Expression and localization of heat shock proteins in rat basophilic leukemia cells: differential modulation by degranulation, thermal or oxidative stress.

BACKGROUND: Rat basophilic leukemia (RBL-2H3) cells are well characterized in terms of morphological and biochemical changes upon activation, and have been extensively used as a model system for studying the mechanisms of the immediate hypersensitivity reaction. To investigate whether overexpression of heat shock/stress proteins (HSP) is involved in the mast cell-dependent reactivity, we examined the adaptive responses of RBL-2H3 cells to classical stress conditions such as heat shock or oxidative injury produced by an aqueous extract of tobacco smoke. METHODS: HSP were determined by flow cytometry and immunocytochemistry. Degranulation was confirmed as the release of beta-hexosaminidase, determined spectrophotometrically, and by electron microscopy experiments. RESULTS: We found that RBL-2H3 cells respond to heat shock or oxidative injury by the synthesis of both the inducible 72 kDa HSP (Hsp70), and the oxidation-specific 32 kDa heme oxygenase (HO)-1. Heat shock induced mainly Hsp70 in a cell growth-dependent manner, whereas oxidative stress induced mainly HO-1 in a cell growth-independent manner. However, heat shock or oxidative stress had no significant effects on degranulation. CONCLUSION: Stress-mediated synthesis of HSP was not associated with RBL-2H3 degranulation and likewise, degranulation did not induce HSP.

Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the Fas-mediated pathway.

The life cycle of many cell types can hinge on the presence of death factors that can control programmed cell death. The Fas-mediated apoptotic pathway has been implicated in controlling apoptosis during spermatogenesis in a number of mammalian species. In the human, the presence of nuclear DNA damage in ejaculated spermatozoa has pointed to a possible role for apoptosis during spermatogenesis. The presence of other molecular markers of apoptosis has, however, not been shown. More importantly, differences in these markers have not been investigated in men with normal and abnormal sperm parameters. In this study we examine for the presence of the cell surface protein Fas in ejaculated human spermatozoa. Ejaculated spermatozoa (55 samples) were labeled with anti-human Fas antibody and the number of spermatozoa displaying Fas were counted using a fluorescence-activated cell sorter (FACS). In 30/31 (96.8%) normal males (>20 million sperm per milliliter), less than 10% of the spermatozoa were Fas positive. In contrast, 14/24 (58.3%) oligozoospermic samples (<20 million sperm per milliliter) contained more than 10% Fas-positive spermatozoa. Similar differences were observed in men whose spermatozoa had poor motility and morphology. These results indicate that apoptosis is a major mechanism in regulating spermatogenesis in the human and that there are clear differences in molecular markers of apoptosis between males with normal and abnormal sperm parameters. We propose that the presence of Fas-labeled spermatozoa in the ejaculate of these men is indicative of an "abortive apoptosis" having taken place, whereby the normal apoptotic mechanisms have misfunctioned, have been overridden, or have not been completed.

Origin of DNA damage in ejaculated human spermatozoa.

The molecular basis of many forms of male infertility is poorly defined. One area of research that has been studied intensely is the integrity of the DNA in the nucleus of mature ejaculated spermatozoa. It has been shown that, in men with abnormal sperm parameters, the DNA is more likely to possess strand breaks. However, how and why this DNA damage originates in certain males and how it may influence the genetic project of a mature spermatozoon is unknown. Two theories have been proposed to describe the origin of this DNA damage in mature spermatozoa. The first arises from studies performed in animal models and is linked to the unique manner in which mammalian sperm chromatin is packaged, while the second attributes the nuclear DNA damage in mature spermatozoa to apoptosis. One of the factors implicated in sperm apoptosis is the cell surface protein, Fas. In this review, we discuss the possible origins of DNA damage in ejaculated human spermatozoa, how these spermatozoa arrive in the ejaculate of some men, and what consequences they may have if they succeed in their genetic project.

Flow cytometry is a rapid and reliable method for evaluating heat shock protein 70 expression in human monocytes.

The increasing interest in stress/heat shock proteins (Hsps) as markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37 degrees C. The heat-inducible increased Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.

The blood supply of the lumbrical muscles.

The blood supply of the lumbrical muscles was studied in 100 upper extremities from fresh human cadavers. Layer by layer dissection revealed the existence of different types of vascularity for the four muscles. The injection of coloured latex or Indian ink solution with gelatin showed the complex arterial network of these muscles together with their various sources of blood supply. Four separate sources of blood supply for each of the muscles were found: the superficial palmar arch (SPA), the common palmar digital artery, the deep palmar arch (DPA) and the dorsal digital artery. It was established that there were no anastomoses between the blood vessels of the tendons of the flexor digitorum profundus muscle and those of the lumbrical muscles. Considerable differences were observed in the details of the blood supply of the individual lumbrical muscles.

In-situ competition between protamine and fluorochromes for sperm DNA.

In this study we investigated the relationship between the presence of bound protamine on mouse and human sperm DNA and the level of chromomycin A3 (CMA3) and 4'6-diamidino-2-phenylindole (DAPI) fluorescence. This was accomplished by performing a competition assay between salmon protamine and fluorochromes on decondensed spermatozoa that had their nuclear proteins extracted and were fixed on slides. Various concentrations (0, 0.005, 0.0225, 0.05, 0.225, 0.5 and 5 mg/ml) of salmon protamine were added to either the CMA3 or DAPI staining solutions. Fluorescence emission measurements of stained sperm nuclei were then performed using a microfluorometer. When the treated decondensed sperm heads were stained with either CMA3 or DAPI all spermatozoa were found to fluoresce intensely. The addition of protamines to the spermatozoa led to an elimination of CMA3 fluorescence, while the intensity of DAPI staining was decreased to approximately 50% at the highest concentrations of protamine. The addition of increasing amounts of salmon protamine also induced the sperm nuclei to regain their initial condensed appearance. This study shows that protamine retains a strong affinity for sperm DNA in situ and that CMA3 fluorescence is a strong indicator of the protamination state of spermatozoa.

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.

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.

Heat-shock proteins in host-pathogen interactions: implications for cystic fibrosis.

The expression of heat-shock proteins by both pathogen and host cells during the phagocytosis of Staphylococcus aureus and Pseudomonas aeruginosa, two bacterial species that colonize the airways of patients with cystic fibrosis, probably contributes to pulmonary inflammation in cystic fibrosis. Here, we discuss the likely signals for heat-shock-protein induction within host and bacterial cells.

Exposure of monocytes to heat shock does not increase class II expression but modulates antigen-dependent T cell responses.

Expression of heat shock (HS) proteins (HSP) increases after exposure to elevated temperatures or other types of injury, such as oxidative injury. Because of their function as 'molecular chaperones', HSP are suggested to participate in antigen processing and presentation. We have previously reported that HS modulates antigen presentation in a human EBV-transformed B cell line. Here we investigated the effects of HS on MHC class II expression and on antigen processing and presentation by human monocytes. Monocytes were isolated from peripheral blood of normal human volunteers, purified by adherence, then exposed to temperatures ranging from 37 to 45 degrees C for 20 min, allowed to recover for 2 h at 37 degrees C and used for immunofluorescence or as antigen presenting cells in autologous and heterologous lymphocyte proliferation assays. No increase in class II expression was detected as assessed by flow cytometry. Monocytes (3 x 10(4)) and lymphocytes (1 x 10(5)) were co-cultured for 5 days in the presence of several antigens [diphtheria toxoid, tetanus toxoid or purified peptide derivative (PPD)] and labeled with 1 microCi [3H]thymidine for 16 h. Pre-exposure to HS (44 degrees C) significantly (P < 0.001) increased T cell responses to diphtheria toxoid, whereas the effect on the responses to other antigens (tetanus toxoid or PPD) were not significant. HS did not increase heterologous T cell responses nor T cell proliferation induced by the non-processed superantigens such as staphylococcal enterotoxin B. The effect of HS was inhibited by actinomycin B and thus appeared dependent upon HSP synthesis. HSP-mediated increases in antigen processing may potentiate the ongoing immune response at inflammatory sites.

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.