Jean-Paul Giroud

Effects of interleukin-6 on cytochrome P450-dependent mixed-function oxidases in the rat

Intravenous treatment of male rats with recombinant human interleukin-6 (rhIL6) at 50, 100 and 200 micrograms/kg (corresponding to 4, 8 and 16 x 10(4) U/animal, respectively) reduced the activities of hepatic microsomal cytochrome P450-dependent monoxygenases to varying degrees. Ethylmorphine-N-demethylase activity fell to 53% of control values, an effect similar to that induced by 2.5 mg/kg Escherichia coli lipopolysaccharide (LPS). Ethoxycoumarin-O-deethylase activity was also sensitive to inhibition, whereas IL6 had little effect on the activities of other P450-dependent enzymes, including ethoxyresorufin-O-deethylase. Pentoxyresorufin dealkylase activity, which is representative of the cytochrome P450 IIB 1/2 subfamily, was unaffected by IL6 whereas LPS reduced it to 33.7% of control values. Another hepatocyte-related parameter, serum concentration of alpha 1-acid glycoprotein (AGP), was increased by up to 3.5-fold over baseline by IL6 and 10-fold by LPS. Recombinant human interleukin-1 beta (rhIL1 beta) (10 micrograms/kg, corresponding to 5 x 10(4) U/rat) and recombinant human tumor necrosis factor alpha (rhTNF) (150 micrograms/kg corresponding to 24 x 10(4) U/rat) were both as potent as LPS (2.5 mg/kg) in increasing serum AGP levels and reducing hepatic microsomal monoxygenase activities. IL6 did not potentiate the effects of rhIL1 beta. Hepatic microsomal glucuronyltransferase activities were little affected by LPS and unaffected by rhIL6. Finally, rhIL6 was more potent after i.p. injection than after i.v. or s.c. injection. These results suggest that the effects of LPS, TNF and IL1 on the mixed-function oxidase system in vivo may be due partly to an induction of IL6 in vivo. The different sensitivities of the enzymes to IL6 but not to IL1 or TNF may be due to the involvement of two distinct mechanisms.

Peptides and histamine release from rat peritoneal mast cells

Various vasoactive peptides were compared for their histamine releasing effects on rat mast cells. Neurotensin, substance P (SP), and kallidin were the most active natural peptides, followed by bradykinin; neurokinin A and B, bombesin, angiotensin and tuftsin were practically inactive. Several kinins and tachykinin-related peptides were tested in an attempt to characterize the receptors mediating histamine liberation. The order of potency of the kinins was the following: kallidin greater than [Tyr(Me)8]bradykinin = bradykinin greater than [desArg10]kallidin greater than desArg9-bradykinin, the same as that found in smooth muscle possessing receptors of the B2 type. Tachykinin-related peptides were potent stimulants and followed the order: [D-Tryp7,9,10]SP-(1-11) greater than [D-Pro2,D-Tryp7,9,10]SP-(1-11) greater than SP-(1-11) greater than SP-(1-9) greater than [D-Pro4,D-Tryp7,9,Leu11]SP-(4-11) greater than SP-(1-7) greater than SP-(4-11) greater than neurokinin A = neurokinin B, indicating that: (a) undecapeptide antagonists of SP behave as superagonists; (b) both N- and C-terminal portions of SP-(1-11) are essential for activity; and (c) receptors for the tachykinins mediating histamine release appear to be of the SP-P type.

Increase in tumor necrosis factor‐α production linked to the toxicity of indomethacin for the rat small intestine

1 The toxic effects of nonsteroidal anti‐inflammatory drugs for the lower gastrointestinal tract share certain features with inflammatory processes, suggesting that the release of inflammation cytokines such as TNF‐α may damage the intestine. 2 Rats received a s.c. injection of indomethacin. Then, jejunum‐ileum was taken up for the quantification of ulcerations, production of TNF‐α, nitrites and PGE2 ex vivo and activity of calcium‐independent NO synthase and myeloperoxydase. Activation of NO metabolism and myeloperoxydase were measured as potential effectors of TNF‐α. 3 Jejunum‐ileum from rats having received indomethacin (10 mg kg−1) produced TNF‐α ex vivo. Cytokine production was associated with the onset of macroscopic ulcerations of the small intestine, and preceded nitrite production and tissue activity of myeloperoxidase. 4 Similar intestinal ulcerations and upregulation of TNF‐α were obtained with flurbiprofen (30 mg kg−1), chemically unrelated to indomethacin. 5 TNF‐α production was proportional to the indomethacin dose (from 3–20 mg kg−1) and correlated with the surface area of ulcerations and nitrite production, 24 h after indomethacin administration. 6 Pretreatment of rats with RO 20‐1724, a type‐IV phosphodiesterase inhibitor which inhibits TNF‐α synthesis, substantially reduced jejunum‐ileum ulcerations, TNF‐α and nitrite production and tissue enzyme activities. 7 These findings provide evidence that TNF‐α is increased in indomethacin‐induced intestinal ulcerations and support suggestions that TNF‐α is involved at an early stage of nonsteroidal anti‐inflammatory drug toxicity for the small intestine.

Network of inflammatory cytokines and correlation with disease activity in ulcerative colitis

Objective:The inflammatory component of most human inflammatory chronic diseases implicates the production of proinflammatory cytokines. Tumor necrosis factor α (TNFα) and interleukin 1β (IL1β) seem to play an important role in ulcerative colitis (UC) in relevant experimental models. Moreover, antiTNF therapy seems promising experimentally and clinically. However, these cytokines, and TNFα more particularly, are hardly seen in vivo in such patients. The mediators of choice, correlated with disease activities or drug efficacy, remain unclear. To characterize in vivo the network of colonic cytokines in patients with UC, and the contribution of the various cytokines to disease activity we performed this study, using the colonic perfusion method.Methods:A 20-cm colon length was perfused. Perfusate samples were collected for cytokine determination by enzyme-linked immnoassays. Nineteen perfusions were performed in mild to moderate UC, including two successive perfusions in four patients. Six healthy control patients and four having Crohns disease (CD) with rectal involvement were studied. Endoscopic score, leukocyte scintigraphy, and systemic markers of inflammation were simultaneously quantified.Results:Large amounts of IL1β, TNFα, IL6, and IL8 were produced in UC patients with a highly significant correlation between TNFα, IL1β and IL8 two by two. Multivariate factorial analysis indicated that IL1β showed the best correlation with disease activity. Locally produced IL6 was strongly associated with circulating platelet counts. Moreover, production of inflammatory cytokines was associated with similar variations of disease activity in the four patients with two successive perfusions performed. The level of inflammatory cytokines in CD was lower than in UC; TNFα, IL1β, and IL6 were not found in any control patients.Conclusions:UC appears to be a chronic inflammatory disease characterized by high production of all four proinflammatory cytokines (IL1β, TNFα, IL6, and IL8). These results suggest that colonic perfusion may be a suitable method to evaluate the local anticytokine properties of new drugs, in correlation with disease activity and systemic markers of inflammation.

Tachykinins: effects on motility and metabolism of rat polymorphonuclear leucocytes.

Chemotactic and chemoluminescent activities of substance P, substance K, kassinin and the substance P fragments SP 4-11, SP 7-11, SP 1-4 have been investigated in order to identify the minimum active molecular structure responsible for rat polymorphonuclear activation. Substance P, SP 4-11 and SP 7-11 stimulated directed locomotion (chemotaxis) and were found to be active also in the chemoluminescence assay while SP 1-4 had no effect. Moreover, all peptides, except substance K and SP 1-4, inhibited the chemotactic response of polymorphonuclears to the peptide formyl-methionyl-leucyl-phenylalanine and, to a minor extent, also to leukotriene B4. A maximum of activity was observed with the C-terminal sequence SP 4-11. Substance K was found to be inactive. Kassinin exhibited a weak chemotactic effect and exerted a slight inhibition of attracting activity of peptide-formyl-methionyl-leucyl-phenylalanine. Considering that substance P and related peptides are active only at very high concentrations, it cannot be affirmed that these agents activate specific receptors. If receptors are involved, they would be of the SP-P type, since substance K is inactive.

Modulation of human polymorphonuclear neutrophil functions by α1acid glycoprotein

Abstractα1-Acid glycoprotein (α1-AGP), a naturally occurring human plasma protein and acute-phase reactant, was extracted by a two-step procedure from sera collected from four healthy men. Its activity was testedin vitro on human polymorphonuclear (PMN) functions (migration, aggregation, O2− generation). α1,-AGP was not chemoattractant but inhibited the PMN response to the chemoattractant formylmethionyl-leucyl-phenylalanine without affecting spontaneous migration (Boyden and agarose methods of assessment). At concentrations between 0.15 and 0.45 mg/ml, α1AGP exerted an aggregating effect with a maximal effective concentration of 0.3 mg/ml. α1-AGP inhibited superoxide generation by PMNs stimulated either by opsonized zymosan or phorbol myristate acetate. This inhibition varied according to the intensity of the stimulation. At low stimulus concentrations, a dose-dependent inhibition of membrane-associated PMN responsiveness to soluble or particulate stimuli was observed. These findings suggest that α1-AGP may be able to prevent PMN activation in the course of inflammatory processesin vivo.

Anti-tumor necrosis factor properties of non-peptide drugs in acute-phase responses

Dexamethasone (sodium phosphate), pentoxifylline, fusidic acid (sodium salt), pentamidine (isethionate) and R-phenylisopropyladenosine (R-PIA) were tested for their anti-tumor necrosis factor (TNF) activities in an endotoxin-induced shock rat model. All the drugs reduced serum TNF concentrations in a dose-dependent manner, whereas their effects on serum interleukin-6 levels differed. Doses that reduced TNF levels by 50% were 0.012 mg/kg for dexamethasone, 0.06 mg/kg for R-PIA, 0.24 mg/kg for pentamidine, 6.5 mg/kg for fusidic acid and 15 mg/kg for pentoxifylline. Administration of the drugs to rats before intraplantar injection of carrageenan reduced paw edema by 50-70%. Injection of a monoclonal anti-TNF antibody reproduced the inhibitory effect. Moreover, the time course of tissue-associated TNF following carrageenan injection was compatible with mediation of edema by TNF. Results obtained for this acute, non-immunological inflammatory reaction strongly suggest that the model is TNF-dependent. Our results reinforce the idea that TNF is a crucial target in the therapeutics of inflammatory reactions. These drugs, which are able to cross cell barriers, might have clinical applications in localized and/or chronic diseases in which TNF is involved.

Substance-P-like levels in inflammatory exudates

Levels of SP-like immunoreactivity were assessed by enzyme immunoassay in exudates induced in the rat by intrapleural injection of either calcium pyrophosphate (CaPP) or carrageenan. SP-like levels increased during the first hour, up to approximately 2 ng/ml, and remained significantly higher than control values from 1 to 6 h after the induction of pleurisy by CaPP. With carrageenan as the irritant, SP-like levels rose during the first 4 h, up to 3 ng/ml, and remained significantly higher than control values from 4 to 24 h. In terms of the total volume of exudate induced by carrageenan, total amounts increased up to 8 ng/rat at 16 h after the beginning of the reaction. Our data demonstrate a detectable release of SP-like material in these pleural exudates and suggest its involvement in the inflammatory response, either directly, or through other mediators, or simply by acting on nociceptive fibers and inducing vascular changes.

Staurosporine, a protein kinase inhibitor, up-regulates the stimulation of human neutrophil respiratory burst by N-formyl peptides and platelet activating factor

Staurosporine (STAR), a potent protein kinase C (PKC) antagonist, was found to modulate the chemoattractant-induced respiratory burst of human polymorphonuclear leukocytes (PMNs) according to drug concentration. Low STAR concentrations from 10 to 200 nM potentiated the N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet activating factor (Paf)-induced respiratory burst, affecting both the initial rate and the total amount of superoxide anion generated. The maximal increase occurred in the presence of 100 nM STAR and optimal fMLP concentration and reached 60-100% of control values. Above 250 nM, STAR inhibited the respiratory burst with an IC50 of 360 and 320 nM for fMLP and Paf, respectively. The respiratory burst induced by PKC activators such as phorbol myristate acetate or phorbol 12, 13 dibutyrate was inhibited effectively by STAR, with a low IC50 (25 nM) for both stimuli. Thus, the use of low STAR concentrations points to two possible roles of PKC in the regulation of NADPH oxidase activity, i.e. a positive regulation in phorbol ester-treated cells and a negative regulation in chemoattractant-stimulated PMNs.

Effects of tripeptides derived from milk proteins on polymorphonuclear oxidative and phosphoinositide metabolisms

The tripeptide GLF (glycyl-leucyl-phenylalanine) was isolated from human milk proteins. This peptide increased phagocytosis by human and murine macrophages and protected mice against Klebsiella pneumoniae infection. Specific binding sites on human polymorphonuclear leukocytes (PMNs) have been demonstrated recently. The aim of the present research was to study the action of this peptide on rat and human PMN oxidative burst and to investigate the consequences of cell stimulation on polyphosphoinositide hydrolysis. A biphasic stimulating concentration-dependent effect of GLF on PMN chemiluminescence and superoxide anion generation was demonstrated. One of the peaks of the oxidative response occurred around 10(-9) M, which correlates with the Kd of high affinity receptors of GLF. The other maximum, around 10(-4) M, might be due to the hydrophobic nature of the tripeptide. O2- generation mimicked the phorbol myristate acetate response: after a lag period of 2-5 min, O2- release gradually increased for 10-15 min until a plateau was reached. Furthermore, GLF enhanced phosphoinositide breakdown with maximal IP3 production at 10(-7) M. Various analogs of GLF were synthesized in order to define the relative importance of the different amino acids and their position in the tripeptide molecule: glycyl-phenylalanine-leucine was devoid of biological properties but enhanced the activity of GLF on the metabolic burst at high concentrations; peptides leucyl-leucyl-phenylalanine and leucyl-leucyl-tyrosine, which displaced GLF from its specific membrane receptors, exerted stimulating effects on PMN oxidative and phosphoinositide metabolisms. It is quite conceivable that these short peptides, which may be generated in the newborn during digestion and which are able to stimulate phagocytic cells, are implicated in the defense of the neonate immature organism against infection.