S. Poole

Anti-inflammatory and analgesic effects of the phosphodiesterase 4 inhibitor rolipram in a rat model of arthritis.

There has been much interest in strategies which modulate tumour necrosis factor-alpha (TNF-alpha) levels and/or function in rheumatoid arthritis. The elevation of intracellular levels of cyclic AMP in leukocytes by phosphodiesterase 4 inhibitors is accompanied by significant inhibition of the production of TNF-alpha. Nevertheless, these drugs may enhance the hyperalgesia induced by a range of inflammatory mediators, including TNF-alpha. In the present study, we examined the effects of the phosphodiesterase 4 inhibitor rolipram on the local inflammatory infiltrate and hyperalgesia in a rat model of adjuvant-induced arthritis. Rolipram (3 mg/kg) was administered by oral gavage from day 10 to 14 after disease induction. Pretreatment with rolipram abrogated oedema formation and significantly inhibited hyperalgesia. Histopathological analysis revealed a marked inhibition of cellular influx as well as bone and cartilage destruction. Serum and local TNF-alpha levels were suppressed in treated animals whereas there were little changes in interleukin-1beta levels. Although cyclic AMP elevating agents may affect nociceptor threshold to increase the hyperalgesic responses acutely, they also possess significant anti-inflammatory activity, which may hinder local mediator release and/or action. The anti-inflammatory effects of rolipram predominate during this chronic arthritis model in the rat.

Cytokine Degradation by Biofilms of Porphyromonas gingivalis

Abstract. The aim of this study was to determine whether biofilms of Porphyromonas gingivalis could proteolytically degrade the cytokines interleukin (IL)-1β, IL-6, or IL-1 receptor antagonist (IL-1ra). Biofilms were grown on membrane filters on the surface of Wilkins-Chalgren blood agar. The biofilms were removed from the plates, and solutions containing 2.5 μg/ml of each cytokine were added. Following incubation for up to 4.0 h, supernatants from the biofilms were subjected to SDS-PAGE. The separated proteins were transferred by Western blotting to PVDF membranes and probed with peroxidase-conjugated antibodies recognizing both the intact cytokines and their degradation products. After 2 h, no intact IL-1β, IL-6, or IL-1ra were detectable. Cytokine proteolysis also occurred in the presence of horse serum. These results demonstrate that biofilm-grown P. gingivalis can degrade both pro- and anti-inflammatory cytokines and so may be able to perturb cytokine networks in vivo by eliminating cytokines from the local environment.

Endogenous interleukin-10 is required for the defervescence of fever evoked by local lipopolysaccharide-induced and Staphylococcus aureus-induced inflammation in rats

We tested the hypothesis that endogenous interleukin (IL)‐10 limits the fever induced by a Gram‐negative bacterial toxin (Escherichia coli lipopolysaccharide, LPS) and a Gram‐positive bacterial toxin (Staphylococcus aureus), when these toxins are injected into a subcutaneous air pouch (i.po.) in rats. Injection of LPS or S. aureus caused fevers that were reduced in amplitude and duration by simultaneous administration of rat recombinant IL‐10. The inhibition of fever by IL‐10 was accompanied by a significant reduction in the toxin‐evoked increases in concentrations of immunoreactive IL‐6 at the site of inflammation and of IL‐6 and IL‐1 receptor antagonist in the circulation. Conversely, neutralisation of endogenous IL‐10 in the pouch increased the amplitude and dramatically increased the duration of toxin‐evoked fever, and augmented toxin‐induced increases in pouch tumour necrosis factor‐α, IL‐1β, and especially IL‐6. Our data support a crucial regulatory role for endogenous IL‐10 in limiting the fever responses during both Gram‐negative and Gram‐positive infections.

Molecular chaperones stimulate bone resorption.

Abstract. Molecular chaperones, also known as heat shock proteins (hsp), are intracellular proteins found in all cells that catalyze protein folding. We have discovered that one class of bacterial molecular chaperone, the chaperonins, are potent inducers of bone resorption. To address the question of whether the osteolytic activity of the chaperonins was unique to this protein class, or was a common attribute of molecular chaperones generally, we have examined a number of bacterial and mammalian molecular chaperones for activity in the murine calvarial bone resorption assay. All the Escherichia coli molecular chaperones (groEL, groES, and dnaK) were active. The osteolytic activity of groEL was inhibited by indomethacin and the natural antagonist of interleukin-1 receptor antagonist (IL-1ra) but was unaffected by neutralization of tumor necrosis factor (TNF) or inhibition of 5-lipoxygenase. Mammalian molecular chaperones of molecular mass 27, 47, 70, and 90 kDa were also tested and, with the exception of the 47 kDa protein, all showed activity in the murine calvarial assay. Molecular chaperones appear, therefore, to have the capacity to modulate the cellular processes in bone explant cultures, resulting in resorption of the calcified matrix. The possibility that these proteins could play a role in the normal or pathological remodeling of bone is discussed.

Human chaperonin 60 (Hsp60) stimulates bone resorption: structure/function relationships

It is established that the molecular chaperone, chaperonin 60, from various bacteria and from Homo sapiens has cell-cell signalling activity and is able to induce proinflammatory cytokine synthesis. We previously reported that chaperonin 60 proteins from Gram-negative bacteria, but not mycobacteria, have the capacity to resorb cultured murine calvarial bone. We now report that lipopolysaccharide-low human recombinant chaperonin 60 (Hsp60) is a relatively weak cytokine-inducing agonist but is a potent stimulator of murine calvarial bone resorption. The osteolytic activity of Hsp60 was significantly inhibited by indomethacin, interleukin-1 receptor antagonist, and osteoprotegerin, but 5-lipoxygenase inhibitors were less effective. Analysis of Hsp60 truncation mutants revealed that N-terminal mutants (Delta1-137, Delta1-358, and Delta1-465) retained bone resorbing activity. In contrast, a C-terminal truncation mutant (Delta1-26 + Delta466-573) was inactive. This suggests that the active domain in this protein is found within residues 466-573. It is now established that Hsp60 is present in the blood of the majority of the population with the normal range encompassing levels able to activate bone cells. The possibility exists that this protein could play a role in bone remodelling.

Antinociceptive activity of peptides related to interleukin-1β-(193–195), Lys-Pro-Thr

A series of closely related peptide analogues of Lys-Pro-Thr [(interleukin-1 beta-(193-195)] have been investigated in two models of antinociception in mice (acetic acid-induced abdominal constrictions and formalin tests) and compared with morphine, aspirin and indomethacin. Formalin-induced nociceptive responses in the mouse showed early (0-5 min) and late (15-30 min) phases of peak activity. Lys-D-Pro, Lys-D-Pro-Thr, Lys-D-Pro-Arg, Lys-D-Pro-Val, morphine and aspirin, were antinociceptive in both phases after intraperitoneal (i.p.) and oral (p.o.) administrations. Lys-D-Pro-Leu inhibited the early phase response only after i.p. injection. Lys-D-Pro-Val-NH2, Lys-D-Pro-Gln, Lys-D-Pro-Tyr, Lys-D-Pro-Asn, Asp-Lys-D-Pro-Val and indomethacin were active only against the late phase (ED50 values of 64, 32, 44, 94, 67 and 25 mg/kg i.p., respectively). Several of the peptides showed good bio-availability, e.g. Lys-D-Pro-Asn (ED50: 10 mg/kg i.p.; 11.4 mg/kg p.o.) in the abdominal constrictions test, where two modes of action were apparent, non-opioid and opioid; non-opioid (naloxone-insensitive antinociception) mechanisms were illustrated by Lys-D-Pro-Thr and Lys-D-Pro-Asn; opioid (naloxone-sensitive antinociception) mechanisms by Lys-D-Pro-Val and Lys-D-Pro-Leu. These data identify orally active antinociceptive peptides in both antinociceptive tests with varied relative potency profiles to morphine, indomethacin and aspirin in the mouse formalin test.