Interactions between proteins implicated in exocytosis and voltage-gated calcium channels

Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N- and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescence confocal microscopy at the frog neuromuscular junction confirmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the alpha 1A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the alpha 1A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium influx close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel.     

Molecular chaperones,stress proteins and redox homeostasis

Protection against oxidative stress is highly interrelated with the function of the most ancient cellular defense system, the network of molecular chaperones, heat shock, or stress-proteins. These ubiquitous, conserved proteins help other proteins and macromolecules to fold or re-fold and reach their final, native conformation. Redox regulation of protein folding becomes especially important during the preparation of extracellular proteins to the outside oxidative milieu, which should take place in a gradual and step-by-step controlled manner in the endoplasmic reticulum or in the periplasm. Several chaperones, such as members of the Hsp33 family in yeast and the plethora of small heat shock proteins as well as one of the major chaperones, Hsp70 are able to act against cytoplasmic oxidative damage. Abrupt changes of cellular redox status lead to chaperone induction. The function of several chaperones is tightly regulated by the surrounding redox conditions. Moreover, our recent data suggest that chaperones may act as a central switchboard for the transmission of redox changes in the life of the cell.     

IL-7 up-regulates the expression of IL-8 from resting and stimulated human blood monocytes.

Blood monocytes are important cellular sources of a vast array of bioactive substances, including regulatory and chemotactic cytokines. The regulation of these cytokines is of critical importance to the expression of acute and chronic inflammatory responses. IL-7, a T and B cell-activating cytokine, has recently been shown to have stimulatory effects on the expression of several monocyte-derived proinflammatory cytokines. We now describe the induction of IL-8 mRNA and extracellular protein from human blood monocytes by IL-7. The up-regulation of IL-8 mRNA by IL-7 was not altered by concomitant treatment with cycloheximide, suggesting that the direct stimulatory effects of IL-7 were not dependent upon de novo protein synthesis. In addition, IL-7 significantly potentiated the production of IL-8 from LPS-, TNF-, and IL-1-treated peripheral blood monocytes. Our findings suggest that IL-7 may play a critical role in the modulation of macrophage-derived cytokine expression and may function in vivo as an important proinflammatory cytokine.     

Biphasic platelet-activating factor synthesis by human monocytes stimulated with IL-1-beta, tumor necrosis factor, or IFN-gamma

The capacity of IL-1-beta, TNF, and IFN-gamma to stimulate platelet-activating factor (PAF) synthesis by human monocytes is examined in our report. All three cytokines induced PAF synthesis in a novel biphasic pattern with peaks of PAF synthesis 1 to 2 and 6 to 8 h after stimulation of the monocytes. In contrast, calcium ionophore A23187 elicited a single peak of early PAF synthesis. PAF in the early peak was largely retained intracellularly whereas PAF in the late peak was largely released into culture fluids. Combinations of cytokines were subadditive or antagonistic in inducing PAF synthesis. Cycloheximide inhibited the late peak of PAF synthesis indicating that protein synthesis is required for synthesis of the phospholipid PAF. Specific antibodies to TNF or IL-1-beta inhibited the late peak of PAF synthesis induced by IFN-gamma indicating that late PAF synthesis is dependent on cytokine synthesis. The quantities of PAF produced by cytokine-activated monocytes are sufficient to activate human monocytes. Thus, these studies suggest that PAF may mediate in part monocyte activation by cytokines.     

Extracellular Hsp72, an endogenous DAMP,is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4

Background

Neutrophils play an important role in the pathophysiology of RSV, though RSV does not appear to directly activate neutrophils in the lower airways. Therefore locally produced cytokines or other molecules released by virally-infected airway epithelial cells are likely responsible for recruiting and activating neutrophils. Heat shock proteins (HSPs) are generally regarded as intracellular proteins acting as molecular chaperones; however, HSP72 can also be released from cells, and the implications of this release are not fully understood.

Methods

Human bronchial epithelial cells (16HBE14o-) were infected with RSV and Hsp72 levels were measured by Western blot and ELISA. Tracheal aspirates were obtained from critically ill children infected with RSV and analyzed for Hsp72 levels by ELISA. Primary human neutrophils and differentiated HL-60 cells were cultured with Hsp72 and supernatants analyzed for cytokine production. In some cases, cells were pretreated with polymyxin B prior to treatment with Hsp72. IκBα was assessed by Western blot and EMSA''s were performed to determine NF-κB activation. HL-60 cells were pretreated with neutralizing antibody against TLR4 prior to Hsp72 treatment. Neutrophils were harvested from the bone marrow of wild type or TLR4-deficient mice prior to treatment with Hsp72.

Results

Infection of 16HBE14o- with RSV showed an induction of intracellular Hsp72 levels as well as extracellular release of Hsp72. Primary human neutrophils from normal donors and differentiated HL-60 cells treated with increasing concentrations of Hsp72 resulted in increased cytokine (IL-8 and TNFα) production. This effect was independent of the low levels of endotoxin in the Hsp72 preparation. Hsp72 mediated cytokine production via activation of NF-κB translocation and DNA binding. Using bone marrow-derived neutrophils from wild type and TLR4-mutant mice, we showed that Hsp72 directly activates neutrophil-derived cytokine production via the activation of TLR4.

Conclusion

Collectively these data suggest that extracellular Hsp72 is released from virally infected airway epithelial cells resulting in the recruitment and activation of neutrophils.     

Heat shock protein 70 is a potent activator of the human complement system

According to new hypotheses, extracellular heat shock proteins (Hsps) may represent an ancestral danger signal of cellular death or lysis-activating innate immunity. Recent studies demonstrating a dual role for Hsp70 as both a chaperone and cytokine, inducing potent proinflammatory response in human monocytes, provided support for the hypothesis that extracellular Hsp is a messenger of stress. Our previous work focused on the complement-activating ability of human Hsp60. We demonstrated that Hsp60 complexed with specific antibodies induces a strong classical pathway (CP) activation. Here, we show that another chaperone molecule also possesses complement-activating ability. Solid-phase enzyme-linked immunosorbent assay was applied for the experiments. Human Hsp70 activated the CP independently of antibodies. No complement activation was found in the case of human Hsp90. Our data further support the hypothesis that chaperones may messenger stress to other cells. Complement-like molecules and primitive immune cells appeared together early in evolution. A joint action of these arms of innate immunity in response to free chaperones, the most abundant cellular proteins displaying a stress signal, may further strengthen the effectiveness of immune reactions.     

Fas (CD95) induces proinflammatory cytokine responses by human monocytes and monocyte-derived macrophages

Fas (CD95, APO-1) is regarded as the prototypical cell death receptor of the TNFR superfamily. Fas-induced apoptosis is generally considered to be a noninflammatory process, contributing to the silent resolution of immune and inflammatory responses. However, accumulating evidence indicates that Fas may also induce cellular activation signals. We hypothesized that Fas could activate proinflammatory cytokine responses by normal human monocytes and macrophages. Monocytes were isolated by negative immunoselection from the PBMC fraction of venous blood from healthy volunteers, and monocyte-derived macrophages were cultivated in vitro. Both monocytes and monocyte-derived macrophages released TNF-alpha and IL-8 following Fas ligation, and conditioned medium from Fas-activated monocytes and macrophages induced the directed migration of neutrophils in a chemotaxis assay. Fas-induced monocyte cytokine responses were associated with monocyte apoptosis, nuclear translocation of NF-kappaB, and cytokine gene expression and were blocked by caspase inhibition but not by inhibition of IL-1beta signaling. In contrast, Fas-induced macrophage cytokine responses occurred in the absence of apoptosis and were caspase independent, indicating maturation-dependent differences in the Fas signaling pathways that lead to proinflammatory cytokine induction. Rather than contributing to the resolution of inflammation, Fas ligation on circulating monocytes and tissue macrophages may induce proinflammatory cytokine responses that can initiate acute inflammatory responses and tissue injury.     

Administration of recombinant IL-4 to humans regulates gene expression, phenotype, and function in circulating monocytes.

As a multifunctional cytokine that can augment certain T cell responses, IL-4 is being evaluated currently as a possible therapeutic agent in the treatment of cancer patients. In this report, PBMC were isolated from such patients before and after IL-4 therapy and were analyzed for phenotypic and functional alterations. Differential blood counts showed that the relative percentages of lymphocytes and, to a lesser degree, monocytes decreased after treatment with IL-4. However, when phenotypically analyzed by FACS, monocyte numbers generally increased, whereas there was a decrease in lymphocyte numbers. Monocytes taken from patients before therapy and cultured with and without LPS exhibited normal patterns of monokine-specific (IL-1 beta and TNF-alpha) mRNA expression, as well as secretion of peptide. The addition of rIL-4 to these cultures, however, resulted in the down-regulation of both gene expression and peptide release. Although monocytes taken from post-therapy patients also displayed normal patterns of monokine gene expression and cytokine release after stimulation with LPS, they were no longer inhibited by the addition of exogenous rIL-4 in vitro. These data suggest that monocytes become refractory to the inhibitory effects of IL-4 after in vivo exposure to this cytokine. However, when these monocytes were examined for expression of CD14, CD32, and CD64, exogenous IL-4 was observed to decrease markedly the appearance of these markers, regardless of whether the cells were obtained before or after therapy. Furthermore, monocytes from post-therapy patients exhibited reduced production of PGE2 and superoxide anion, compared with cells obtained before therapy. This effect persisted in culture independent of the further addition of exogenous IL-4. These data suggest a dichotomy between the IL-4-dependent mechanisms that regulate monokine production and those that regulate certain other monocyte functions. The exact mechanisms that govern these two pathways are not known. These results have important clinical implications for the use of IL-4 as an immunotherapeutic agent, as well as providing insight into the physiologic role of IL-4.     

A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures

Like other bacteria, Bacillus subtilis possesses a family of homologous small acidic proteins (CspB, CspC and CspD, identity > 70%) that are strongly induced in response to cold shock. We show that deletion of cspC or cspD genes did not result in a detectable phenotype; in contrast, csp double mutants exhibited severe reduction in cellular growth at 15°C as well as at 37°C, including impairment of survival during the stationary phase. Two-dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37°C and after cold shock, suggesting that CSPs down-regulate production of members from this protein family. A cspB/C/D triple mutant (64BCDbt) could only be generated in the presence of cspB in trans on a plasmid that was not lost, in spite of lack of antibiotic pressure, indicating that a minimum of one csp gene is essential for viability of B . subtilis . After cold shock, synthesis of CspB in 64BCDbt was drastically lower than in wild-type cells accompanied by cessation in growth and strong reduction in general protein synthesis. As CspB, CspC and CspD are shown to bind to RNA in a co-operative and interactive manner, CSPs are suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures.     

Endotoxin-neutralizing effects of histidine-rich peptides

Inflammatory responses of human peripheral blood monocytes to the Gram-negative endotoxin lipopolysaccharide (LPS) are enhanced by structurally diverse substances, such as anionic polysaccharides or cationic polypeptides. Only a few substances are known to effectively blunt LPS-induced monocyte activation. We now show that synthetic poly-L-histidine (Hn) binds to LPS and abrogates the release of the proinflammatory cytokine interleukin-8 (IL-8) in LPS-stimulated human whole blood. LPS-induced stimulation of monocytes was strictly pH-dependent with only minor amounts of IL-8 secreted in acidic blood. Maximum levels of IL-8 secretion occurred at a strongly basic pH. Hn inhibition of the release of IL-8 from LPS-stimulated monocytes was observed under acidic, neutral and physiological conditions. With increasing alkalosis, the effectiveness of Hn was gradually lost, suggesting that protonated, but not deprotonated, Hn was effective in inhibiting LPS-induced monocyte responses. Histidine-rich protein 2 from the malaria parasite, Plasmodium falciparum, inhibited the ability of LPS to evoke an inflammatory response in CD14-transfected THP-1 cells. Further, a short synthetic peptide derived from human histidine- and proline-rich glycoprotein also exhibited LPS-inhibitory effects in CD14 transfectants. Taken together, these observations demonstrate the capacity of histidine-rich peptides, irrespective of their origin, to neutralize LPS-induced proinflammatory host responses.     

Hsp72 induces inflammation and regulates cytokine production in airway epithelium through a TLR4- and NF-kappaB-dependent mechanism

Heat shock proteins are generally regarded as intracellular proteins acting as molecular chaperones; however, Hsp72 is also detected in the extracellular compartment. Hsp72 has been identified in the bronchoalveolar lavage fluid (BALF) of patients with acute lung injury. To address whether Hsp72 directly activated airway epithelium, human bronchial epithelial cells (16HBE14o-) were treated with recombinant Hsp72. Hsp72 induced a dose-dependent increase in IL-8 expression, which was inhibited by the NF-kappaB inhibitor parthenolide. Hsp72 induced activation of NF-kappaB, as evidenced by NF-kappaB trans-activation and by p65 RelA and p50 NF-kappaB1 binding to DNA. Endotoxin contamination of the Hsp72 preparation was not responsible for these effects. Next, BALB/c mice were challenged with a single intratracheal inhalation of Hsp72 and killed 4 h later. Hsp72 induced significant up-regulation of KC, TNF-alpha, neutrophil recruitment, and myeloperoxidase in the BALF. A similar challenge with Hsp72 in TLR4 mutant mice did not stimulate the inflammatory response, stressing the importance of TLR4 in Hsp72-mediated lung inflammation. Last, cultured mouse tracheal epithelial cells (MTEC) from BALB/c and TLR4 mutant and wild-type mice were treated ex vivo with Hsp72. Hsp72 induced a significant increase in KC expression from BALB/c and wild-type MTEC in an NF-kappaB-dependent manner; however, TLR4 mutant MTEC had minimal cytokine release. Taken together, these data suggest that Hsp72 is released and biologically active in the BALF and can regulate airway epithelial cell cytokine expression in a TLR4 and NF-kappaB-dependent mechanism.     

Transforming growth factor-beta 1 rapidly induces Hsp70 and Hsp90 molecular chaperones in cultured chicken embryo cells.

In this report we show that: (1) molecular chaperones in the heat shock protein (hsp) family are a new class of cellular proteins induced by Transforming Growth Factor-beta 1 (TGF beta), a cytokine present in serum, (2) rapid induction of Hsc70 precedes a general increase in protein synthesis and may be a preparatory event, (3) TGF beta is a potent regulator of overall protein synthesis in chicken embryo cells (CEC), and (4) isoforms of Hsp90 with different biochemical properties exist, raising the possibility that they may have different functions. TGF beta can substitute for serum in stimulating synthesis of members of the Hsp90 and Hsp70 families of stress proteins, whereas other cytokines, including PDGF, FGF, and EGF, were not effective nor did they enhance the stimulatory effect of TGF beta on the hsp's. Analysis of the induction of hsp's using one- and two-dimensional polyacrylamide gel electrophoresis indicated that members of the Hsp70 family of molecular chaperones were induced rapidly by TGF beta, reaching maximum rates of accumulation by 5 hours of treatment. Total protein synthesis increased more slowly, undergoing an approximately twofold increase in 24 hours. Using a modified protocol for two-dimensional gel electrophoresis, the Hsp90 protein family was separated into four isoelectric forms, two of which were phosphorylated (Hsp90-2 and -4). These phosphorylated isoforms turned over faster than the unphosphorylated forms of Hsp90. All four isoforms were heat inducible, but only Hsp90-2 and -3 were induced rapidly by TGF beta, again within 5 hours of treatment. The effects of serum on these protein families were similar to those of TGF beta, suggesting that this cytokine may be the serum component primarily responsible for up-regulating members of the Hsp90 and Hsp70 families. We hypothesize that cells rapidly increase their chaperoning capacity for newly synthesized polypeptides in preparation for an increase in the rate of synthesis of proteins up-regulated by TGF beta.     

CD40 signaling of monocyte inflammatory cytokine synthesis through an ERK1/2-dependent pathway. A target of interleukin (il)-4 and il-10 anti-inflammatory action

Ligation of CD40 on monocytes through its interaction with CD40 ligand (CD154) present on activated T helper cells, results in activation of monocyte inflammatory cytokine synthesis and rescue of monocytes from apoptosis induced through serum deprivation. Both of these consequences of CD40 stimulation have been shown to be dependent on the induction of protein tyrosine kinase activity. CD40-mediated activation of protein tyrosine kinase activity and subsequent inflammatory cytokine production are abrogated by treatment of monocytes with the T helper type 2 cytokines interleukin 4 (IL-4) and interleukin 10 (IL-10). In the current study we demonstrate that stimulation of monocytes through CD40 resulted in the phosphorylation and activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinases, whereas phosphorylation of mitogen-activated protein kinases family members p38 and c-Jun N-terminal kinase was not observed in response to this stimuli over the time course examined. PD98059, an inhibitor of the upstream activator of ERK1/2, the MAP/ERK kinase MEK1/2, suppressed IL-1beta and tumor necrosis factor-alpha production in a dose-dependent fashion. Pretreatment of monocytes with IL-4 and IL-10 inhibited CD40-mediated activation of ERK1/2 kinase activity when used individually, and are enhanced in effectiveness when used in combination. Together, the data demonstrate that CD40-mediated induction of IL-1beta and tumor necrosis factor-alpha synthesis is dependent on a MEK/ERK pathway which is obstructed by signals generated through the action of IL-4 and IL-10.     

Hsp70 in the atrial neuroendocrine units of the snail, Achatina fulica

Heat shock proteins (Hsps) are evolutionary conserved peptides well known as molecular chaperones and stress proteins. Elevated levels of extracellular Hsps in blood plasma have been observed during the stress responses and some diseases. Information on the cellular sources of extracellular Hsps and mechanisms regulating their release is still scanty. Here we showed the presence and localization of Hsp70 in the neuroendocrine system in the atrium of the snail, Achatina fulica. The occurrence of the peptide in snail atrium lysate was detected by Western blot analysis. Immunoperoxidase and immunogold staining demonstrated that Hsp70-immunoreactivity is mainly confined to the peculiar atrial neuroendocrine units which are formed by nerve fibers tightly contacted with large granular cells. Immunolabelling intensity differed in morphologically distinct types of secretory granules in the granular cells. The pictures of exocytosis of Hsp70-immunolabeled granules from the granular cells were observed. In nerve bundles, axon profiles with Hsp70-immunoreactive and those with non-immunoreactive neurosecretory granules were found. In addition, Hsp70-like material was also revealed in the granules of glia-interstitial cells that accompanied nerve fibers. Our findings provide an immuno-morphological basis for a role of Hsp70 in the functioning of the neuroendocrine system in the snail heart, and show that the atrial granular cells are a probable source of extracellular Hsp70 in the snail hemolymph.     

IL-4 inhibits the expression of IL-8 from stimulated human monocytes

Peripheral blood monocytes are important mediators of inflammation via the generation of various bioactive substances, including the recently isolated and cloned chemotactic peptide IL-8. Through cytokine networking, monocyte-derived cytokines are capable of inducing IL-8 expression from non-immune cells. IL-4, a B and T lymphocyte stimulatory factor, has recently been shown to inhibit monocyte/macrophage function, including the ability to suppress monocyte-generated cytokines. We describe the in vitro inhibition of IL-8 gene expression and synthesis from LPS, TNF, and IL-1 stimulated peripheral blood monocytes by IL-4. IL-4 suppressed IL-8 production from stimulated monocytes in a dose-dependent fashion, with partial suppression observed at IL-4 concentrations as low as 10 pg/ml. The IL-4-induced suppressive effects were observed even when IL-4 was administered 2 h post-LPS-stimulation. The IL-4-induced inhibition of IL-8 mRNA expression was dependent on protein synthesis, as the suppressive effects of IL-4 were significantly negated by the addition of cycloheximide. Our findings suggest that IL-4 may be an important endogenous regulator of inflammatory cell recruitment, and adds further support to the potential role of IL-4 as a down-regulator of monocyte immune function.     

Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco

To detect microbial infection multicellular organisms have evolved sensing systems for pathogen-associated molecular patterns (PAMPs). Here, we identify bacterial cold shock protein (CSP) as a new such PAMP that acts as a highly active elicitor of defense responses in tobacco. Tobacco cells perceive a conserved domain of CSP and synthetic peptides representing 15 amino acids of this domain-induced responses at subnanomolar concentrations. Central to the elicitor-active domain is the RNP-1 motif KGFGFITP, a motif conserved also in many RNA- and DNA-binding proteins of eukaryotes. Csp15-Nsyl, a peptide representing the domain with highest homology to csp15 in a protein of Nicotiana sylvestris exhibited only weak activity in tobacco cells. Crystallographic and genetic data from the literature show that the RNP-1 domain of bacterial CSPs resides on a protruding loop and exposes a series of aromatic and basic side chains to the surface that are essential for the nucleotide-binding activity of CSPs. Similarly, these side chains were also essential for elicitor activity and replacement of single residues in csp15 with Ala strongly reduced or abolished activity. Most strikingly, csp15-Ala10, a peptide with the RNP-1 motif modified to KGAGFITP, lacked elicitor activity but acted as a competitive antagonist for CSP-related elicitors. Bacteria commonly have a small family of CSP-like proteins including both cold-inducible and noninducible members, and Csp-related elicitor activity was detected in extracts from all bacteria tested. Thus, the CSP domain containing the RNP-1 motif provides a structure characteristic for bacteria in general, and tobacco plants have evolved a highly sensitive chemoperception system to detect this bacterial PAMP.     

Bacteriophages immunomodulate the response of monocytes

Bacteriophages are present in fluids from cirrhosis patients. However, their effect on the immune response is unknown. In this work, we explore the role of phages in the phenotype, function, and cytokine production of monocytes. We stimulated healthy monocytes with five different butanol-purified phage suspensions infective for Gram-negative and Gram-positive bacteria. We studied the expression of the monocyte markers involved in lipopolysaccharide recognition (LPS; CD14), antigen presentation (HLA-DR) and co-stimulation (CD86), and the concentration of induced cytokines (TNF-α, IFN-α, and IL-10) by phages. To confirm the direct role of phages without the interference of contaminating soluble LPS in phage suspensions, polymyxin B was added to the cell cultures. Phagocytosis experiments were assessed by flow cytometry using labeled phage suspensions. We observed that butanol-purified phages reduced the surface levels of CD14 and CD86 in monocytes and increased the secreted levels of TNF-α and IL-10 compared with the control sample containing only butanol buffer. All phage suspensions showed downregulation of HLA-DR expression but only Staphylococcus aureus phage contaminated with Escherichia coli reached statistical significance. The addition of polymyxin B did not restore the monocytic response induced by phages, suggesting that the effect was not caused by the presence of LPS. Monocytes were able to phagocyte phages in a dose- and time-dependent manner. To conclude, the phagocytosis of butanol-purified phages altered the phenotype and cytokine production of monocytes suggesting they become tolerogenic.