A newly developed PLD1 inhibitor ameliorates rheumatoid arthritis by regulating pathogenic T and B cells and inhibiting osteoclast differentiation.

Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays multiple roles in inflammation. We investigated the therapeutic effects of the newly developed PLD1 inhibitors A2998, A3000, and A3773 in vitro and in vivo rheumatoid arthritis (RA) model. A3373 reduced the levels of LPS-induced TNF-α, IL-6, and IgG in murine splenocytes in vitro. A3373 also decreased the levels of IFN-γ and IL-17 and the frequencies of Th1, Th17 cells and germinal-center B cells, in splenocytes in vitro. A3373 ameliorated the severity of collagen-induced arthritis (CIA) and suppressed infiltration of inflammatory cells into the joint tissues of mice with CIA compared with vehicle-treated mice. Moreover, A3373 prevented systemic bone demineralization in mice with CIA and suppressed osteoclast differentiation and the mRNA levels of osteoclastogenesis markers in vitro. These results suggest that A3373 has therapeutic potential for RA.

rhPLD2 suppresses chronic inflammation reactions in a guinea pig asthma model.

BACKGROUND: Asthma is a complex inflammatory disorder of the airways, and research on alternative therapeutic strategies has attracted attention. This study aimed at hypersusceptibility and toxicity of recombinant human phospholipase D2 (rhPLD2) in guinea pigs. We determined the behavioral responses in the model of immediate hypersensitivity animals and changes of eosinophil levels following use of the drugs. Special attention was given to the effects of rhPLD2 in vivo on the guinea pig with chronic persistent asthma and the mechanism involved. METHODS: To investigate the effect of rhPLD2 on the expression of protein kinase C (PKC), and to examine the activity of signal transducer and activator of transcription 1 and 5a in the lung of the guinea pig with chronic asthma. Guinea pigs with chronic asthma were divided into five groups: a saline group, a dexamethasone 5.0 mg group, and rhPLD2 (1.5, 2, or 3 mg) groups. Non-sensitized animals were as normal control group. PKC expression was measured by immunohistochemistry, alterations of STAT1 and STAT5a were detected by TransAM transcription factor assay kits. RESULTS: rhPLD2 (3.0 mg) decreased PKC expression to baseline and inhibited STAT1 activity compared with that of the saline group (p < 0.01). CONCLUSION: The rhPLD2 may suppress the chronic inflammatory reaction through down-regulating PKC expression and STAT1/STAT5a activity in the lung. The rhPLD2 may be a suitable therapeutic target for asthma.

Two new phospholipase D isoforms of Loxosceles laeta: cloning, heterologous expression, functional characterization, and potential biotechnological application.

Toxin phospholipases-D present in the venom of Loxosceles spiders is the principal responsible for local and systemic effects observed in the loxoscelism. In this study, we describe the cloning, expression, functional evaluation, and potential biotechnological application of cDNAs, which code for two new phospholipase D isoforms, LIPLD1 and LIPLD2, of the spider Loxosceles laeta. The recombinant protein rLIPLD1 had hydrolytic activity on sphingomyelin and in vitro hemolytic activity on human red blood cells, whereas rLIPLD2 was inactive. The purified recombinant proteins and the venom are recognized by polyclonal anti-rLIPLD1 and rLIPLD2 sera produced in animals and conferred immunoprotection against the venom. These new isoforms reinforce the importance of the multigene family of phospholipases-D present in Loxosceles spiders. A highly immunogenic inactive isoform such as rLIPLD2 raises important expectation for its use as a potential immunogenic inducer of the immunoprotective response to the toxic action of the venom of Loxosceles laeta.

Different sensitivity of ATP + Mg + Na (I) and Pi + Mg (II) dependent types of ouabain binding to phospholipase A2.

The effect of phospholipase A2 and of related agents on ouabain binding and Na, K-ATPase activity were studied in intact and detergent-treated membrane preparations of rat brain cortex and pig kidney medulla. It was found that phospholipase A2 (PLA2) may distinguish or dissociate ouabain binding complexes I (ATP + Mg + Na) and II (Pi + Mg), stimulating the former and inhibiting the latter. Procedures which break the permeability barriers of vesicular membrane preparations, such as repeated freezing-thawing, sonication or hypoosmotic shock failed to mimic the effect of PLA2, indicating that it was not acting primarily by opening the inside-out oriented vesicles. The detergent digitonin exhibited similar effects on ouabain binding in both ATP + Mg + Na and Pi + Mg media. Other detergents were ineffective. The ability of PLA2 to distinguish between ouabain binding type I and II can be manifested even in SDS-treated, purified preparations of Na, K-ATPase. The number of ATP + Mg + Na-dependent sites is unchanged, while the Pi + Mg-dependent sites are decreased in number in a manner similar to that seen in original membranes. This inhibition is completely lost in the reconstituted Na, K-ATPase system, where the ATP- as well as Pi-oriented ouabain sites are inhibited by PLA2.

Interactions between membranes and cytolytic peptides.

The physico-chemical and biological properties of cytolytic peptides derived from diverse living entities have been discussed. The principal sources of these agents are bacteria, higher fungi, cnidarians (coelenterates) and the venoms of snakes, insects and other arthropods. Attention has been directed to instances in which cytolytic peptides obtained from phylogenetically remote as well as from related sources show similarities in nature and/or mode of action (congeneric lysins). The manner in which cytolytic peptides interact with plasma membranes of eukaryotic cells, particularly the membranes of erythrocytes, has been discussed with emphasis on melittin, thiolactivated lysins and staphylococcal alpha-toxin. These and other lytic peptides are characterized in Table III. They can be broadly categorized into: (a) those which alter permeability to allow passage of ions, this process eventuating in colloid osmotic lysis, signs of which are a pre-lytic induction or latent period, pre-lytic leakage of potassium ions, cell swelling and inhibition of lysis by sucrose. Examples of lysins in which this mechanism is involved are staphylococcal alpha-toxin, streptolysin S and aerolysin; (b) phospholipases causing enzymic degradation of bilayer phospholipids as exemplified by phospholipases C of Cl. perfringens and certain other bacteria; (c) channel-forming agents such as helianthin, gramicidin and (probably) staphylococcal delta-toxin in which toxin molecules are thought to embed themselves in the membrane to form oligomeric transmembrane channels.