C.J.A.M. Tak

Nicotine inhibits the in vitro production of interleukin 2 and tumour necrosis factor-α by human mononuclear cells

Smoking protects against ulcerative colitis (UC), and treatment with nicotine patches has a beneficial symptomatic effect in patients with UC. To find an explanation for this response to nicotine in UC, we assessed the effects of nicotine on cytokine production by mononuclear cells (MNC). MNC were isolated from peripheral blood from healthy volunteers. Non-adherent MNC were preincubated with varying concentrations of nicotine or prednisolone for 24 h followed by addition of phytohemagglutinin (10 micrograms/ml). The concentrations of interleukin 2 (IL-2) and tumour necrosis factor-alpha (TNF alpha) in the supernatants were determined by ELISA. Nicotine as well as prednisolone caused a significant inhibition of IL-2 and TNF alpha production. The maximum inhibition caused by nicotine was about 50% of that caused by prednisolone and was reached at concentrations equivalent to nicotine levels measured in plasma of smokers. These results indicate that nicotine exerts its immunoregulatory role through modulation of the cytokine production by non-adherent mononuclear cells.

Serum levels of tumor necrosis factor determine the fatal or non-fatal course of endotoxic shock

The role of tumor necrosis factor alpha (TNF alpha) in endotoxin-induced shock was investigated in pigs receiving 5 micrograms kg-1 of Escherichia coli endotoxin (LPS) during 60 min of continuous infusion into the superior mesenteric artery. LPS concentration in aortic plasma, as determined by a chromogenic Limulus amoebocyte lysate (LAL) test, reached a peak of approximately 1000 ng l-1 during LPS infusion, and declined rapidly after discontinuation of the infusion. Serum TNF levels were determined by a bioassay using the L929 murine transformed fibroblast line. Eight of the 17 animals infused with LPS died within 30 min after beginning LPS administration, while the other 9 pigs survived beyond the experimental observation period of 3 h, although they were in a state of shock. No difference in LPS concentration was found between the survivors and the non-survivors. However, the serum TNF levels in non-survivors were significantly higher than in survivors when measured at 30 min after beginning LPS administration. In survivors, the peak increase in serum TNF levels was measured at 60 min after the beginning of LPS injection and returned rapidly to the baseline values. Although the role of TNF inducing rapid death seems to be dominant, the hemodynamic, hematology and blood chemistry disturbances seen during shock continued in survivors long after the return of TNF to baseline levels. These findings indicate that besides TNF other mediators are also involved in the LPS infusion-induced shock.

Interactions between cytokines and eicosanoids: a study using human peritoneal macrophages

To examine the interactions between the main pro-inflammatory cytokines and eicosanoids produced by human inflammatory cells, human peritoneal macrophages (hp-M phi) were isolated from ascitic fluid of patients with portal hypertension. Interactions between interleukin-1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha (TNF-alpha), leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) were studied by addition or inhibition of several cytokines and eicosanoids: human recombinant IL-1 beta (hrIL-1 beta) addition, LTB4 addition and 5-lipoxygenase inhibition (6-hydroxy-2-(4-sulfamoylbenzylamino)-4,5,7-trimethylbenzothiaz ole hydrochloride (E6080)), PGE2 addition and cyclooxygenase inhibition (indomethacin). In hp-M phi hrIL-1 beta stimulated the LTB4 production, while the PGE2 production was inhibited. HrIL-1 beta had no significant effect on IL-6 production in hp-M phi. LTB4 did not regulate IL-1 beta and IL-6 production. Increasing PGE2 down regulated the TNF-alpha production, but did not effect the IL-1 beta and IL-6 production.

Levels of soluble intercellular adhesion molecule 1, eicosanoids and cytokines in ascites of patients with liver cirrhosis, peritoneal cancer and spontaneous bacterial peritonitis

The levels of the eicosanoids leukotriene B4, prostaglandin E2, prostacycline and thromboxane B2, the cytokines interleukin-1 beta, interleukin-6 and tumour necrosis factor-alpha and soluble intercellular adhesion molecule 1 were measured in ascites and plasma samples of patients with liver cirrhosis (53), peritoneal cancer (26) and spontaneous bacterial peritonitis (10) to assess their value as a possible diagnostic and prognostic parameter in the course of the disease. Soluble intercellular adhesion molecule 1, of the eicosanoids prostaglandin E2 and leukotriene B4, and the protein concentration in ascites were all significantly elevated in ascites of patients with peritoneal cancer in comparison to ascites of patients with liver cirrhosis. In ascites of patients with spontaneous bacterial infection interleukin-6 concentration was significantly elevated and the protein concentration was significantly lower in comparison to the other two groups. None of these parameters, however, seems to be of practical use as a diagnostic parameter, as there is an overlap between all the levels of these mediators in ascites of liver cirrhosis, peritoneal cancer and spontaneous bacterial peritonitis group. Soluble intercellular adhesion molecule 1 levels were much higher in plasma than in ascites, in contrast to interleukin-6 levels which were much higher in ascites than in plasma. Soluble intercellular adhesion molecule 1 in ascites correlated with soluble intercellular adhesion molecule 1 in plasma (r = 0.6926, P = 0.0001). Soluble intercellular adhesion molecule 1, interleukin-6 and the number of polymorphonuclear cells in peritoneal fluid correlated during episodes of infection in patients with a peritonitis. For this reason soluble intercellular adhesion molecule 1 and interleukin-6 could be of prognostic value for patients with peritonitis.

Intestinal permeability and contractility in murine colitis.

We developed an in vitro organ bath method to measure permeability and contractility simultaneously in murine intestinal segments. To investigate whether permeability and contractility are correlated and influenced by mucosal damage owing to inflammation, BALB/c mice were exposed to a 10% dextran sulphate sodium (DSS) solution for 8 days to induce colitis. The effect of pharmacologically induced smooth muscle relaxation and contraction on permeability was tested in vitro. Regional permeability differences were observed in both control and 10% DSS-treated mice. Distal colon segments were less permeable to 3H-mannitol and 14C-PEG 400 molecules compared with proximal colon and ileum. Intestinal permeability in control vs. 10% DSS mice was not altered, although histologic inflammation score and IFN-gamma pro-inflammatory cytokine levels were significantly increased in proximal and distal colon. IL-1beta levels were enhanced in these proximal and distal segments, but not significantly different from controls. Any effect of pharmacologically induced contractility on intestinal permeability could not be observed. In conclusion, intestinal permeability and contractility are not correlated in this model of experimentally induced colitis in mice. Although simultaneous measurement in a physiological set-up is possible, this method has to be further validated.

Production of inflammatory mediators by human macrophages obtained from ascites

Ascites is a readily available source of human macrophages (M phi), which can be used to study M phi functions in vitro. We characterized the mediators of inflammation produced by human peritoneal M phi (hp-M phi) obtained from patients with portal hypertension and ascites. The production of the cytokines interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) was found to be lipopolysaccharide (LPS) concentration dependent (0-10 micrograms/ml) with a maximal production at 10 micrograms/ml and also dependent on the time of exposure to the stimulus (0-36 h). IL-1 beta, IL-6 and TNF-alpha production after LPS administration reached a plateau at 24 h. In vitro stimulation for 24 h with LPS does not influence the eicosanoid production from endogenous arachidonate. 13 min of exposure of the cells to the calcium ionophore A23187 gives a significant increase in eicosanoid production from both exogenous and endogenous arachidonate. The main eicosanoids produced are the 5-lipoxgenase products LTB4 and 5-hydroxyeicosatetraenoic acid (HETE). The increase in production of the other eicosanoids is not significant. The eicosanoid production depends on the stimulus concentration. The optimal A23187 concentration is 1 microM. Oxygen radical production was measured in the M phi by a flowcytometric method. The fluorescence intensity of phorbol 12-myristate 13-acetate stimulated and dihydro-rhodamine 123 loaded hp-M phi increases significantly after 15 min. We conclude that LPS stimulation of hp-M phi from liver disease results in similar production of IL-1 beta, IL-6 and TNF-alpha, but that the profile of the eicosanoid production of these M phi stimulated with LPS and A23187 differs from M phi of other origin and species.

A comparison between two methods for measuring tumor necrosis factor in biological fluids

The current study was undertaken to compare two methods for the efficiency of measuring tumor necrosis factor (TNF-α) in biological fluids, which is species undependent, reliable, sensitive, simple and not expensive. We have compared the MTT tetrazolium cytotoxic assay [1,2] and the3H-thymidine (3H-TdR) incorporation cytostatic assay for measuring the anti-tumor activity of human recombinant TNF-α, of human colonic tissue and of supernatants ofin vitro stimulated human and rat peritoneal macrophages. Two target cell-lines, namely murine myelomonocytic leukaemia WEHI-164- and L-929-transformed murine fibroblast cell-lines, were used in the MTT assay. The L-929 line was also used in the3H-TdR assay. WEHI-164 was more sensitive than the L-929 cell-line in the MTT cytotoxic assay. Furthermore, the MTT assay was more sensitive to TNF-α than the3H-TdR assay. Both methods can be used for the detection of anti-tumor activity in biological fluids but the MTT cytotoxic method has the advantage of being more sensitive and more simple.

Activity of human peritoneal macrophages against a human tumor: role of tumor necrosis factor-α, 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.

Somatostatin does not attenuate intestinal injury in dextran sodium sulphate-induced subacute colitis.

FRom several in vitro and in vivo studies involvement of somatostatin (SMS) in intestinal inflammation emerge. Acute colitis induced in rats is attenuated by the long-acting SMS analogue octreotide. We studied the potential beneficial effect of SMS on non-acute experimental colitis. BALB/c mice received either saline, SMS-14 (36 or 120 microg daily) or octreotide (3 microg daily) subcutaneously delivered by implant osmotic pumps. A non-acute colitis was induced by administration of dextran sodium sulphate (DSS) 10% in drinking water during 7 days. DSS evoked a mild, superficial pancolitis, most characterized by mucosal ulceration and submucosal influx of neutrophils. Neither SMS-14 nor octreotide reduced mucosal inflammatory score or macroscopical disease activity, although reduction of intestinal levels of interleukin-1beta (IL-1beta), IL-6 and IL-10 during DSS was augmented both by SMS and octreotide. A slight increase of neutrophil influx was seen during SMS administration in animals not exposed to DSS. In conclusion, SMS or its long-acting analogue did not reduce intestinal inflammation in non-acute DSS-induced colitis. According to the cytokine profile observed, SMS-14 and octreotide further diminished the reduction of intestinal macrophage and Th2 lymphocyte activity.

Indomethacin stimulation of macrophage cytostasis against MOPC-315 tumor cells is enhanced by endogenous metabolites of lipoxygenase and counteracted by prostaglandin E2

ConclusionsWe have shown that indomethacin stimulation of macrophage cytostasis against MOPC-315 tumor cells can be counteracted by PGE2 and by the lipoxygenase inhibitor NDGA. The results are congruent with the earlier finding, which showed that LTD4 reinforces the effect of indomethacin on macrophage cytostasis, which thus is modulated by the balance between cyclooxygenase and lipoxygenase metabolites.