R- and S-isomers of nonsteroidal anti-inflammatory drugs differentially regulate cytokine production.

2-arylpropionic acids, a well known class of non-steroidal anti-inflammatory drugs (NSAIDs), exist as a racemic mixture of their enantiomeric forms, with S-isomers primarily responsible for inhibition of prostaglandin (PG) production and of inflammatory events. In this study we show that S-isomers are also responsible for the paradoxical up-regulation of tumor necrosis factor (TNF) induced by ketoprofen, flurbiprofen and ibuprofen in murine peritoneal macrophages stimulated by bacterial endotoxin (LPS). This effect is in close correlation with cyclooxygenase inhibitory capacity of S-isomers and, from Northern blot analysis, seems to be mediated by the up-regulation of TNF mRNA. In addition, up-regulation of TNF production by S-isomers is associated with inhibition of interleukin-10 (IL-10) production. Conversely, we have observed that S-enantiomers reduce IL-6 production at a concentration 100 times higher than that able to inhibit cyclooxygenase activity. The unwanted pro-inflammatory effects of S-isomers through TNF and IL-10 production could therefore hinder their analgesic effect, that is, at least in part, related to IL-6 inhibition. In addition, TNF amplification by S-isomers could be correlated to the clinical evidence of their gastric toxicity. On the other hand, R-isomers did not affect TNF and IL-10 production even at cyclooxygenase-blocking concentration, while they reduced IL-6 production to the same levels as S-isomers. It is concluded that the regulation of cytokine production by S-isomers of 2-arylpropionic acids could partially mask their therapeutic effects and could be correlated to the clinical evidence of their higher gastric toxicity. On the other hand, IL-6 inhibition without the unwanted effects on TNF and IL-10 production shown by R-isomers could be correlated to the analgesic effect reported for R-2-arylpropionic acids.

Characterization and properties of dominant-negative mutants of the ras-specific guanine nucleotide exchange factor CDC25(Mm).

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation depends on the competing action of GTPase activating proteins and guanine nucleotide exchange factors (GEF). The properties of two dominant-negative mutants within the catalytic domains of the ras-specific GEF, CDC25(Mm), are described. In vitro, the mutant GEF(W1056E) and GEF(T1184E) proteins are catalytically inactive, are able to efficiently displace wild-type GEF from p21(ras), and strongly reduce affinity of the nucleotide-free ras x GEF complex for the incoming nucleotide, thus resulting in the formation of a stable ras.GEF binary complex. Consistent with their in vitro properties, the two mutant GEFs bring about a dramatic reduction in ras-dependent fos-luciferase activity in mouse fibroblasts. The stable ectopic expression of the GEF(W1056E) mutant in smooth muscle cells effectively reduced growth rate and DNA synthesis with no detectable morphological changes.

Differential contribution of R and S isomers in ketoprofen anti-inflammatory activity: role of cytokine modulation.

Among nonsteroidal anti-inflammatory drugs (NSAIDs), 2-arylpropionic acids exist as a racemic mixture of its enantiomeric forms, with S-enantiomers primarily responsible for inhibition of prostaglandin synthesis and of inflammatory events. The aim of this study was to compare the anti-inflammatory effects of R- and S-ketoprofen in vitro and in vivo. S-Ketoprofen efficiently inhibited carrageenan-induced edema formation, but it could also amplify the LPS-induced production of the inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1), in close correlation with its ability to inhibit prostaglandin synthesis. Because these inflammatory cytokines are among the factors involved in carrageenan-induced inflammation and also are possibly involved in gastric damage, enhanced cytokine production could partially mask the analgesic effect of S-ketoprofen, and it can be associated with the clinical evidence of its gastric toxicity. On the other hand, R-ketoprofen contributes to the overall activity of the racemate, by playing the main role in ketoprofen-induced analgesia. Unlike the S-isomer, R-ketoprofen did not induce a significant increase of cytokine production even at cyclooxygenase-blocking concentrations. It is concluded that the R-isomer directly contributes to the anti-inflammatory effects of ketoprofen, being more analgesic, and because it does not amplify inflammatory cytokine production.

Inhibitory activity of IL-1 receptor antagonist depends on the balance between binding capacity for IL-1 receptor type 1 and IL-1 receptor type II.

A series of mutants of human IL-1 receptor antagonist (IL-1ra) has been designed by comparison of IL-1ra and IL-1beta structures in order to increase receptor antagonist capacity. Upon in vitro and in vivo assay of IL-1 antagonism, the IL-1ra mutants DoB 0039 (N91-->R), DoB 0040 (T109-->A) and DoB 0041 (N91/T109-->R/A) could inhibit IL-1beta effects more efficiently than wild-type IL-1ra, with DoB 0041 being the most active. Analysis of the receptor-binding capacity of the IL-1ra mutants showed that all three mutants could inhibit binding of IL-1alpha or IL-1beta to IL-1RI-bearing cells more efficiently than wild-type IL-1ra. Conversely, binding of IL-1beta to IL-1RII-bearing cells could be inhibited by DoB 0041 much less efficiently than by wild-type IL-1ra. It is known that the two types of IL-1 receptors (IL-1RI and IL-1RII) play different roles in the regulation of IL-1 activity, with IL-1RI being solely responsible for cell triggering upon IL-1 binding, whereas IL-1RII acts as a scavenger of IL-1 and can thus be considered as a natural IL-1 inhibitor. Thus, the enhanced inhibitory capacity of DoB 0041 as compared with wild-type IL-1ra is explained in terms of better binding to the activating receptor IL-1RI and poorer interaction with the inhibitory receptor IL-1RII.

Transfected type II interleukin-1 receptor impairs responsiveness of human keratinocytes to interleukin-1.

Of the two known types of specific receptors for interleukin (IL)-1, the function of the type II IL-1 receptor (IL-1RII) is still elusive. IL-1RII is allegedly devoid of signaling capacity and is therefore thought to act by trapping and inhibiting IL-1. To directly assess the functional role of IL-1RII, a human keratinocyte cell line has been stably transfected with a cDNA coding for IL-1RII, and its responsiveness to IL-1 has been compared with that of nontransfected cells. Parental cells express IL-1RI and are responsive to low doses of IL-1, whereas transfected cells overexpress IL-1RII, both in its membrane and soluble form, and show a dramatically impaired response to IL-1. Selective block of IL-1RII restores the ability of transfected keratinocytes to respond to IL-1, indicating that the overexpressed IL-1RII is in fact uniquely responsible for their refractoriness to IL-1. The main mechanism of unresponsiveness in transfected keratinocytes appears to be the capture and neutralization of IL-1 by the soluble form of IL-1RII.