Uno Johansson

The lysosomal protease cathepsin D mediates apoptosis induced by oxidative stress

It has been suggested that lysosomes and the lysosomal proteases cathepsin D and B act as proapoptotic mediators of apoptosis, in addition to mitochondrial release of cytochrome c and the activation of the caspase family of proteases. We found that cathepsin D was implicated in the onset of apoptosis in fibroblasts exposed to oxidative stress generated by redox cycling of naphthazarin (NZ)(5,8‐dihydroxy‐1,4‐naphthoquinon). At the start of NZ treatment, the intracellular reduced glutathione concentration was diminished and cathepsins D, B, and L were all translocated from lysosomes to the cytosol before any biochemical or morphological signs of apoptosis were detected. Increase in cathepsin D activity and in the level of p53 protein, a transcription factor for cathepsin D, was observed before activation of caspase‐3. Moreover, pretreatment with the cathepsin D inhibitor pepstatin A or the caspase‐3 inhibitor Ac‐DEV‐DCHO prevented apoptosis, although the increase of cathepsin D activity was still detected when caspase‐3 was inhibited. Cathepsin B activity decreased following oxidative stress, and inhibition of the protease did not affect the apoptotic process. We suggest that translocation of lysosomal proteases is an early event in NZ‐induced apoptosis and that the release and increased activity of cathepsin D allow this protease to exert an apoptosis‐mediating effect upstream of the caspase cascade.

Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential-sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (delta psi(m)) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of delta psi(m), and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

Lysosomal membrane permeabilization during apoptosis--involvement of Bax?

Bcl‐2 family members have long been known to control permeabilization of the mitochondrial membrane during apoptosis, but involvement of these proteins in lysosomal membrane permeabilization (LMP) was not considered until recently. The aim of this study was to investigate the mechanism underlying the release of lysosomal proteases to the cytosol seen during apoptosis, with special emphasis on the role of Bax. In human fibroblasts, exposed to the apoptosis‐inducing drug staurosporine (STS), the release of the lysosomal protease cathepsin D to the cytosol was observed by immunocytochemistry. In response to STS treatment, there was a shift in Bax immunostaining from a diffuse to a punctate pattern. Confocal microscopy showed co‐localization of Bax with both lysosomes and mitochondria in dying cells. Presence of Bax at the lysosomal membrane was confirmed by immuno‐electron microscopy. Furthermore, when recombinant Bax was incubated with pure lysosomal fractions, Bax inserted into the lysosomal membrane and induced the release of lysosomal enzymes. Thus, we suggest that Bax is a mediator of LMP, possibly promoting the release of lysosomal enzymes to the cytosol during apoptosis.

Cytosolic acidification and lysosomal alkalinization during TNF-α induced apoptosis in U937 cells

Apoptosis is often associated with acidification of the cytosol and since loss of lysosomal proton gradient and release of lysosomal content are early events during apoptosis, we investigated if the lysosomal compartment could contribute to cytosolic acidification. After exposure of U937 cells to tumor necrosis factor-α, three populations; healthy, pre-apoptotic, and apoptotic cells, were identified by flow cytometry. These populations were investigated regarding intra-cellular pH and apoptosis-associated events. There was a drop in cytosolic pH from 7.2 ± 0.1 in healthy cells to 6.8 ± 0.1 in pre-apoptotic, caspase-negative cells. In apoptotic, caspase-positive cells, the pH was further decreased to 5.7 ± 0.04. The cytosolic acidification was not affected by addition of specific inhibitors towards caspases or the mitochondrial F0F1-ATPase. In parallel to the cytosolic acidification, a rise in lysosomal pH from 4.3 ± 0.3, in the healthy population, to 4.8 ± 0.3 and 5.5 ± 0.3 in the pre-apoptotic- and apoptotic populations, respectively, was detected. In addition, lysosomal membrane permeability increased as detected as release of cathepsin D from lysosomes to the cytosol in pre-apoptotic and apoptotic cells. We, thus, suggest that lysosomal proton release is the cause of the cytosolic acidification of U937 cells exposed to TNF-α.

Adverse immunological effects and autoimmunity induced by dental amalgam and alloy in mice.

Dental amalgam fillings are the most important source of mercury exposure in the general population, but their potential to cause systemic health consequences is disputed. In this study, inbred mice genetically susceptible to mercury‐induced immune aberrations were used to examine whether dental amalgam may interfere with the immune system and cause autoimmunity. Female SJL/N mice were implanted in the peritoneal cavity with 8‐100 mg silver amalgam or silver alloy for 10 weeks or 6 months. Chronic hyperimmunoglobulinemia, serum IgG autoantibodies targeting the nucleolar protein fibrillarin, and systemic immune‐complex deposits developed in a time‐ and dose‐dependent manner after implantation of amalgam or alloy. Splenocytes from mice implanted with amalgam or alloy showed an increased expression of class II molecules. The functional capacity of splenic T and B cells was affected in a dose‐dependent way: 10 weeks of low‐dose and 6 months of high‐dose amalgam implantation strongly increased mitogen‐induced T and B cell proliferation, whereas 10 weeks of high‐dose implantation decreased the proliferation. Not only mercury but also silver accumulated in the spleen and kidneys after amalgam implantation. In conclusion, dental amalgam implantation in a physiological body milieu causes chronic stimulation of the immune system with induction of systemic autoimmunity in genetically sensitive mice. Implantation of silver alloy not containing mercury also induced autoimmunity, suggesting that other elements, especially silver, have the potential to induce autoimmunity in genetically susceptible vertebrates. Accumulation of heavy metals, from dental amalgam and other sources, may lower the threshold of an individual metal to elicit immunological aberrations. We hypothesize that under appropriate conditions of genetic susceptibility and adequate body burden, heavy metal exposure from dental amalgam may contribute to immunological aberrations, which could lead to overt autoimmunity.—Hultman, P., Johansson, U., Turley, S. J., Lindh, U., Enestrom, S., Pollard, K. M. Adverse immunological effects and autoimmunity induced by dental amalgam and alloy in mice. FASEB J. 8, 1183‐1190 (1994)

Ultraviolet A and B affect human melanocytes and keratinocytes differently. A study of oxidative alterations and apoptosis

Abstract:  Ultraviolet (UV) radiation is an etiologic agent for malignant melanoma and non‐melanoma skin cancer, but the spectral range responsible for tumor induction is still to be elucidated. In this study, we compared effects of UVA and UVB irradiation on normal human melanocytes (MCs) and keratinocytes (KCs) in vitro. We demonstrate that UVA irradiation induces immediate loss of reduced glutathione (GSH) in both MCs and KCs. Exposure to UVA also causes reduced plasma membrane stability, in both cell types, as estimated by fluorescein diacetate retention and flow cytometry. Furthermore, we noted reduction in proliferation and higher apoptosis frequency 24 h after UVA irradiation. UVB irradiation of KCs caused instant reduction of reduced GSH and impaired plasma membrane stability. We also found decline in proliferation and increased apoptosis after 24 h. In MCs, on the other hand, UVB had no effect on GSH level or plasma membrane stability, although increased apoptotic cell death and reduced proliferation was detected. In summary, MCs and KCs showed similar response towards UVA, while UVB had more pronounced effects on KCs as compared to MCs. These results might have implications for the induction of malignant melanoma and non‐melanoma skin cancer.

The Genotype Determines the B Cell Response in Mercury–Treated Mice

Background: Mercury causes in mouse strains of the H–2s haplotype an autoimmune syndrome with antibodies to the nucleolar protein fibrillarin and systemic immune complex (IC) deposits. Other strains, like BALB/C (H–2d), develop only IC deposits, and most strains are resistant. However, mercury activates the murine immune system and causes lymphoproliferation in most strains: H–2s strains are high–responders also in this respect, while the relation between lymphoproliferation and autoimmune manifestations is unclear for other strains. We examined the B cell response to mercury in order to better understand the relation between lymphoproliferation and systemic autoimmunity, using the high–responder H–2s strains (A.SW and SJL), the intermediate responder strain BALB/C (H–2d), and the A.TL (H–2tl) and DBA/2 (H–2d) strains which are resistant to systemic autoimmunity. Methods: During 4–7 weeks of subcutaneous mercuric chloride injections, the number of B cells and the expression of cell surface activation and proliferation markers was monitored by flow cytometry. The number of cytoplasmic Ig+ splenocytes was determined by direct immunofluorescence technique on slides, and serum Ig isotype levels as well anti–ssDNA and anti–DNP antibodies were determined by ELISA. Serum ANA were monitored weekly by indirect immunofluorescence technique. Results: Mercury–treated A.SW and SJL mice (H–2s) developed an increased expression of the proliferation marker CD71 on B cells, an increased number of B cells in the spleen, and an early, strong, but transient increase in serum Ig concentrations of Th1– as well as Th2–regulated Ig isotypes. Mercury–treated H–2s mice rapidly developed a polyclonal B cell response including the IgM isotype, but also antinucleolar antibodies (ANoA) of the IgG isotype with a clumpy pattern, characteristic for antifibrillarin antibodies. The IgG ANoA response was of a long duration and high titer. The A.TL strain (H–2tl) showed only a slight, restricted B cell activation. The BALB/C strain developed a slight, transient B cell activation dominated by IgG1 and IgE, and antinuclear antibodies (ANA). The DBA/2 strain showed only a minimal B cell response without ANA. Conclusion: Mercury induces an early, transient, polyclonal B cell activation linked to the H–2A or H–2K locus in H–2s strains on the A background. This polyclonal response differs from the long–lasting, high–titered IgG autoantibody response to a nucleolar antigen with characteristics of fibrillarin in H–2s strains, which indicates that these responses arise from separate mechanisms. Another group of strains, exemplified by BALB/C (H–2d), responds to mercury with a slight, transient, Th2–dominated B cell response, a restricted antibody specificity targeting the cell nucleus, and systemic IC deposits. Another H–2d strain, DBA, is essentially resistant to mercury, illustrating the importance of non–H–2 genes for regulating the response to mercury.

Murine Silver-Induced Autoimmunity: Silver Shares Induction of Antinucleolar Antibodies with Mercury, but Causes Less Activation of the Immune System

BACKGROUND Mercury and silver induce antinucleolar autoantibodies (ANoA) targeting the 34-kDa nucleolar protein fibrillarin in susceptible mouse strains. Mercury has the ability to cause a general activation of the immune system, but antigen-specific mechanisms following direct or indirect interaction between mercury and fibrillarin are now believed to play a crucial role in the autoimmune pathogenesis. Our previous studies showed that silver neither induced the systemic immune complex deposits nor the increase of serum immunoglobulins seen after mercury treatment. The main objective of this study was to examine the relation between activation of the immune system and the induction of ANoA. METHODS During 4 weeks of subcutaneous silver nitrate injections into mice of the susceptible A.SW and SJL strains and the resistant A.TL strain, the number of T and B cells as well as the expression of cell surface activation and proliferation markers were monitored by flow cytometry. The number of cytoplasmic Ig+ splenocytes was determined by direct immunofluorescence technique on slides, and serum Ig levels as well as anti-ssDNA anti anti-DNP antibodies were determined by ELISA. Serum ANoA were monitored by the indirect immunofluorescence technique. RESULTS Silver caused in the susceptible strains a weaker and later activation and proliferation of T and B cells than mercury, and no significant polyclonal B cell activation. In contrast, the ANoA titer was not different from that seen in mercury-treated mice of the same strains. Silver-treated mice of the A.TL strain showed neither activation of the immune system nor ANoA. CONCLUSIONS Despite being as effective as mercury inducing ANoA, silver caused only a slight activation of the immune system. This demonstrates that the massive activation of the immune system in mercury treatment is not necessary for the induction of ANoA, and indicates that (auto)antigen-specific mechanisms are likely to play a key role in mercury- and silver-induced murine autoimmunity.

Lipofuscin-induced autofluorescence of living neonatal rat cardiomyocytes in culture

Lipofuscin-specific autofluorescence of living, cultured rat neonatal myocardial myocytes was studied with respect to intensity and spectral characteristics. The autofluorescence emission spectrum changed over time indicating continuously ongoing intramolecular reorganisation of the pigment. Moreover, as compared with formaldehyde-fixed material native lipofuscin produced a considerable red-shifted autofluorescence, indicating formaldehyde-induced molecular modification or quenching of certain parts of the spectrum. The relationship between oxidative stress and lipofuscinogenesis was confirmed because the rate of lipofuscin accumulation was increased when cells were grown at increasing ambient oxygen concentrations.

Gold causes genetically determined autoimmune and immunostimulatory responses in mice

Natrium aurothiomaleate (GSTM) is a useful disease‐modifying anti‐rheumatic drug, but causes a variety of immune‐mediated adverse effects in many patients. A murine model was used to study further the interaction of GSTM with the immune system, including induction of systemic autoimmunity. Mice were given weekly intramuscular injections of GSTM and controls equimolar amounts of sodium thiomaleate. The effects of gold on lymphocyte subpopulations were determined by flow cytometry. Humoral autoimmunity was measured by indirect immunofluorescence and immunoblotting, and deposition of immunoglobulin and C3 used to assess immunopathology. Gold, in the form of GSTM, stimulated the murine immune system causing strain‐dependent lymphoproliferation and autoimmunity, including a major histocompatibility complex (MHC)‐restricted autoantibody response against the nucleolar protein fibrillarin. GSTM did not cause glomerular or vessel wall IgG deposits. However, it did elicit a strong B cell‐stimulating effect, including both T helper 1 (Th1)‐ and Th2‐dependent isotypes. All these effects on the immune system were dependent on the MHC genotype, emphasizing the clinical observations of a strong genetic linkage for the major adverse immune reactions seen with GSTM treatment.