated wells which captured all the free antigen molecules. These molecules were quantified using a standard curve established with known concentrations of biotinylated antigen, also incubated in wells coated with the high level of antibody.

Ricin toxicity and intracellular routing in tumoral HT-29 cells. I. Ricin routing and toxicity are related to the state of differentiation of HT-29 cells.

We previously showed that ricin, which is more cytotoxic to undifferentiated than to differentiated tumoral HT-29 cells, enters these cells by different routes. The final steps of ricin endocytosis were investigated in order to identify the translocation site from which ricin exerts its toxicity. Toxicity measurements and kinetic experiments followed by subcellular fractionation were run in parallel. In differentiated cells, from 20 min of internalization, radiolabeled ricin was found in a Golgi-enriched fraction. At 60 min, which corresponds to the lag time for ricin toxicity, the amount of radioactivity located in this fraction decreased without any concomitant increase in the other fractions. In undifferentiated cells, from 20 min of incubation, radiolabeled ricin was detected in the ER-enriched fractions. At 30 min, the lag time for ricin toxicity, the amount of radioactivity detected in these fractions decreased without any concomitant increase in the Golgi-enriched fraction. Monensin, which was used to confirm the passage of ricin through the Golgi, greatly increased ricin toxicity and diminished the lag time only in differentiated cells. Brefeldin A inhibited ricin toxicity when added before the end of the lag time in both cell populations and reduced the amount of ricin detected, respectively, in the Golgi- and ER-enriched fractions in differentiated and undifferentiated cells. We propose that ricin enters the cytosol from the Golgi apparatus and essentially from the ER in differentiated and undifferentiated HT-29 cells, respectively, and that these different intracellular routings might explain the differential toxicity of ricin.

Organization of the endoplasmic reticulum-Golgi system is related to the state of enterocytic differentiation of human HT-29 cells.

The organization of the endoplasmic reticulum (ER)-intermediate compartment (IC)-Golgi system was studied in tumoral HT-29 cells. Depending on the culture conditions, these cells are either undifferentiated or exhibit enterocytic differentiation after reaching confluence. In differentiated HT-29 cells, these organelles were organized as in most cell types. They displayed a classical structure and appeared associated with microtubules, as nocodazole altered both their structure and intracellular localization. Likewise, membrane dynamics of the Golgi appeared normal: as in many other cells, brefeldin A (BFA) induced retrograde transport from the Golgi to the ER, demonstrated by tubulation of the Golgi elements and shift of the galactosyltransferase activity from the Golgi- to the RER-enriched fraction, isolated by subcellular fractionation. In contrast, atypical features were observed in undifferentiated HT-29 cells: the Golgi structure exhibited abnormal swellings; the IC elements were very rare. Only cytochalasin D altered the structure and intracellular localization of the three organelles, suggesting that they were associated with microfilaments instead of microtubules. The membrane dynamics were unusual: brefeldin A led to a vesiculation of the Golgi elements with a slowed-down retrograde transport of galactosyltransferase. HT-29 cells engaged in the differentiation process, but which were still undifferentiated, showed mainly the features of undifferentiated cells, with a few characteristics of differentiated cells. These results indicate that the structure of the Golgi apparatus, IC and ER, their relationships to cytoskeletal elements and membrane dynamics depend on the state of differentiation of HT-29 cells. Although they are tumoral, differentiated HT-29 cells exhibit features observed in non-tumoral polarized epithelial cells. On the contrary, undifferentiated HT-29 cells display important abnormalities that may be related to their metastatic properties.

Atypical microtubule organization in undifferentiated human colon cancer cells.

We previously reported that undifferentiated colonic cancer HT-29 cells, unlike the differentiated ones, exhibit unusual organelle distributions and atypical vesicle trafficking patterns, which are microtubule-independent and microfilament-dependent. In the present study, we have analyzed the microtubule network in both phenotypes, using confocal microscopy, and determined the expression levels of some microtubule-associated proteins by quantitative immunoblotting. Differentiated cells exhibited the microtubular organization of polarized epithelial cells. Non-polarized undifferentiated cells presented an atypical microtubule organization as microtubules were localized mainly at the cell 'top'. Immunoblot analysis indicated the absence or low content of several structural and motor microtubule-associated proteins in undifferentiated cells, compared to differentiated cells. This may explain in part their atypical microtubular organization. This study agrees with a crucial role for microfilaments in the intracellular organization of undifferentiated HT-29 cancer cells, while differentiated HT-29 cells exhibit intracellular organization similar to that of normal enterocytic cells, although they are also tumoral.

Involvement of the [uPAR:uPA:PAI-1:LRP] complex in human myogenic cell motility.

The urokinase-type plasminogen activator system is a proteolytic system involved in tissue remodeling and cell migration. At the cell surface, receptor (uPAR)-bound urokinase (uPA) binds its inhibitor PAI-1, localized in the matrix, and the complex is internalized by endocytic receptors, such as the low-density lipoprotein receptor-related protein (LRP). We previously proposed a nonproteolytic role for the uPA system in human myogenic cell differentiation in vitro, i.e., cell fusion, and showed that myogenic cells can use PAI-1 as an adhesion matrix molecule. The aim of this study was to define the role of the uPA system in myogenic cell migration that is necessary for fusion. Using a two-dimensional motility assay and microcinematography, we showed that any interference with the [uPAR:uPA:PAI-1] complex formation, and interference with LRP binding to this complex, markedly decreased myogenic cell motility. This phenomenon was reversible and independent of plasmin activity. Inhibition of cell motility was associated with suppression of both filopodia and membrane ruffling activity. [uPAR:uPA:PAI-1:LRP] complex formation involves high-affinity molecular interactions and results in quick internalization of the complex. It is likely that this complex supports the membrane ruffling activity involved in the guidance of the migrating cell toward appropriate sites for attachment.

Ricin toxicity and intracellular routing in tumoral HT-29 cells. II. Differential ricin toxicity from the apical and basolateral surfaces of differentiated HT-29 cells.

We previously showed that ricin, a toxin commonly used in the construction of immunotoxins, was more toxic to undifferentiated than to differentiated HT-29 tumoral cells. This results from differences in the intracellular routings of the toxin. As these studies concerned the entry through the apical pole of differentiated polarized HT-29 cells, we investigated and compared the intracellular routing of ricin from the apical and basolateral membranes of differentiated HT-29 cells and the toxicity of ricin depending on the pole of administration. For this purpose, we developed the culture of polarized HT-29 cells on porous membrane filters and demonstrated that differentiated HT-29 cells can establish a leakproof monolayer. Ricin is 2.5-fold less toxic when it is added at the basolateral than at the apical pole of the cells, which may result from different observations: (1) less ricin is bound at the basolateral membrane than at the apical one, leading to a lesser internalization of the toxin; (2) ricin sorting in the apical and basolateral endocytic compartments of HT-29 cells differs: apically internalized ricin is targeted intracellularly while basolaterally internalized ricin uses mainly the transcytotic pathway; using NH4Cl and monensin, we observed that ricin follows the same pathway from both sides of the cells, namely the endosomal system, to reach the Golgi apparatus from which toxin intoxication occurs; (3) kinetics studies showed that a delay exists before an efficient intoxication by the basolateral pole is observed. The use of monensin at low concentration in order to perturb only the Golgi functions indicated that this delay could account for a different presentation of the toxin toward the membrane of the apical and basolateral endocytic compartments. Together, our results showed that, in differentiated HT-29 cells, if the pathways carrying ricin from the apical and basolateral membranes to the Golgi apparatus appear identical, ricin exerts differentially its toxicity depending upon the surface of administration, i.e., the apical or the basolateral surface of the cells.

Differential intracellular distribution and activities of mu- and m-calpains during the differentiation of human myogenic cells in culture.

Calpains are intracellular calcium-dependent cystein proteases active at neutral pH. There have been found in human adult myogenic cells (i.e. satellite cells) 2 forms of calpains requiring either micromolar Ca2+: mu-calpain, or millimolar Ca2+: m-calpain. Calpains could be involved in both intracellular proteolysis and cytoskeleton reorganization required for myogenic cell fusion. We showed significant differences in calpains distribution during differentiation of myogenic cells. Using mono- and polyclonal antibodies against both types of calpains, we localized mu-calpain and m-calpain in cultured human satellite cells. mu-calpain was detected in the nuclei of myoblasts and in the cytoplasm of myotubes. m-calpain was only present in the cytoplasm, and was concentrated near the nuclear envelope. Biochemical assays for calpain activities showed that the amounts of these proteinases were modulated during cell growth and differentiation. m-calpain activity was high at the proliferation phase (day 4 of culture) and reached a maximum with the beginning of fusion (day 8) and decreased slightly when the number of myotubes increased (day 12). This activity profile suggests that m-calpain could play a role in the initiation of fusion of satellite cells. The activity of mu-calpain increased regularly with cell growth, the maximum being reached when the cells differentiate, i.e. when its intracellular localization shifted from the nucleus to the cytoplasm. We conclude that the activity and the intracellular localization of the 2 forms of calpains differ with the state of differentiation of myogenic cells.

In vitro evaluation of human muscle satellite cell migration prior to fusion into myotubes.

We developed a short-time assay to evaluate muscle satellite cell migration, based on the fact that during myogenic differentiation, myoblasts migrate preferentially towards high cellular density areas where myotubes would form. This assay consists of a computer-assisted count of cells within a randomly chosen field, performed every hour for eight hours, and compared with the cell number at the start time of the experiment. Nine primary myoblast cultures were tested in triplicate. The method relies on several requisites. (1) Negligible cell proliferation: cell division was nearly absent in 8 h experiments. (2) Directional cell movement: a major flow of cells, either entering or exiting the fields, was constantly observed. 'Counter-flows', detected by visual counting, involved minor percentages of cells. (3) Constant migration rate: a linear increase in cell count variations over 8 h and a very high degree of intra-assay homogeneity were observed. Individual primary cell culture characteristics (depending on characteristics of the different donors) were the sole factor with a significant impact on migration rate. Automatic cell counting conveniently assessed the inhibitory effect of GRGDTP, an inhibitor of integrin-mediated cell adhesion. The method described here is rapid, does not require heavy equipment, and allows studies under serum-free conditions required to test molecules interfering with cell migration, in the course of the in vitro myogenic process.

Interleukin-1 expression in inflammatory myopathies: evidence of marked immunoreactivity in sarcoid granulomas and muscle fibres showing ischaemic and regenerative changes.

The most frequent autoimmune adult inflammatory myopathies are dermatomyositis, polymyositis, inclusion body myositis, and sarcoid myopathy. Interleukin-1 (IL-1) is a pleiotropic molecule, implicated in the inflammatory process, but also in tissue protection and remodelling. We evaluated the immunocytochemical expression of [L,-1alpha and beta in frozen muscle biopsy specimens from patients with dermatomyositis (15 cases), polymyositis (five cases), inclusion body myositis (five cases) and sarcoid myopathy (five cases). Positive immunoreactivities, were observed in both inflammatory cells and muscle fibres. Specificity of the immunostaining was assessed by Western blot experiments. IL-1 positive inflammatory cells were rare in polymyositis and inclusion body myositis, moderately abundant in dermatomyositis, and prominent in sarcoid myopathy granulomas. In sarcoid myopathy, 24.6 +/- 4.1% inflammatory cells were IL-1alpha-positive and 45.2 +/- 2.6% were IL-1beta-positive. IL-1 positive muscle fibres were mainly observed in dermatomyositis, usually remote from inflammatory infiltrates. Positive immunostaining for IL-1 was observed in fibres showing ischaemic punched-out vacuoles, that correspond to areas of myosinolysis, in atrophic perifascicular fibres, and in fibres located within healing microinfarcts. All NCAM-positive regenerating fibres were IL-1 positive. We conclude that: (i) IL-1 is expressed in granulomas of sarcoid myopathy, which is in keeping with the role ascribed to IL-1 in the formation of granulomas: (ii) IL-1 is expressed by muscle fibres undergoing ischaemic damage: and (iii) IL-1 expression by muscle fibres is associated with myofibrillar protein breakdown and regeneration.

Interleukin-1 expression in normal motor endplates and muscle fibers showing neurogenic changes.

Histological features of neurogenic muscle involvement include type grouping, muscle fiber atrophy and target fibers. In zidovudine-induced myopathy and dermatomyositis, immunoreactivity for interleukin (IL)-1 has been reported in diseased muscle fibers involving myofibrillar breakdown and atrophy. Since IL-1 is a signal for muscle proteolysis, we studied myofiber expression of IL-1 in neurogenic muscle involvement, specially in atrophic myofibers and target fibers which are associated with myofilament breakdown. Muscle biopsy samples from patients with normal (5 cases) or neurogenic muscle involvement (25 cases) were studied by enzyme histochemistry and immunohistochemistry. In normal muscles, immunoreactivity for IL-1beta was restricted to the postsynaptic domain of motor endplates and that for IL-1alpha had a similar localization but was faint. Immunoreactivity for IL-1alpha and -beta was observed, respectively, in 42.5% and 75.5% of target fibers, in 8.5% and 10.4% of dark angulated fibers, in 0% and 0.3% of non-atrophic type-grouped fibers, in 14.2% and 16.5% of moderately atrophic fibers, and in 65% and 20.9% of severely atrophic fibers. Immunoblot study showed the presence of both proIL-1 (31 kDa) and mature IL-1 (17.5 kDa). From this study, we conclude that IL-1 is normally expressed in the muscular domain of neuromuscular junctions; that IL-1 is mainly expressed in neurogenic target fibers; and that IL-1 expression by muscle fibers in pathological conditions seems to be associated with myofibrillar protein breakdown and regeneration.

Zidovudine-induced mitochondrial disorder with massive liver steatosis, myopathy, lactic acidosis, and mitochondrial DNA depletion.

Zidovudine is known to be responsible for a mitochondrial myopathy with ragged-red fibres and mitochondrial DNA depletion in muscle. Lactic acidosis alone or associated with hepatic abnormalities has also been reported. A single report mentioned the concomitant occurrence of muscular and hepatic disturbances and lactic acidosis in a patient receiving zidovudine, but muscle and liver tissues were not studied. A 57-year-old man with AIDS, who had been treated with zidovudine for 3 years, developed fatigue and weight loss. Serum creatine kinase and hepatic enzyme levels were high. Lactic acidosis was present. Liver biopsy showed diffuse macrovacuolar and microvacuolar steatosis. After withdrawal of zidovudine, creatine kinase, aspartate aminotransferase, and alanine aminotransferase levels normalised within 5 days, and lactacidaemia decreased. Acidosis persisted. The patient became confused and febrile and died 8 days after detection of high blood lactic acid. A muscle sample obtained at autopsy showed mitochondrial abnormalities with ragged-red fibres and lipid droplet accumulation. Southern blot analysis showed depletion of mitochondrial DNA, affecting skeletal muscle and liver tissue. No depletion was found in myocardium and kidney. This case emphasises that zidovudine treatment can induce mitochondrial multisystem disease, as revealed in our case by myopathy, liver steatosis and lactic acidosis.