Basophil activation test with indomethacin to assess hypersensitivity to non-steroidal anti-inflammatory drugs: a preliminary study.

BACKGROUND: The basophil activation test has been investigated for diagnosing hypersensitivity to non-steroidal anti-inflammatory drugs (NSAIDs). This has not yet been done in relation to indomethacin. OBJECTIVE: First seek to establish the viable concentrations of indomethacin and the diluent propylene glycol (PPG) in relation to basophils then test this in patients with hypersensitivity to NSAIDs. MATERIALS & METHODS: The ideal concentrations of PPG and indomethacin were assessed by incubating them with basophils from an atopic donor and evaluating the intensity of expression of CD63 molecules by means of flow cytometry. We also evaluated the cell viability directly using the trypan blue in seven controls. Then indomethacin was tested in ten patients with hypersensitivity to NSAIDs compared with eight persons in control group. RESULTS: In relation to the toxicity of propylene glycol, concentrations less than or equal to 0.5% are safe. There was no cytotoxicity or nonspecific stimulation from using indomethacin at concentrations of 10 mcg/mL, 1 mcg/mL and 0.1 mcg/mL. Then indomethacin was tested at concentration of 10 mcg/mL diluted in 0.5% propylene glycol in both groups. There was no statistical difference in the intensity of activation of basophils comparing the group of patients with hypersensitivity to NSAIDs and the control group. CONCLUSIONS: As a diluent for indomethacin, PPG should be used at concentrations less than or equal to 0.5%. The indomethacin at concentration of 10 mcg/mL was not able to differentiate patients with and without hypersensitivity to NSAIDs.

Anti-inflammatory mechanism of action of azithromycin in LPS-stimulated J774A.1 cells.

Azithromycin is a macrolide antibiotic with well-described anti-inflammatory properties which can be attributed, at least partially, to its action on macrophages. We have previously shown, with 18 different macrolide molecules, that IL-6 and PGE2 inhibition correlates with macrolide accumulation, as well as with their binding to phospholipids in J774A.1 cells. The present study was performed in order to substantiate the hypothesis that biological membranes are a target for macrolide anti-inflammatory activity. By analyzing the effect of azithromycin on overall eicosanoid production, we found that in LPS-stimulated J774A.1 cells, azithromycin, like indomethacin, inhibited the synthesis of all eicosanoids produced downstream of COX. Upstream of COX, azithromycin inhibited arachidonic acid release in the same way as a cPLA2 inhibitor, while indomethacin had no effect. Further comparison revealed that in LPS-stimulated J774A.1 cells, the cPLA2 inhibitor showed the same profile of inhibition as azithromycin in inhibiting PGE2, IL-6, IL-12p40 and arachidonic acid release. Therefore, we propose that the anti-inflammatory activity of azithromycin in this model may be due to interactions with cPLA2, causing inadequate translocation of the enzyme or disturbing physical interactions with its substrates.

[The increase of bioavailability and anti-inflammatory effect of indomethacin loaded into phospholipid nanoparticles].

The ultrafine formulation on the base of plant phosphatidylcholine and antiinflammatory remedy indomethacin with nanoparticles less than 50 nm was obtained. Drug bioavailability after its peroral administration to rats was more than 2 fold higher as compared with free indomethacin. Increased antiinflammatory activity of indomethacin in phospholipids nanoparticles as compared with its free form was shown in two models of inflammation - adjuvant arthritis in rats and conconavalin A induced edema in mice. The increased bioavailability of indomethacin after administration of its phospholipid formulation allows to decrease a dose for achievement of therapeutic effect, that reduces risks of occurrence of collateral displays.

Effects of T cell-induced colonic inflammation on epithelial barrier function.

BACKGROUND: Epithelial barrier disturbance is thought to contribute to the pathogenesis of inflammatory bowel diseases; however, it remains unclear whether it is a primary defect participating to the onset of inflammation or only a consequence of sustained inflammation. METHODS: A time course study of epithelial barrier functions and immune mediators was performed in the CD4(+)CD45RB(hi) T cell transfer model of colitis using Ussing chambers. RESULTS: In nonreconstituted severe combined immunodeficiency (SCID) mice, no epithelial dysfunction was observed. However, after transfer of CD4(+)CD45RB(hi) T cells or total CD4(+) T cells, colon of SCID mice displayed a decreased epithelial resistance, even before overt microscopic inflammation had occurred. Sustained colitis of CD4(+)CD45RB(hi) T cell reconstituted mice was also associated with enhanced subepithelial resistance, enhanced paracellular permeability, and decreased net ion transport. All these reflect a disturbance of barrier function and may contribute to diarrhea. Epithelial resistance was positively correlated with interleukin 10 (IL-10) and transforming growth factor beta (TGF-beta) levels and net ion transport inversely correlated with tumor necrosis factor alpha (TNF-alpha) levels, pointing to the protective effect of IL-10 and TGF-beta and to a damaging effect of TNF-alpha. Indomethacin, a nonselective COX inhibitor, decreased epithelial resistance independent of T cells and inflammation, but its effect was more pronounced in inflamed colon. CONCLUSIONS: Induction of colitis by transfer of CD4(+)CD45RB(hi) T cells in SCID mice leads to changes in the colonic epithelium before colitis develops. Decreased epithelium resistance might contribute to the development of colitis; however, it is not sufficient to lead to chronic inflammation.

Interactions between osteosarcoma cell lines and dendritic cells immune function: An in vitro study.

Dendritic cells (DCs) might be partly responsible for the defective immune response in tumor bearing hosts, but no study in osteosarcoma patients is still available. Therefore, we investigated in vitro whether human osteosarcoma cell lines have an inhibitor effect on different types of DCs: CD14+DCs, DC1 and DC2. DCs derived from healthy donors were cultured with osteosarcoma cell lines and appropriate cytokine cocktails and analysed for the expression of co-stimulatory molecules (CD40, CD80, CD83, CD86, HLA-DR). Each interaction resulted in a lower phenotypic expression of the DCs maturation markers, especially on DC2. Moreover, the addition of various cytokines and compounds (rhIL-12, CD40L, Indometacin) induced the DC1 and DC2 subsets towards the Th1 pattern as shown by ELISA. Osteosarcoma highly interferes with an in vitro DCs immune function as antigen presenting cells. The understanding of tumor biology underlines the need for a specific osteosarcoma immunotherapy able to reverse this immune-surveillance inhibition.

Effect of prenatal administration of NSAIDs on the immune response in juvenile and adult rats.

In order to investigate the effect of non-steroidal anti-inflammatory drugs (NSAIDs) on the development of rat immunity, indomethacin (IND; 0.25, 0.5, or 1.0 mg/kg/day), acetyl salicylic acid (ASA; 90, 180, or 360 mg/kg/day), or diclofenac sodium salt (DSS; 0.5, 1.0, or 2.0 mg/kg/day) suspended in 0.5% methylcellulose aqueous solution, was orally administered once daily to five pregnant Sprague-Dawley (IGS) rats per group on days 18-21 of gestation. After parturition, the serum IgM and IgG levels, the spleen weight, and the number of spleen cells were measured in 3- and 8-week-old pups. Afterwards, immunophenotyping analysis of splenocytes or peripheral blood lymphocytes and T-dependent antibody response were performed. The number of spleen cells in 3-week-olds increased when 1.0 mg/kg of IND and 180 mg/kg of ASA were administered. Immunophenotyping analysis using flow cytometry (FCM) indicated that the proportion and number of CD45RA(+) cells increased, and the proportion of CD3(-) NKR-P1A(+) cells decreased in males when dosed with IND at 1.0 mg/kg or ASA at 180 mg/kg. The serum anti-KLH IgG antibody titer decreased in the males of the IND 1.0 mg/kg dosing group, the serum levels of anti-KLH IgM, total IgM, and IgG were not changed at all. These changes disappeared in 8-week-old pups. There were no effects on any of the parameters in the 3- and 8-week-olds of the DSS treatment group. These results suggest that IND or ASA administration to dams during late gestation either causes a change in the lymphocyte subsets, or that they suppress the T-dependent antibody response in juvenile males. Both of these changes eventually recover to intact levels later on during development. These results will contribute to the development of a technique for the assessment of developmental immunotoxicity and generate data on the effect of prenatal administration of NSAIDs on the developmental immune system in pups.

Thrombin, but not bradykinin, stimulates proliferation in isolated human osteoblasts, via a mechanism not dependent on endogenous prostaglandin formation.

Osteolysis or osteosclerosis often occurs in bone tissue adjacent to chronic inflammatory processes. Numerous cytokines and inflammatory mediators have been implicated as osteoclast-activating agents, explaining inflammation-induced bone resorption. In many cases, the cause of the sclerosis seen in these lesions is less thoroughly investigated. We have studied the effects of thrombin and bradykinin, 2 inflammatory mediators, on the rate of proliferation in isolated human osteoblasts (hOBs). Thrombin, at and above 1 U/mL, stimulated the rate of thymidine incorporation into hOBs. The absolute cell number also increased, as measured by an assay based on the detection of cell metabolism. A synthetic peptide ligand for the thrombin receptor enhanced the rate of [3H]thymidine incorporation in hOBs, indicating that thrombin-induced proliferation is mediated via the tetheric thrombin receptor. The thrombin-induced proliferation was not affected by indomethacin, excluding prostanoids as mediators of this effect. Bradykinin did not affect either the rate of thymidine incorporation, or number of cells in long-term cultures of hOBs. In conclusion, the inflammatory mediator, thrombin, stimulates proliferation in isolated human osteoblasts probably via the recently described G-protein-coupled tetheric thrombin receptor. Thrombin may therefore be involved as a mediator of inflammation-induced sclerosis and bone formation.

Use of type I and type IV hypersensitivity responses to define the immunopharmacological profile of drugs.

Administration of antigen suspended in incomplete Freund's adjuvant supplemented with either heat-killed Mycobacterium tuberculosis (complete Freund's adjuvant, CFA) or Bordetella pertussis toxin sensitizes animals so that subsequent antigen challenge leads to delayed-type (DTH) or immediate type hypersensitivity (ITH) responses, named type IV and type I, respectively. Appropriate timing of administration of drugs with respect to immunization or antigen challenge allowed to detect predominantly immunosuppressive, antiinflammatory or antianaphylactic activities. Among the reference drugs tested, only cyclosporin A (CsA) and dexamethasone (Dex) markedly inhibited DTH reaction, due to their immunosuppressive and antiinflammatory activities, respectively, whereas leflunomide and indomethacin resulted less potent. On the other hand, only dexchlorpheniramine, a histamine-receptor antagonist, afforded significant protection against anaphylactic shock, a form of ITH. Two new chemical entities were studied according to this protocol: ITF 1697, a chemically stabilized C-reactive protein-derived tetrapeptide, and ITF 2018, a leflunomide analogue. Data obtained with these new compounds showed that ITF 1697 has antianaphylactic activity, while ITF 2018 is endowed, mainly, with antiinflammatory activity. These results show that, through appropriate timing of administration, established in vivo models of immunologically mediated disease states allow an accurate profiling of the effects of pharmacologically active molecules and the detection of unsuspected activities for new drugs.

Recombinant interleukin-1 promotes leishmaniasis in susceptible mice.

Interleukin-1 exacerbates leishmanial lesions in Leishmania major-infected BALB/c mice. Indomethacin can modulate the effect of IL-1, so at least part of the IL-1 effect on disease progression is due to the induction of prostaglandin synthesis.

Role of interleukin 8 on leucocyte-endothelial cell adhesion in intestinal inflammation.

BACKGROUND: An important action of interleukin 8 (IL8) is stimulation of granulocytes. The object of this study was to assess the contribution of IL8 to the leucocyte-endothelial cell interactions associated with intestinal inflammation in the rat. METHODS: Two indomethacin injections (48 and 24 hours prior to the experiments) induced a longlasting ileitis in rats. The number of adherent and emigrated leucocytes, leucocyte rolling velocity, and shear rate were monitored in normal and inflamed mesenteric postcapillary venules. Some animals received a monoclonal antibody (MAb) against IL8 or CD11b/CD18 at 24 and 12 hours prior to the experiment. RESULTS: Indomethacin elicited a seven-fold increase in leucocyte adherence and a 5.4-fold increase in leucocyte emigration, while leucocyte rolling velocity was reduced by nearly 80%. The indomethacin induced increases in leucocyte adherence and emigration were significantly reduced (by 57% and 67%, respectively) while leucocyte rolling velocity was increased (to 63% of control) by the IL8-specific MAb. The level of inhibition seen with the IL8 MAb was similar to that associated with administration of a MAb directed against the leucocyte adhesion molecule CD11b/CD18. CONCLUSIONS: IL8 contributes to the leucocyte-endothelial cell interactions elicited in mesenteric venules by indomethacin.

Endotoxin stimulates interleukin-6 production in intestinal epithelial cells. A synergistic effect with prostaglandin E2.

OBJECTIVE: To test the hypothesis that endotoxin stimulates the release of interleukin-6 (IL-6) from intestinal epithelial cells and that this effect of endotoxin is regulated by prostaglandin E2 (PGE2). DESIGN: A rat intestinal crypt cell line, IEC-6, was cultured in the presence of lipopolysaccharide (LPS), 0.1 to 1.0 microgram/mL, and/or PGE2, 1 mumol/L. In other experiments, indomethacin, 20 mumol/L, was added to LPS-treated cells to block the effects of prostaglandins. Control wells contained medium alone. Levels of IL-6 were determined by the B9 murine hybridoma bioassay. Polymerase chain reaction was performed on RNA from control and LPS-treated cells to examine IL-6 message. RESULTS: Lipopolysaccharide and PGE2 induced IL-6 release from IEC-6 cells in a dose- and time-dependent fashion, and the substances interacted synergistically. Addition of indomethacin blunted the effect of endotoxin on IL-6 production, consistent with a stimulatory role of PGE2. Polymerase chain reaction demonstrated increased IL-6 messenger RNA in endotoxin-treated cells. CONCLUSIONS: Endotoxin and PGE2 stimulate IL-6 production in IEC-6 cells and interact synergistically. The endotoxin-stimulated IL-6 release may be regulated at the transcriptional level.

In vitro effects of live and killed Brucella abortus on bovine cytokine and prostaglandin E2 production.

Live and gamma-irradiated-killed Brucella abortus strain 2308 increased interleukin 1 (IL-1), but not interleukin 2 (IL-2), interferon-gamma (IFN-gamma), or prostaglandin E2 (PGE2) production when incubated with normal bovine peripheral blood mononuclear cells (PBMC). Live B. abortus was more effective than killed B. abortus in stimulating IL-1 production by normal PBMC. Both live and killed B. abortus were equally effective in suppressing IL-2 and IFN-gamma production by Concanavalin A-stimulated PBMC. Incubation of PBMC with the cyclo-oxygenase inhibitor, indomethacin, blocked PGE2 synthesis, but did not further enhance IL-1 production or prevent suppressed IL-2 and IFN-gamma production that was induced by live and killed B. abortus. These results suggest that B. abortus-induced suppression of IL-2 and IFN-gamma production did not appear to be mediated by the suppressive prostaglandin, PGE2, or other cyclo-oxygenase metabolites.

Some biochemical characteristics of the early phase of immunomodulation.

The study estimates some biochemical changes of chosen immunological and biochemical parameters after intraperitoneal application of a) immunomodulators of microbial origin--Propionibacterium acnes and Geotrichum candidum, b) chemical substances--indomethacin and phenobarbital. The estimation was concentrated above all to the changes of chemiluminiscence, phagocytic activities, the amount of cAMP in peritoneal macrophages, the amount of liver cytochrome P-450 and cAMP in the liver, spleen and thymus. The experiments also included histological examination of the spleen, thymus, lungs and diaphragm. The effect of Propionibacterium acnes was evident as soon as 24 hrs after application. In the frame of studied parameters, there was manifested the nonspecific activation of the immune system by the mechanisms independent to oxygen. The early changes were accompanied by the increase of cAMP in macrophages. In contrary, the intraperitoneal application of Geotrichum candidum activated the release of oxygen radicals from peritoneal macrophages. The amount of the cytochrome P-450 correlated to the intensity of immunomodulation. On the other hand, the induction of cytochrome P-450 by phenobarbital decreased the value of several immunity indices.

Effect of costimulants on interleukin-2-like activities from goat peripheral blood mononuclear cells.

Culture supernatants containing interleukin-2-like activities (CS-IL2) were prepared from goat peripheral blood cells (mononuclear cells 75% and polymorphonuclear cells 25%). These were stimulated with three costimulants, (tetradecanoyl phorbol acetate, indomethacin and calcium ionophore A23187), either alone or in different combinations, in RPMI-1640 medium (containing 0.5% bovine serum albumin (BSA)) with or without serum. After 18 h of incubation with costimulants, concanavalin A (Con A) was added and the incubation was continued for next 48 h. Higher interleukin-2 (IL-2)-like activities were generated in the culture supernatants prepared in RPMI-1640 growth medium containing 0.5% BSA without serum. Further, IL-2-like activities were much higher in culture supernatants obtained by stimulation with all the three costimulants, as well as Con A, than the two costimulants with Con A or any of the costimulants with Con A.

Tumor growth alters macrophage responsiveness to macrophage colony-stimulating factor during reactivity against allogeneic and syngeneic MHC class II molecules.

Tumor-induced changes in macrophage (M phi)2 accessory activities significantly suppress T-cell recognition of allogeneic and syngeneic major histocompatibility complex (MHC) class II molecules. Because these changes are often associated with altered responses to stimulatory and inhibitory cytokines, we investigated the possibility that tumor growth alters the contribution of a macrophage regulatory cytokine, macrophage colony-stimulating factor (M-CSF), during reactivity against allogeneic and syngeneic MHC class II molecules. T-cell reactivity against allogeneic MHC class II molecules was significantly suppressed by tumor-bearing host (TBH) M phi in the presence of M-CSF. M-CSF-induced suppression was independent of TBH M phi prostaglandin E2 (PGE2) synthesis. T-cell reactivity against syngeneic MHC class II molecules increased in the presence of M-CSF when normal host (NH) M phi served as the source of syngeneic molecules. However, T-cell reactivity against syngeneic MHC class II molecules in the presence of M-CSF did not change when TBH M phi served as stimulator/accessory cells. Although T-cell reactivity against NH syngeneic MHC class II molecules was additively increased by M-CSF and indomethacin (a PGE2 synthesis inhibitor) treatment, reactivity against TBH syngeneic MHC class II molecules increased solely through PGE2 synthesis inhibition. Admixtures of both NH and TBH M phi in the absence or presence of M-CSF suggest that tumor-induced suppression was not strictly due to decreased expression of MHC class II molecules. Collectively, these data suggest that TBH M phi are partly suppressive through altered responsiveness to M-CSF.

Comparative effects of Mycobacterium avium glycopeptidolipid and lipopeptide fragment on the function and ultrastructure of mononuclear cells.

Among the various lipids associated with the cell envelope of the Mycobacterium avium complex, the species-specific glycopeptidolipids (GPL) are responsible for distinguishing one serovar from another. In a continuing effort to study the immunomodulatory capabilities of these mycobacterial lipids, we have examined and compared the effects of the GPL and its lipopeptide fragment (beta-lipid) on mononuclear cell function. It was observed that the lymphoproliferative response of murine splenic mononuclear cells to mitogen stimulation was reduced by both the GPL and its lipopeptide fragment. Although the responsiveness appeared to be down-regulated to a greater degree by the beta-lipid, treatment with either GPL or beta-lipid resulted in the release of soluble factors from peritoneal macrophages that caused suppression of the lymphoproliferative responsiveness of splenic mononuclear cells. Flow cytometric analysis of peritoneal macrophages revealed that treatment with the beta-lipid fragment caused a marked decrease in expression of the C3bi complement receptor, Mac-1, on macrophages, whereas treatment with GPL resulted in a marked increase in the expression of Mac-2 receptor on macrophages. Treatment of peritoneal macrophages with either GPL or beta-lipid resulted in the release of tumour necrosis factor (TNF), as determined by an L929 biological cytotoxicity assay. Perturbation of macrophage membrane ultrastructure by both GPL and beta-lipid was confirmed by electron microscopy, and may be a possible explanation for the resulting alterations in mononuclear cell function observed in this study.

Activity of human peritoneal macrophages against a human tumor: role of tumor necrosis factor-alpha, PGE2 and nitrite, in vitro studies.

Human peritoneal macrophages collected from renal patients on continuous ambulatory peritoneal dialysis (CAPD) during inflammation-free periods were induced to express antitumor activity in vitro when cultured in the presence of bacterial lipopolysaccharide (LPS) and even more activity when they were kept in the presence of LPS + IND (indomethacin). The antitumor activity was expressed against a human tumor-cell line, RC43, either in a cell-to-cell contact set-up between the macrophages and the RC43 target cells or when the tumor cells were exposed to supernatants of the cultured macrophages. The antitumor activity of macrophages was correlated to a marked increase in production of tumor necrosis factor-alpha (TNF alpha), not correlated to an increase in nitrite production and inversely correlated to the production of PGE2. The RC43 tumor cells were susceptible to recombinant human TNF alpha, recombinant human IL-1 beta, sodium nitrite and the leukotriene LTB4. The results obtained suggest that activated human macrophages might represent a useful tool for cancer immunotherapy.

Inhibition of IL-2-dependent proliferation by a prostaglandin-dependent suppressor factor.

In the picryl chloride contact sensitivity system in mice, i.v. injections of trinitrobenzene sulfonic acid (TNBSA) prevents elicitation of delayed-type hypersensitivity reactions. This suppression is due in part to a non-specific, PG-dependent factor (TNBSA-F) that is induced by i.v. injection of TNBSA and is produced by pooled spleen and lymph node cells in vitro. Inasmuch as a role for lymphokines such as IL-2 has been postulated in delayed-type hypersensitivity, we determined the in vitro effects of TNBSA-F on the responsiveness of HT-2 target cells to IL-2. TNBSA-F induced a dose-dependent unresponsiveness of HT-2 cells to IL-2. The inhibitory activity was not present in supernatants from lymphoid cells of sham-treated mice. In the presence of indomethacin, spleen, and lymph node cells from TNBSA-immunized mice produced a factor whose activity was much reduced compared to TNBSA-F. This suggested that PG were required for TNBSA-F activity. However, PG alone did not induce the unresponsiveness because TNBSA-F but not sham-treated mice had inhibitory activity despite containing similar levels of PGE2. Rather, the combination of i.v. TNBSA injections and PG synthesis during production of TNBSA-F were required to produce a suppressive TNBSA-F. The inhibitory effect of TNBSA-F was not due to the presence of transforming growth factor-beta, soluble immune-response suppressor, INF-gamma, or JE in the factor preparation. Partial characterization showed a single peak of in vitro TNBSA-F activity (molecular mass approximately 35-55 kDa) by Sephadex G-200 gel filtration chromatography and by HPLC. In addition, TNBSA-F retained its activity after multiple cycles of freeze-thaw and heating for 1 h at 56 degrees C. The inhibitory effects of TNBSA-F on IL-2-induced proliferation suggest that suppression of delayed type hypersensitivity after i.v. administration of TNBSA may, in part, be due to a PG-dependent suppressor factor that inhibits the responsiveness of target cells to IL-2.

Lipopolysaccharide augments HLA-A,B,C molecule expression but inhibits interferon-gamma-induced HLA-DR molecule expression on cultured human endothelial cells.

The effect of bacterial lipopolysaccharide (LPS) on the expression of class I and II major histocompatibility complex (MHC) molecules on the surface of cultured human umbilical vein endothelial cells (HUVEC) was determined by indirect immunofluorescent staining followed by flow cytometric analysis. LPS at concentrations higher than 0.01 micrograms/ml augmented class I MHC (HLA-A,B,C) expression on HUVEC in a concentration-dependent manner. Optimal augmentation, approximately sixfold compared with control, was seen with 10 micrograms/ml of LPS. Time-course experiments indicated that the augmentation was maximal on Day 4. In contrast, LPS had no effect on the induction of class II MHC (HLA-DR) molecules and at concentrations higher than 0.01 micrograms/ml inhibited the interferon-gamma(IFN-gamma)-induced class II MHC expression. The inhibition was about 60% at the concentration of 100 micrograms/ml of LPS. Interleukin-1 (IL-1) had a similar effect as LPS on class I and II MHC expression. However, LPS appeared to affect MHC expression directly and not through production of IL-1 or cyclo-oxygenase pathway products, since anti-IL-1 antibodies or an inhibitor of cyclo-oxygenase pathway products, indomethacin, failed to reverse the effects of LPS. These data stress the role of LPS as a direct modulatory factor of class I and II MHC expression on endothelial cells during the development of immune and inflammatory response against Gram-negative bacteria.