Interferon-beta induces brain-derived neurotrophic factor in peripheral blood mononuclear cells of multiple sclerosis patients.

Interferon-beta (IFN-beta) achieves its beneficial effect on multiple sclerosis (MS) via anti-inflammatory properties. In this study, we assessed the expression of the brain-derived neurotrophic factor (BDNF) in peripheral blood mononuclear cells (PBMC) from relapsing-remitting multiple sclerosis (RRMS) patients treated or not with IFN-beta. Intracellular BDNF was measured by Western blot and ELISA and compared with serum BDNF. We found higher levels of BDNF in PBMC of IFN-beta-treated versus non-treated patients, whereas serum levels of BDNF were similar. We hypothesize that the increased intracellular BDNF secondary to IFN-beta is not released in the periphery. This release is probably not tissue specific but in MS patients, BDNF could be specifically delivered by PBMC at the site of re-activation, i.e. within the central nervous system.

Inhibition of naive Th1 CD4+ T cells by glatiramer acetate in multiple sclerosis.

We investigated whether glatiramer acetate (GA) treatment may affect Th1 differentiation at various T-cell maturation stages. Specifically, we analyzed the effect of in vivo GA treatment on intracellular synthesis of IL-2 and TNF-alpha by naive, memory and effector CD4(+) and CD8(+) T cells by five-colour flow cytometry. Our data indicate that GA treatment downregulates/normalizes an accelerated Th1 differentiation of CD4(+) T cells in RRMS patients at all stages of T-cell maturation. Most notably, we conclude that, by altering naive, unprimed CD4(+) T cells, GA treatment appears to affect T-cell differentiation, at least in part, in an antigen-independent manner.

Role of the prohormone convertase PC3 in the processing of proglucagon to glucagon-like peptide 1.

Proglucagon is processed differentially in pancreatic alpha-cells and intestinal endocrine L cells to release either glucagon or glucagon-like peptide-1-(7-36amide) (tGLP-1), two peptide hormones with opposing biological actions. Previous studies have demonstrated that the prohormone convertase PC2 is responsible for the processing of proglucagon to glucagon, and have suggested that the related endoprotease PC3 is involved in the formation of tGLP-1. To understand better the biosynthetic pathway of tGLP-1, proglucagon processing was studied in the mouse pituitary cell line AtT-20, a cell line that mimics the intestinal pathway of proglucagon processing and in the rat insulinoma cell line INS-1. In both of these cell lines, proglucagon was initially cleaved to glicentin and the major proglucagon fragment (MPGF) at the interdomain site Lys70-Arg71. In both cell lines, MPGF was cleaved successively at the monobasic site Arg77 and then at the dibasic site Arg109-Arg110, thus releasing tGLP-1, the cleavages being less extensive in INS-1 cells. Glicentin was completely processed to glucagon in INS-1 cells, but was partially converted to oxyntomodulin and very low levels of glucagon in AtT-20 cells in the face of generation of tGLP-1. Adenovirus-mediated co-expression of PC3 and proglucagon in GH4C1 cells (normally expressing no PC2 or PC3) resulted in the formation of tGLP-1, glicentin, and oxyntomodulin, but no glucagon. When expressed in alphaTC1-6 (transformed pancreatic alpha-cells) or in rat primary pancreatic alpha-cells in culture, PC3 converted MPGF to tGLP-1. Finally, GLP-1-(1-37) was cleaved to tGLP-1 in vitro by purified recombinant PC3. Taken together, these results indicate that PC3 has the same specificity as the convertase that is responsible for the processing of proglucagon to tGLP-1, glicentin and oxyntomodulin in the intestinal L cell, and it is concluded that this enzyme is thus able to act alone in this processing pathway.

Identification and characterization of alpha 3 beta 1 integrin on primary and transformed rat islet cells.

Dispersed rat islet cells embedded in a matrix of collagen I are known to form aggregates in vitro reminiscent of native islets. Furthermore, it appears that islet function and survival are better maintained in vitro when cells are grown in the presence of extracellular matrix. These studies suggest an important role of cell--matrix interactions in the formation and maintenance of islet structure and function. The molecular basis of these interactions is mostly unknown. In the present study, we confirm the presence of beta 1 integrins on primary and transformed (RIN-2A line) rat islet cells. Perturbation studies in vitro show that beta 1 integrins play a role in islet cell attachment and spreading on bovine extracellular matrix and on the matrix produced by A-431 cells. The alpha 3 integrin subunit is coimmunoprecipitated with beta 1 from extracts of both primary and transformed islet cells, and immunodepletion studies suggest that alpha 3 beta 1 represents nearly half of the total beta 1 integrins expressed on primary islet cells. In situ, alpha 3 and beta 1 are expressed on the surface of all islet cell types, as shown by indirect immunocytochemistry on paraformaldehyde-fixed sections of rat pancreas. In conclusion, the study demonstrates the presence of alpha 3 beta 1 on primary and transformed rat islet cells, and an important role of beta 1 integrins in islet cell attachment and spreading in vitro.

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.

Phagocytosis of Pseudomonas aeruginosa fails to elicit heat shock protein expression in human monocytes.

Phagocytosis represents a powerful stress for the phagocytic cells. Phagocytosis of Staphylococcus aureus induces a stress response associated with the synthesis of specific heat shock/stress proteins (HSP). Here we investigated the stress response of human monocyte-macrophages (m phi) to Pseudomonas aeruginosa, a bacterium found, as for S. aureus, in the airways of patients suffering cystic fibrosis. P. aeruginosa activated in m phi the production of both extra- and intracellular O2-; increased Interleukin-1 beta and actin, but failed to induce host HSP. Neither S. aureus' exotoxins nor the scavenging property of P. aeruginosa's alginate, but the lower toxicity of P. aeruginosa and/or differential activation of proteine kinase C (PKC) by the two bacteria, might explain their differences in host HSP induction. While O2- is insufficient to induce HSP synthesis in m phi, hydroxyl radicals, generated in the presence of exogenous iron, is a likely additional signal, along with PKC activation, for HSP induction during bacterial phagocytosis.

Mitochondria are selective targets for the protective effects of heat shock against oxidative injury.

Heat shock (HS) proteins (HSPs) induce protection against a number of stresses distinct from HS, including reactive oxygen species. In the human premonocytic line U937, we investigated in whole cells the effects of preexposure to HS and exposure to hydrogen peroxide (H2O2) on mitochondrial membrane potential, mass, and ultrastructure. HS prevented H2O2-induced alterations in mitochondrial membrane potential and cristae formation while increasing expression of HSPs and the protein product of bcl-2. Protection correlated best with the expression of the 70-kDa HSP, hsp70. We propose that mitochondria represent a selective target for HS-mediated protection against oxidative injury.

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.

Infection of human and murine macrophages with Leishmania major is associated with early parasite heat shock protein synthesis but fails to induce a host cell stress response.

Heat shock/stress proteins (HSP) represent the most conserved proteins expressed in prokaryotes and eukaryotes. These constitutive and inducible proteins function as molecular chaperones and are part of virulence factors. They participate in self/non-self discrimination and may protect phagocytes from the toxic effects of the reactive oxygen species generated by these cells during bacterial phagocytosis and infection. In this study, we investigated the early stress response of host cells [either human alveolar macrophages (AM) or murine peritoneal macrophages (PM)] during infection by an obligate intracellular parasite (Leishmania major), which lives within phagolysosomes. Immunoblotting with specific antibodies demonstrated that L. major had no effect on host stress protein synthesis, but synthesized high levels of its own stress proteins within AM and PM. The lack of induction of a host cell stress response may relate to the failure of L. major to activate the respiratory burst in these cells, whereas the upshift of L. major HSP within macrophages is part of an adaptive response of the parasite to the host.

DNA damage and stress protein synthesis induced by oxidative stress proceed independently in the human premonocytic line U937.

Heat shock proteins (HSPs) function as stress-inducible molecular chaperones and exert protective effects against cellular injury. The induction of the HSPs is considered to be mediated by the presence of abnormal proteins within the cell and/or by classical second messengers. Several lines of evidence have however suggested a relationship between DNA damage, HSP induction and thermotolerance. We investigated whether DNA alterations could represent a common signal for the induction of stress protein synthesis during heat shock or exposure to reactive oxygen species in the human premonocytic line U937. We measured, in parallel, DNA damage (both strand breaks and fragmentation) and HSP synthesis (by biometabolic labeling and Western blotting) after exposure to heat shock, hydrogen peroxide, bleomycin, cadmium or erythrophagocytosis. Heat shock induced DNA alterations along with HSP synthesis. In contrast, exposure to hydrogen peroxide or bleomycin induced DNA damage, but no HSP synthesis, suggesting that oxidation-induced DNA damage and HSP synthesis proceed independently in U937 cells. Erythrophagocytosis and cadmium induced the classical HSPs but no detectable DNA damage. Since these latter stresses also induced the oxidation-specific stress protein heme oxygenase, we suggest a protective role for heme oxygenase against oxidative DNA damage.

Effects of the Bacterial Extract OM-85 on Phagocyte Functions and the Stress Response.

The effects of the bacterial extract OM-85 on the respiratory burst, intracellular calcium and the stress response have been investigated in human peripheral blood monocytes from normal donors. Activation of the respiratory burst during bacterial phagocytosis has been previously associated with heat shock/stress proteins synthesis. Whereas OM-85 stimulated superoxide production and increased Ca(2+) mobilization, it fared to induce synthesis of classical HSPs. The lack of stress protein induction was observed even in the presence of iron which potentiates both oxidative injury and stress protein induction during bacterial phagocytosis. However OM-85 induced a 75-78 kDa protein, which is likely to be a glucose regulated protein (GRP78), and enhanced intracellular expression of interleukin-lbeta precursor.

Spontaneous heat shock protein synthesis by alveolar macrophages in interstitial lung disease associated with phagocytosis of eosinophils.

Synthesis of heat shock proteins (HSPs) is induced in all cells and tissues after exposure to elevated temperatures, or a variety of other types of injury, including oxidative injury. We have previously reported that stress proteins are induced in monocytes-macrophages during phagocytosis of red blood cells. Receptor-mediated phagocytosis is associated with activation of the respiratory burst, generation of the lipid mediators of inflammation, and increased production of cytokines. Similar activation events have been described in the alveolar macrophage (AM) during pulmonary fibrosis. We therefore analysed the pattern of proteins synthesized by human AMs recovered by bronchoalveolar lavage (BAL) in interstitial lung disease, both under basal conditions and after in vitro exposure to heat or hydrogen peroxide (H2O2). In two out of the 17 cases studied, we observed a high alveolar eosinophilia (10 and 24%, respectively) and phagocytosis, by the AMs, of eosinophilic material. Whereas exposure to heat or H2O2 induced in all AMs the synthesis of the classical HSPs, in these two cases, we found spontaneous synthesis of HSPs and of a 32 kD oxidation-specific stress protein, haeme oxygenase (HO). Exposure of AM to purified eosinophil-derived proteins, such as major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil-derived neurotoxin (EDN), alone or in combination, did not induce stress protein synthesis, further suggesting that phagocytosis is involved in this induction. Stress protein synthesis by AMs may represent a new cellular marker of pulmonary injury and eosinophilic inflammation, and an autoprotective mechanism against oxidative stress.

Phagocytosis of Staphylococcus aureus induces a selective stress response in human monocytes-macrophages (M phi): modulation by M phi differentiation and by iron.

Phagocytosis of microorganisms represents a stress not only for the phagocytosed agent but also for the host cell. We have investigated the stress response induced in human monocytes-macrophages (M phi) phagocytosing inactivated Staphylococcus aureus. Exposure of human M phi to S. aureus induced in these cells (i) a threefold increase in superoxide dismutase activity, (ii) a selective and differentiation-dependent induction of host heat shock protein synthesis (HSP70 but not HSP65), and (iii) de novo synthesis of heme oxygenase, but only when exogenous iron was added to the cultures. The coordinate upregulation of two scavenging enzymes and of HSP70 suggests that all three are part of cellular protective mechanisms against phagocytosis-related oxidative injury to host cells.

Flavonoids, but not protein kinase C inhibitors, prevent stress protein synthesis during erythrophagocytosis.

Erythrophagocytosis induces in monocytes-macrophages the synthesis of stress proteins including the classical heat shock proteins (HSPs) and heme oxygenase (HO). To evaluate the role of oxygen radicals in this induction, we used the antioxidant flavonoids quercetin and kaempferol. These compounds inhibited HSP and HO synthesis, the latter being more sensitive. Quercetin and kaempferol also are inhibitors of protein kinase C (PKC). In order to determine whether inhibition of stress protein synthesis by flavonoids was mediated by their antioxidant properties or by PKC inhibition, we also tested more specific PKC antagonists, staurosporine and H-7. Staurosporine (SS) and H-7 decreased the synthesis of HSP70 and HSP83 but had no effect on HO. These data suggest that (1) erythrophagocytosis-related oxygen radicals are involved in the induction of the stress response in phagocytic cells, (2) the induction of HSPs and HO is differentially regulated, and (3) the effects of flavonoids on HO are linked to their scavenging activity rather than to PKC modulation.

Heat shock proteins: an autoprotective mechanism for inflammatory cells?

Heat shock/stress proteins are synthesized in all cell types under a variety of stressful conditions. Stress associated with ongoing inflammation relates, at least in part, to toxic products locally generated by cells accumulating in the inflamed tissue and organ. These products include oxygen free radicals, cytokines, proteases, chemotactic factors and, in the particular case of the eosinophil, toxic basic proteins. The heat shock response in inflammatory cells appears to be specifically regulated by their own proinflammatory products (oxygen free radicals, cytokines) generated during physiological functions such as phagocytosis, during differentiation, or in certain pathological states such as inflammatory lung diseases. We suggest that in human monocytes-macrophages heat shock proteins belong to the autoprotective equipment against oxidative stress.

Phagocytosis and heat shock response in human monocytes-macrophages.

The heat shock response represents a general physiological cellular reprogramming of gene expression secondary to exposure to multiple different stresses such as heat or oxygen free radicals. It is assumed that this response is turned on in order to protect cells, or to permit better recovery, from the detrimental effects of such stresses. During erythrophagocytosis, heat shock proteins and heme oxygenase synthesis are induced in human monocytes-macrophages. Whereas induction of heme oxygenase appears to be related to the hemoglobin-released iron, the factor(s) responsible for heat shock protein induction remain elusive. The respective roles of phagocytosis itself, oxygen free radicals, calcium, cytoskeletal alterations and oncogene expression are discussed.

Calcium and the heat-shock response in the human monocytic line U-937.

In the human monocytic line U-937, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] increases cytosolic free calcium concentration ([Ca2+]i). 1,25-(OH)2D3 also enhances the synthesis of heat-shock proteins (HSPs) when U-937 cells are exposed to elevated temperatures. To establish whether these two effects of 1,25-(OH)2D3 are related, we examined the effects of calcium on the heat-shock (HS) response, as well as the influence of 1,25-(OH)2D3 on this system. The equilibrium dissociation constant (Kd) of the fluorescent probe used to measure [Ca2+]i, fura-2, at 37 and 45 degrees C was found to be 191 and 234 nM, respectively. Exposure of U-937 cells to 45 degrees C did not increase [Ca2+]i under conditions in which active efflux of the dye was prevented by the organic anion transport inhibitor probenecid (1 mM). In cells preincubated in calcium-free medium, with subsequent addition of 4 mM EGTA before HS, or exposed to the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), the increase in HSPs synthesis was not affected. Cell viability, assessed by [3H]thymidine uptake, was not different between cells exposed to HS in calcium-containing or calcium-free media. Moreover, the effects of 1,25-(OH)2D3 on the HS response were also observed in a calcium-depleted medium, indicating that the effects of 1,25-(OH)2D3 on HSP synthesis were not mediated by [Ca2+]i.