Stefan Tukaj

Melatonin prevents ultraviolet radiation-induced alterations in plasma membrane potential and intracellular pH in human keratinocytes

Melatonin exhibits protective effects against ultraviolet radiation (UVR) via modulation of proinflammatory mediators and its free radical scavenging capacity. To date, several reports presented protective mechanisms of this agent against UVR‐induced alterations in mitochondria and nuclei. This investigation evaluates the potent preventing action of melatonin regarding early‐stage UVR‐mediated perturbations in plasma membrane potential (mbΔψ) and intracellular (cytosolic) pH (pH i) analyzed by flow cytometry. Experiments were carried out in a dose‐ and time‐dependent manner using human keratinocytes [HaCaT and normal human epidermal keratinocytes (NHEK)]. First investigations, which used viability/cytotoxicity assays, showed the gradual mortality with increasing UVR doses and cultivation time. Pre‐incubation with melatonin (10−3 m) prior to UVR exposure reduced lactate dehydrogenase release by 30% (HaCaT) and 28% (NHEK) at the dose of 50 mJ/cm2 after 48 hr (P < 0.001). Furthermore, UVR caused hyperpolarization of mbΔψ immediately (0 hr) after irradiation (25 or 50 mJ/cm2). At the dose of 50 mJ/cm2, cells cultivated for 48 hr manifested a marked increase in mbΔψ by 112% (HaCaT) and 123% (NHEK). The presence of melatonin significantly protected the cells by 12% (HaCaT) and 14% (NHEK) (P < 0.001). Simultaneously, 50 mJ/cm2 induced dramatic acidification reaching after 24 hr the level of 6.40 (without melatonin), 6.56 (with melatonin) for HaCaT and 6.11 (without melatonin), 6.43 (with melatonin) for NHEK. The results presented provide information about the protective mechanisms of melatonin itself on one hand and, combined with data reported so far, confirm the potent antiapoptotic action of melatonin.

Hsp40 proteins modulate humoral and cellular immune response in rheumatoid arthritis patients

Recent research on the heat shock proteins (Hsps) in chronic inflammatory diseases indicates that Hsps may have disease-suppressive activities. Our aim was to characterize immune response directed to bacterial (DnaJ) and human Hsp40s in patients with rheumatoid arthritis (RA). We found elevated levels of anti-DnaJ, anti-Hdj2, and anti-Hdj3 (but not ant-Hdj1) serum antibodies in the RA patients (P ≤ 0.001) compared to healthy controls. In peripheral blood mononuclear cells (PBMCs) culture, all tested Hsp40 proteins significantly inhibited the divisions of CD4+ and CD8+ T cells of the RA patients but not those of the controls. Both DnaJ and Hdj2 stimulated secretion of the main anti-inflammatory cytokine IL-10 by PBMCs of the RA patients (P < 0.05), and of IL-6 by PBMCs of the RA (P < 0.001) and control (P < 0.01) groups. DnaJ reduced TNFα secretion (P < 0.05) by both groups of PBMCs. Our results show for the first time that the RA patients have an increased humoral response to human Hsp40 proteins Hdj2 and Hdj3. This is also the first description of immunomodulatory effect of human Hsp40s on T cells and cytokine secretion in RA, suggesting that Hsp40s act as natural anti-inflammatory agents in RA.

Heat shock protein 90: a pathophysiological factor and novel treatment target in autoimmune bullous skin diseases

The chaperone heat shock protein 90 (Hsp90), a cell stress‐inducible molecule that regulates activity of many client proteins responsible for cellular growth, differentiation and apoptosis, has been proposed as an important therapeutic target in patients with malignancies. More recently, its active participation in (auto)immune processes has been recognized as evidenced by amelioration of inflammatory disease pathways through pharmacological inhibition of Hsp90 in rodent models of autoimmune encephalomyelitis, rheumatoid arthritis and systemic lupus erythematosus. Based on own current research results, this viewpoint essay provides important insights that Hsp90 is also involved as a notable pathophysiological factor in autoimmune blistering dermatoses including epidermolysis bullosa acquisita, bullous pemphigoid and possibly dermatitis herpetiformis. The observed in vitro, ex vivo and in vivo efficacy of anti‐Hsp90 treatment in experimental models of autoimmune bullous diseases and its underlying multimodal anti‐inflammatory mechanisms of interference with key contributors to autoimmune‐mediated blister formation supports the introduction of selective non‐toxic Hsp90 inhibitors into the clinical setting for the treatment of patients with these disorders.

Immunomodulatory effects of heat shock protein 90 inhibition on humoral immune responses

Heat shock protein 90 (Hsp90) inhibition blocks T‐cell‐linked inflammatory disease pathways and exhibits therapeutic activity in autoimmune disease mouse models, including the blistering disease epidermolysis bullosa acquisita. Although we previously showed that preformed autoreactive plasma cells do not seem to be directly affected by anti‐Hsp90 treatment, immunomodulatory effects of Hsp90 inhibition on (auto‐)antibody responses are not yet fully understood. In this study, the Hsp90 blocker 17‐DMAG inhibited proliferation of activated total B cells and their IgG secretion in cultures of human peripheral B cells from healthy subjects, but IgG production was no longer affected when these activated B cells were allowed to differentiate prior to a deferred application of the inhibitor. 17‐DMAG treatment was associated with induction of nuclear and cytoplasmic heat shock factor 1 and Hsp70 in stimulated human B cells, respectively. Type VII collagen (epidermolysis bullosa acquisita)‐immunized mice early treated with 17‐DMAG had reduced total B cells in spleens, a relative increase in splenic regulatory B cell fractions, higher serum IL‐10 concentrations, and lower levels of circulating autoantibodies (paralleled by less pronounced disease induction) compared with vehicle‐treated immunized mice. Autoantibody production was blunted in isolated and autoantigen‐restimulated lymph node cells from immunized mice by either 17‐DMAG or purified autologous splenic regulatory B cells. Thus, in addition to the previously described T cell inhibitory effects of Hsp90 blockade, this treatment potently modulates humoral immune responses at the B cell level, further supporting the introduction of Hsp90 inhibitors into the clinical setting for treatment of autoantibody‐mediated disorders.

Inhibitory effects of heat shock protein 90 blockade on proinflammatory human Th1 and Th17 cell subpopulations.

BackgroundHeat shock protein 90 (Hsp90), a chaperone that regulates activity of many client proteins responsible for cellular growth, differentiation, and apoptosis, has been proposed as an important clinical and preclinical therapeutic target in a number of malignancies and autoimmune diseases, respectively. In this study, we evaluated the effects of pharmacological Hsp90 inhibition on human proinflammatory T cell responses.FindingsUsing anti-CD3 antibody-stimulated human peripheral blood mononuclear cell cultures, we observed that Hsp90 inhibition by non-toxic concentrations of the geldanamycin derivative 17-DMAG significantly blocked T cell proliferation, reduced IFN-γ and IL-17 expression on CD4+ T lymphocytes, and arrested secretion of proinflammatory IFN-γ, TNF-α, and IL-17, cytokines characteristic of Th1 and Th17 cells, respectively. These effects were associated with inhibition of NF-kB activity, upregulation of Hsp70 protein expression, and disruption of T cell-specific nonreceptor tyrosine kinase Lck activation.ConclusionsOur results further support the potential use of Hsp90 inhibitors in patients with autoimmune diseases where uncontrolled Th1 or Th17 activation frequently occurs.

Aberrant expression and secretion of heat shock protein 90 in patients with bullous pemphigoid.

The cell stress chaperone heat shock protein 90 (Hsp90) has been implicated in inflammatory responses and its inhibition has proven successful in different mouse models of autoimmune diseases, including epidermolysis bullosa acquisita. Here, we investigated expression levels and secretory responses of Hsp90 in patients with bullous pemphigoid (BP), the most common subepidermal autoimmune blistering skin disease. In comparison to healthy controls, the following observations were made: (i) Hsp90 was highly expressed in the skin of BP patients, whereas its serum levels were decreased and inversely associated with IgG autoantibody levels against the NC16A immunodominant region of the BP180 autoantigen, (ii) in contrast, neither aberrant levels of circulating Hsp90 nor any correlation of this protein with serum autoantibodies was found in a control cohort of autoimmune bullous disease patients with pemphigus vulgaris, (iii) Hsp90 was highly expressed in and restrictedly released from peripheral blood mononuclear cells of BP patients, and (iv) Hsp90 was potently induced in and restrictedly secreted from human keratinocyte (HaCaT) cells by BP serum and isolated anti-BP180 NC16A IgG autoantibodies, respectively. Our results reveal an upregulated Hsp90 expression at the site of inflammation and an autoantibody-mediated dysregulation of the intracellular and extracellular distribution of this chaperone in BP patients. These findings suggest that Hsp90 may play a pathophysiological role and represent a novel potential treatment target in BP.

Heat shock protein 90 is required for ex vivo neutrophil‐driven autoantibody‐induced tissue damage in experimental epidermolysis bullosa acquisita

A broad range of immunosuppressive and immunomodulatory effects of heat shock protein 90 (Hsp90) blockade has been described in models of autoimmune bullous diseases, but the direct contribution of this chaperone to neutrophil effector pathways in the context of autoantibody‐driven blistering is generally unknown. Therefore, this has been addressed in the current study on the basis of the subepidermal blistering disease epidermolysis bullosa acquisita (EBA) characterized by autoantibodies against type VII collagen, in which a crucial role of neutrophils and both their reactive oxygen species and matrix metalloproteinases in mediating tissue injury has been established. First, the Hsp90 antagonist 17‐DMAG dose‐dependently inhibited dermal‐epidermal separation ex vivo in cryosections of human skin induced by co‐incubation of EBA patient autoantibodies with neutrophils from healthy blood donors. Next, 17‐DMAG dose‐dependently suppressed production and release of reactive oxygen species by human neutrophils induced by both fMLP ± LPS and EBA‐specific immune complexes. In addition, co‐immunoprecipitation studies revealed that extracellular Hsp90 interacted with secreted matrix metalloproteinases 2 and 12 in sera of EBA patients, suggesting that these basement membrane‐degrading proteolytic enzymes are client proteins of Hsp90 and dependent on its chaperone function. Our findings add to the knowledge of the multimodal anti‐inflammatory effects of Hsp90 blockade and implicate that Hsp90 is closely involved in the effector mechanisms of neutrophil‐driven autoantibody‐induced tissue damage, thus being a relevant therapeutic target in patients with neutrophil‐mediated autoimmune diseases such as inflammatory types of EBA.

Adaptation strategies of two closely related Desmodesmus armatus (green alga) strains contained different amounts of cadmium: a study with light-induced synchronized cultures of algae.

During the Desmodesmus armatus cell cycle, 8-celled coenobia of 276-4d strain accumulated a much lower amounts of cadmium than unicells of B1-76 strain. Cadmium reduced growth and photosynthesis in the cells of strain B1-76, but not those of 276-4d strain. Cells of 276-4d strain revealed a higher activity of superoxide dismutase (SOD) isoforms, in particular the activity and protein content of Fe-SOD. Cu/Zn-SOD was earlier and much stronger induced by cadmium in 276-4d than in B1-76 strain, whereas Fe- and Mn-SOD activity and Fe-SOD synthesis were induced only in 276-4d strain. Cadmium did not affect the heat shock protein 70 synthesis in B1-76 strain, but significantly stimulated this process in 276-4d strain. The level of glutathione increased 30-fold during cell development of Cd-exposed 276-4d strain, while in B1-76 it increased about 12 timed. Matured cells of both strains exposed to cadmium produced comparable amounts of phytochelatins and other thiol peptides, but their production in young cells of B1-76 strain was much higher than in 276-4d strain. In conclusion, a complex of internal detoxification mechanisms appeared to be more efficient in cells of 276-4d strain than B1-76 one.

Hsp90 blockade modulates bullous pemphigoid IgG-induced IL-8 production by keratinocytes

Bullous pemphigoid (BP) is the most common subepidermal autoimmune blistering skin disease characterized by autoantibodies against the hemidesmosomal proteins BP180 and BP230. The cell stress chaperone heat shock protein 90 (Hsp90) has been implicated in inflammatory responses, and recent evidence suggests that it represents a novel treatment target in autoimmune bullous diseases. The aim of the study was to investigate the contribution of Hsp90 to the proinflammatory cytokine production in keratinocytes induced by autoantibodies to BP180 from BP patient serum. HaCaT cells were treated with purified human BP or normal IgG in the absence or presence of the Hsp90 blocker 17-DMAG and effects on viability, interleukin 6 (IL-6) and IL-8 (cytokines critical for BP pathology), NFκB (their major transcription factor), and Hsp70 (marker of effective Hsp90 inhibition and potent negative regulator of inflammatory responses) were investigated. We found that BP IgG stimulated IL-6 and IL-8 release from HaCaT cells and that non-toxic doses of 17-DMAG inhibited this IL-8, but not IL-6 secretion in a dose- and time-dependent fashion. Inhibition of this IL-8 production was also observed at the transcriptional level. In addition, 17-DMAG treatment blunted BP IgG-mediated upregulation of NFκB activity and was associated with Hsp70 induction. This study provides important insights that Hsp90 is involved as crucial regulator in anti-BP180 IgG-induced production of keratinocyte-derived IL-8. By adding to the knowledge of the multimodal anti-inflammatory effects of Hsp90 blockade, our data further support the introduction of Hsp90 inhibitors into the clinical setting for treatment of autoimmune diseases, especially for BP.

Increased migratory properties of aortal smooth muscle cells exposed to calcitriol in culture.

The aim of the present study was to examine the effect of 1,25(OH)2D3 (calcitriol) on SMC (smooth muscle cell) migration, especially in the context to atherogenesis. SMCs were obtained from the aortas of newborn Wistar rats by enzymatic digestion. Different aspects of cell behavior during migration in culture were examined by phase contrast, fluorescence and electron microscopy (TEM, SEM) and supported by flow cytometric and biochemical analyses. Morphological studies revealed that supra-physiological (1-100 nmol/l) concentrations of calcitriol inhibit SMC differentiation, therefore these cells display several hallmarks of the synthetic state. Dynamic changes in actin cytoskeleton organization were a critical event in SMC shape, adhesion and spreading. Calcitriol diminished stress fibers assembly and focal adhesions formation. Reduced expression of beta1-integrin receptors on SMC surface after exposition to calcitriol coincided with increased proliferative and migratory activities of these cells. Moreover, after calcitriol stimulation, the ability of SMCs to the production of proinflamatory cytokines IFN-gamma, TNF-alpha and IL-6 was inhibited. The results from these comparative investigations indicate that 1,25(OH)2D3 inhibit differentiation and facilitate SMC migration in culture. It has been also suggested that such responses of SMCs to calcitriol play a beneficial role in fibrous cap formation during atherosclerotic process.