[Induction of apoptosis of splenic lymphocytes in mice by accelerated carbon ions]. / Induction de l'apoptose dans les lymphocytes spléniques de souris par un faisceau d'ions carbone.

To assess the capacity of heavy ions to induce apoptosis in lymphocytes, mice have been irradiated with accelerated carbon ions (95 MeV/nucleon) at doses ranging from 0.1 to 4 Gy. Their spleens were removed 24 h later and gently dissociated to prepare a single cell suspension. Mononuclear cells were then maintained in culture at 37 degrees C, and the occurrence of apoptosis in these cells was analysed 24 h later. Lymphocytes were also irradiated in vitro, in the presence of Ac-DEVD-CHO, a potent caspase-3 and -7 inhibitor. Results from three experiments performed at the Grand Accelerateur National d'Ions Lourds (GANIL, Caen, France) are reported here. They indicate that carbon ions induce a marked, dose-dependent, reduction of the spleen weight and cellularity. However, in sharp contrast with spleen cells prepared from X-ray irradiated mice, only a slight increase of apoptosis is evidenced in cultured lymphocytes from mice irradiated with heavy ions. The significance of such results is discussed. So far, few data exist concerning the biological effects of heavy ions, in particular their capacity to induce apoptosis in lymphocytes; the present study provides useful clues for further investigations.

Role of nitric oxide in depressed lymphoproliferative responses and altered cytokine production following thermal injury in rats.

Immunodeficiency follows extensive burns. We investigated some underlying mechanisms in rats, 10 days after a full-thickness skin burn affecting 20% of total body surface area. In both normal and burned rats the splenocyte proliferative response to Con A was linearly and negatively correlated with nitric oxide (NO) production. In all burned rats, the proliferative response was depressed by more than 80% and NO production corresponded to a nitrite concentration above 20 microM. Proliferative responses in burned rats were fully restored in the presence of 250 microM NG-monomethyl-L-arginine (NMMA). A time course study of NO production in response to Con A, LPS, anti-CD3, and IFN-gamma showed that splenic macrophages from burned rats responded to direct and indirect stimuli more rapidly and more intensively than normal macrophages. In the second part of this work, the effect of the overproduction of NO on the synthesis of immunoregulatory and proinflammatory cytokines was investigated. Although it was inhibited, IFN-gamma production by splenocytes from burned rats remained sufficient for NO synthase induction and was restored by NMMA. Concomitantly, IL-2 concentration was enhanced but returned to normal in the presence of NMMA. TNF production was halved after burn injury and NMMA partially restored it. In contrast, IL-6 production was enhanced and increased further in the presence of NMMA. Therefore, cytokines were differently affected by burn injury and variously regulated by NO.

Kinetic evaluation of nitric oxide production in pleural exudate after induction of two inflammatory reactions in the rat.

NO generation in the course of two acute, non immune, inflammatory reactions (pleurisy induced by rat isologous serum and carrageenan) was assessed by means of nitrite measurement in pleural exudate from 0.5 to 24 h. NO release varied time-dependently, similarly for the two inflammatory reactions. A first, but transient, peak was reached in 30 min while a second peak, more sustained, began at the fourth hour and was maximum at the tenth. Kinetic evolution of NO release was consistent with activation, in a first step, of a constitutive NO synthase probably from endothelial origin (inhibited by 2-Methyl-2-Thiopseudourea sulfate but not by dexamethasone) and with activation, in a second wave, of inducible NOS from endothelial and exudative cells. NO release was potentiated by administration per os of L-Arginine and seems to be involved in the evolution of acute inflammatory reactions and oxygen metabolite production.

Release of mast cell mediators and nitrites into knee joint fluid in osteoarthritis--comparison with articular chondrocalcinosis and rheumatoid arthritis.

In order to address the issue of the role of mast cells and nitric oxide (NO) in joint effusions occurring in the course of osteoarthritis (OA), synovial fluids collected from the knee of patients with OA, articular chondrocalcinosis and rheumatoid arthritis (RA) were studied for number of mast cells, and histamine, tryptase, phospholipase A2 and nitrite content. Mast cell counts are elevated in synovial fluid from OA patients when compared with RA. Histamine content in synovial fluid parallels the number of mast cells. Tryptase levels are elevated in OA in comparison with both other conditions, but do not reach the level of significance. Identical phospholipase A2 levels are recorded in three groups. Nitrite concentrations are also higher in synovial fluid from OA patients when compared with RA patients. These results suggest that mast cells in association with various inflammatory cells, may contribute to inflammation and cartilage breakdown in OA.

Acute-phase response, interleukin-6, and alteration of cyclosporine pharmacokinetics.

OBJECTIVE: Administration of interleukin-6 partially reproduces the inhibitory effects of the acute-phase response on cytochrome P450-dependent drug metabolism. The aim of the study was to determine whether endogenous cytokine has such an effect in patients treated by cyclosporine, which is metabolized by the cytochrome P4503A subfamily. METHODS: Blood cyclosporine and serum interleukin-6 levels were determined in six patients undergoing bone marrow transplantation, as long as they received cyclosporine by continuous infusion. Two serum acute-phase proteins, C-reactive protein and alpha 1-acid glycoprotein, and two cyclosporine metabolites, AM1 and AM9, were also determined. RESULTS: At the time of marrow infusion, levels of specific markers of inflammation were low. A peak in interleukin-6 level was then observed a mean of 10.8 days after transplantation, closely associated with variations in C-reactive protein levels. A parallel twofold increase in AM1 concentrations was observed, followed by a three-fold increase in cyclosporine levels, which peaked 4.8 days after interleukin-6. The times of peak cyclosporine and AM1 levels correlated with the time of peak interleukin-6 levels. AM9 was detectable in three patients but concentrations fell when interleukin 6 became detectable. CONCLUSIONS: An inflammatory reaction could be an important source of intraindividual variability in cyclosporine pharmacokinetics, possibly through an inhibition of cytochrome P4503A-dependent enzyme activities by endogenous interleukin-6. Blood AM1 accumulation might be explained by a secondary metabolic step that is highly sensitive to the inhibitory effect of interleukin-6.

Restoration of postburn impaired lymphocyte responsiveness by nonsteroidal anti-inflammatory drugs is independent of prostaglandin E2 inhibition.

Prostaglandin E2 (PGE2) has been implicated in postburn immunosuppression, which is responsible for septic complications. In the present work, seven non-steroidal anti-inflammatory drugs (NSAIDs), differing by their capacity to inhibit the cyclooxygenase pathway, were compared for their ability to restore T lymphocyte proliferative responses evaluated 4 days after thermal injury in rats. Salicylic acid, 5-aminosalicylic acid, and niflumic acid, given daily, fully restored spleen cell responses to concanavalin A (Con A) and phytohemagglutinin. These drugs were active only at doses that were below the anti-inflammatory doses and did not modify normal spleen cell responses. In these conditions, indomethacin slightly restored lymphocyte reactivity, whereas acetylsalicylic acid, ketoprofen, and piroxicam were ineffective. PGE2 production by Con A-stimulated spleen cells from untreated burned rats and after treatment with niflumic acid or 5-aminosalicylic acid did not correlate with the intensity of the proliferative response. Indomethacin, niflumic acid, and 5-aminosalicylic acid were added in vitro to spleen cells from normal and burned rats, at concentrations from 10(-7) to 10(-4) M. PGE2 production was strongly depressed by indomethacin and niflumic acid and not modified by 5-aminosalicylic acid. The proliferative response of normal spleen cells was depressed in a concentration-dependent manner by niflumic acid and slightly inhibited at the highest concentrations of indomethacin. In contrast, indomethacin concentration dependently restored the burn-impaired proliferative response, whereas niflumic acid further depressed it and 5-aminosalicylic acid had no effect. These results demonstrate that only some NSAIDs are able to restore T lymphocyte reactivity impaired after thermal injury and that this property is not related to inhibition of PGE2 production.

Inhibition of human monocyte TNF production by adenosine receptor agonists.

Adenosine receptor agonists and agents enhancing pericellular concentrations of adenosine possess antiinflammatory properties. In the present study, we found that R-phenylisopropyladenosine (R-PIA), 5'-N-ethylcarboxamido adenosine (NECA), other agonists of adenosine receptors and dipyridamole, an adenosine uptake inhibitor, inhibited tumor necrosis factor (TNF) production by endotoxin-stimulated human monocytes in a concentration-dependent manner with no inhibition of interleukin-6. The rank order of agonist potency is characteristic of neither A1 nor A2 receptors and suggests the involvement of another receptor subtype. The effect of R-PIA on TNF was in part abolished by the antagonist 8-sulfophenyltheophylline. In endotoxin-treated rats, R-PIA pretreatment (2.5 mg/kg) reduced serum TNF levels by 98%, with no modification of serum IL6 levels. TNF inhibition could be an important mechanism by which adenosine analogs exert their antiinflammatory action.

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.

Nitric oxide mediates the depression of lymphoproliferative responses following burn injury in rats.

Among the multiple biological activities of nitric oxide (NO) an immunoregulatory role consisting of the mediation of macrophage suppressive activity, has recently been evidenced. In the present work, we investigated whether NO was implicated in immunosuppression following burn injury. Thermal injury affecting 20-25% of the total body surface area in Wistar rats, provoked a biphasic depression of spleen cell proliferative responses to phytohemagglutinin (PHA) and concanavalin A (Con A). We show that these responses are fully restored on day 4 after burn and only by 55% on day 10 when spleen cells were stimulated in the presence of NG-monomethyl-L-arginine (NMMA), a potent inhibitor of the macrophage inducible NO synthase. Nitrite content in culture supernatant, as an indicator of NO release (in the absence of NMMA), was significantly augmented in Con A-stimulated spleen cells from burned rats as compared to normal spleen cells. These results show for the first time that NO is implicated, at least in part, in an immunosuppression state which is not linked to an infectious disease.

Modification of inflammatory processes by phenobarbital in rats.

Enzyme-inducing drugs such as phenobarbital (PB) increase serum concentrations of an acute-phase protein, alpha 1-acid glycoprotein (AGP), in man, dogs, and rats via an unknown mechanism. We studied the effects of PB on components of an acute inflammatory reaction in rats in order to determine if PB acts only on this biological marker of inflammation or is capable of altering the clinical course of inflammatory processes. Local carrageenan injection induces a similar time-dependent plantar edema and increases serum AGP levels in Sprague-Dawley (SD) and Dark Agouti (DA) rats. Pretreatment with PB for seven days modified neither parameter in SD rats while plantar edema was aggravated and serum AGP levels were increased in DA rats. The sedative-hypnotic properties of PB were not involved, since a single administration of this drug had no action in DA rats. On the other hand, chronic PB administration reduced the severity of an autoimmune disease, type II collagen-induced arthritis, in DA rats. These data indicate that PB, a potent inducer a cytochrome P-450-dependent enzymes, modifies the course of the inflammatory process. Preliminary results with macrophage transfer experiments suggest that this response to PB could be mediated by stimulated macrophages.

Acute non-immunological inflammation inhibits natural killer (NK) activity in the lungs and enhances metastatic development.

A non-immunological acute inflammatory reaction, induced in rats by intrapleural injection of calcium pyrophosphate microcrystals, decreased natural killer activity of lung intracapillary leucocytes (LICL), and, to a lesser extent, of spleen cells. NK activity was significantly depressed as early as 2 h after pleurisy induction, maximally suppressed at 72 h (when the inflammation had resolved) and returned to near-normal values by day 10. This decrease in NK activity was demonstrated using YAC-I lymphoma cells and syngeneic fibrohistiocytoma (P77) cells, as targets. The inhibition of NK activity of LICL was accompanied by an impaired destruction of P77 tumour cells injected i.v. 3 days after the onset of pleurisy. This was shown by an increase in the number of radiolabelled tumour cells surviving in the lungs at 24 h and of tumour nodules developed in lung parenchyma at 3 weeks. Suppression of NK activity was not due to a decrease in the proportion of large granular lymphocytes (LGL) in the LICL population. Suppressor macrophages may be involved, at least in part, since a partial restoration of LICL cytotoxicity was obtained after depletion of plastic-adherent cells. The effect of inflammation was reproduced in normal rats by injecting inflammatory serum or PGE2, suggesting a possible role of circulating mediators.

When should the immune clock be reset? From circadian pharmacodynamics to temporally optimized drug delivery.

Immune defenses are organized along both 24-h and yearly time scales. Two circadian systems have been isolated in man, which can be desynchronized: (1) the circulation of T, B, or NK lymphocyte subsets in peripheral blood and (2) the density of epitope molecules (CD3, CD4, ...) at their surface, which may relate to cell reactivity to antigen exposure. The in vitro response of murine splenocytes to interleukin 2, interferon (IFN), or cyclosporin A strongly depended upon circadian time of exposure. Temporally optimized delivery of biologic response modifiers (BRM) may be guided by immunologic marker rhythms. An alternative yet complementary strategy was sought with IFN: since high doses were shown as more effective than low doses against several malignancies, this drug was given at the presumed less toxic time, so that its dose could be increased. Continuous drug delivery was circadian modulated in 8 cancer patients. Dose intensities twice to fourfold higher than those usually recommended were safely infused to ambulatory patients. Chronotherapy with BRM may represent a necessary step for optimizing the immunologic control of malignancies.

Local and systemic effects of an acute inflammation on eicosanoid generation capacity of polymorphonuclear cells and macrophages.

Acute non-specific inflammation was induced in rats by injection of isologous serum into the pleural cavity. Pleural and peritoneal cells were collected at various times after pleurisy induction and tested for production of leukotriene B4 (LTB4), prostaglandin E2 (PGE2) and prostacyclin (PGI2) after in-vitro stimulation with calcium ionophore A23187. Cells obtained by lavage of pleural and peritoneal cavities of normal rats were used as controls. Increased production of LTB4, PGE2 and PGI2 by pleural cells was observed 3 days after pleurisy induction, but with a significant depression of PGI2 release at 3 h. As the relative proportions of polymorphonuclear cells (PMN) and macrophages in the inflammatory exudate varied during the development of inflammation, these cells were examined separately for LTB4 production. PMN and macrophages contributed equally to the liberation of this mediator in normal and inflamed rats. Similar qualitative and quantitative changes in LTB4 production by pleural cells were observed, irrespective of the type of irritant used (isologous serum, dextran, carrageenan, microcrystals). In contrast, intrapleural injection of saline had no significant effect. In order to determine whether local inflammation may influence mediator release by phagocytic cells at remote sites, peritoneal cells were collected 3 or 72 after pleurisy induction. The production of LTB4, PGE2 and PGI2 was increased at 72 h. Mediator production by peritoneal macrophages was observed in both normal and inflamed rats. In conclusion, acute non-specific inflammation provoked increased arachidonic acid metabolite generation by phagocytes both locally and at a distance: this occurred more than 24 h after pleurisy resolution.

Imuthiol influences on cytotoxic T cells and NK activity in +/+ and athymic nude BALB/c mice.

The effects of imuthiol (sodium ditiocarb, DTC) on the expression of cytotoxic responses (CTL) and natural killer (NK) activity were evaluated in aged and young euthymic mice, and in nu/nu BALB/c mice. Imuthiol generated CTL and concomitantly reduced NK activity in nu/nu mice, suggesting that the agent can generate T cells in athymic nude animals. Treatment for up to 4 months augmented spleen NK and CTL activities in young or aged euthymic mice, but the generation of CTL in old animals was increased by long-term treatments better than by a single injection. The capacity of imuthiol to activate specific and nonspecific cytotoxic functions in euthymic mice may contribute to enhancement of resistance in vivo against transformed cells after treatment with this agent.

Modulation of immune responses in mice by oral administration of niflumic acid.

The in vivo effects of non-steroidal anti-inflammatory drugs (NSAIDs) on the host immune system are still poorly understood. However, through inhibition of prostaglandin synthesis, NSAIDs may exhibit immunomodulating properties. The present work was aimed at evaluating the influence of niflumic acid on immune responses when administered orally for 7 consecutive days to 8-week-old inbred mice. Immunological tests were performed 24 h after the arrest of the treatment. At a dosage of 50 mg/kg/day, niflumic acid exerted noticeable immunostimulating effects, as shown by an increase in plaque-forming cell numbers after in vivo immunization with sheep red blood cells, an augmentation of spleen cell proliferation responses to stimulation with T- or B-cell mitogens and of T-cell cytotoxic response to allogenic cells. Phagocytosis-induced chemiluminescence of peritoneal macrophages was also enhanced whereas interleukin-1 production by these cells was depressed, but without concomitant modification in interleukin-2 production by T-cells. Increasing the niflumic acid dosage to 75 mg/kg resulted in the disappearance of the immunostimulatory effects on lymphocytes responses. Macrophage activities were affected similarly in mice receiving 50 mg/kg. These results demonstrate that niflumic acid is able to stimulate in vivo several immunological functions and, consequently, to maintain host immune defenses. Interestingly, it depressed interleukin-1 production, known to play a major role in the inflammatory process.

Circannual rhythm in natural killer activity and mitogen responsiveness of murine splenocytes.

Seasonal variations were observed in murine splenic natural killer (NK) cell activity and also in murine lymphocyte responsiveness to mitogens, namely, concanavalin A, phytohemagglutinin, and lipopolysaccharide. The maximum and minimum splenic NK cell activities were observed in January-February and July-August, respectively. Conversely, maxima and minima of lymphoproliferative responses to all the three mitogens occurred in April-June and January-February, respectively. Such variations, when inferential statistics are used, appeared to be accounted for by circannual and other low-frequency (infradian) bioperiodicities. More specifically, the circannual rhythm in murine NK cell activity was demonstrated in data from a total of 356 mice collected over a period of 5 years. The various components of the immune system are characterized by a multifrequency time structure. The understanding of the organization of the immune system along the yearly scale may have bearings on that of the seasonal incidence of numerous infectious diseases and on the success/failure of immunotherapy.

In vivo immunopharmacological properties of tuftsin (Thr-Lys-Pro-Arg) and some analogues.

Tuftsin (Thr-Lys-Pro-Arg) is part of the Fc fragment of a leukophilic IgG and is a stimulator of the phagocytic activity of macrophages and polymorphonuclear cells (PMN) when cleaved from its carrier molecule. Tuftsin was shown to stimulate in vitro all PMN and macrophage functions examined through binding to specific cell surface receptors. In the present work, we provide further evidence that synthetic tuftsin administered to mice may act as an immunomodulator and that its effects on immune functions may result from a primary action on macrophages. After i.v. injection at a dosage of 25 micrograms/mouse, tuftsin stimulated effector (phagocytosis) and regulatory (IL1 production) functions of macrophages and potentiated DTH reaction. Lymphocyte functions (proliferative response to mitogens, T cell-mediated cytotoxicity, IL2 and gamma IFN production) were depressed at times at which macrophage activities were maximally enhanced, suggesting that negative regulatory functions of these latter cells were also stimulated. Tuftsin analogues were synthetized representing substitution or derivatization of the threonyl residue. The relative potencies of these analogues in augmenting phagocytosis-induced chemiluminescence of macrophages were tuftsin greater than or equal to (Gly1)-tuftsin greater than for-tuftsin greater than (for-Met1)-tuftsin greater than (Met1)-tuftsin. Concerning potentiation of DTH reaction the order was (Gly1)-tuftsin greater than or equal to (for-Met1)tuftsin greater than tuftsin greater than (Met1)-tuftsin greater than for-tuftsin. In contrast to tuftsin, none of the analogues induced depression of spleen cell reactivity to mitogens. In addition, (for Met1)-tuftsin administration resulted in an increased production of IL2 and IFN by ConA-stimulated spleen cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Early changes in immune parameters induced by an acute nonantigenic inflammation in mouse: influence of imuthiol.

Calcium pyrophosphate (CaPP)-induced pleurisy, may represent one of the simplest expressions of inflammation in that the irritant is a non-diffusible, non-antigenic and non-pyrogenic agent. Spleen or lymph node T or B cell numbers and activities, as well as NK activity, were modified at distance by CaPP-pleurisy. An intense increase in blood polymorphonuclear cells was also triggered by the inflammatory process. Treatment with imuthiol (sodium diethyldithiocarbamate), an agent known to be active on the T-cell lineage, restored towards control values the inflammatory response and tended to normalize white blood cell percentages altered by the inflammatory process. The findings suggest imuthiol could be employed as a virtually nontoxic and non-steroidal anti-inflammatory agent.