Porphyromonas gingivalis antagonises Campylobacter rectus induced cytokine production by human monocytes

Porphyromonas gingivalis and Campylobacter rectus are two major bacterial species implicated in the pathogenesis of periodontitis. P. gingivalis can antagonise the inflammatory response to other periodontal pathogens, a property commonly attributed to its lipopolysaccharide (LPS). The aim of this study was to investigate the capacity of P. gingivalis to antagonise C. rectus induced cytokine stimulation from human monocytes, and to investigate the involvement of its LPS. Primary human monocytes and Monomac-6 cells were challenged with culture supernatants from P. gingivalis and C. rectus, and levels of IL-1beta, IL-6 and IL-8 produced were measured by ELISA after 6h incubation. Purified P. gingivalis LPS was also added alone or in combination with C. rectus culture supernatant. Both species significantly stimulated the production of all three cytokines from the two cell lines, but P. gingivalis was considerably weaker inducer. Co-stimulation of the cells with P. gingivalis and C. rectus suppressed the cytokine-stimulatory capacity of the latter. P. gingivalis LPS alone was sufficient to antagonise IL-6 and IL-8, but not IL-1beta stimulation by C. rectus. In conclusion, mixed infections may impair host immune responses by reducing pro-inflammatory cytokine levels, which may be of relevance to the pathogenesis of periodontitis.     

Increased production of tumor necrosis factor and prostaglandin E2 by monocytes in cancer patients and its unique modulation by their plasma

Summary We investigated the role of monocytes in the production of tumor necrosis factor (TNF) and prostaglandin E₂ (PGE₂) in 77 cancer patients with malignancies of the digestive tract, using 30 normal individuals and 18 noncancer patients as controls. Monocytes were incubated with lipopolysaccharide for 20 h, and TNF production and PGE₂ production were analyzed by bioassays. Elevated levels of TNF (>512 U/ml) and PGE₂ (>8 ng/ml) production were demonstrated in many cancer patients when these factors were induced in the medium with 10% fetal bovine serum. The elevated level of TNF was seen to be restricted for the most part to patients with malignancies. Thus, 51 out of 59 cancer patients (86%), consisting of 44 primary cancer patients and 15 recurrent cancer patients, showed an increased level of TNF. In contrast, almost all of 18 postoperative cancer patients showed TNF levels comparable to those of normal individuals. Furthermore, 16 primary cancer patients were also demonstrated to have reduced levels of TNF production by monocytes after curative operation. When 10% cancer-patient plasma was added to the induction culture, TNF production by monocytes was drastically suppressed in the cancer patients. Interestingly, the same addition of plasma induced a prominent enhancement of PGE₂ production in the cancer patients. The plasma of noncancer patients did not modulate production of these factors. No TNF activity was found in the plasma of cancer patients, but such plasma did contain an increased level of PGE₂ (100–300 pg/ml). Although PGE₂ (>2ng/ml) was able to suppress TNF production by monocytes, the addition of 10% plasma PGE₂ was not enough to induce suppression. An unknown factor(s) in the plasma of cancer patients may uniquely modulate the elevated TNF and PGE₂ production in these patients.     

Blood prostaglandin A (PGA) levels in normal human subjects

Blood prostaglandin A levels were measured in ten healthy subjects under different conditions of collection and storage.Plasma levels ranged from 1.20 to 1.81 ng/ml (M ± SE = 1.50 ± 0.10) in 5 females to 1.23 to 1.68 ng/ml (1.45 ± 0.09) in 5 males, when centrifuged and frozen immediately after collection. Storate at 4°C for varying times up to 24 hours and at 22°C (room temperature) up to 4 hours did not affect mean plasma concentrations significantly, but increased the range obtained to 1.00 to 2.47 ng/ml for both male and female groups.Serum concentrations differed in males and females and were lower than corresponding mean plasma values for males and higher for females. Mean serum concentrations were 1.77 ± 0.08 ng/ml in females and 1.18 ± 0.05 ng/ml in males and did not change significantly up to 24 hours of storage at 4°C.These results suggest that prostaglandin A assayed in both plasma and serum under the conditions described is stable and should allow for greater flexibility in sampling under different experimental conditions.     

Inhibition of phospholipase activity in human monocytes by IFN-gamma blocks endogenous prostaglandin E2-dependent collagenase production

Previous studies from our laboratory have demonstrated that exposure of human monocytes to a stimulant, such as Con A, results in the production of the enzyme collagenase through PGE2-dependent pathway. Inasmuch as rIFN-gamma has been shown to modulate monocyte/macrophage PG synthesis, we examined the effect of rIFN-gamma on the activation sequence leading to collagenase production. The addition of rIFN-gamma (10 to 1000 U/ml) to Con A-stimulated monocytes resulted in a dose-dependent inhibition of PGE2 and collagenase synthesis. The suppression of collagenase production by rIFN-gamma was related to its ability to reduce PGE2 levels as demonstrated by the restoration of collagenase activity by the addition of PGE2. HPLC analysis of the arachidonic acid (AA) metabolites released by monocytes showed that rIFN-gamma caused a reduction in the release of AA and products of the cyclooxygenase and lipoxygenase pathways. These data indicated that rIFN-gamma decreased eicosanoid production by inhibiting the release of AA from phospholipids. This conclusion was supported by the reduction in membrane bound phospholipase activity in rIFN-gamma-treated monocytes. Moreover, the inhibition by rIFN-gamma of PGE2 and collagenase was reversed by the addition of phospholipase A2. Our findings demonstrate that rIFN-gamma inhibits phospholipase activity in activated monocytes and as a result blocks PGE2-dependent collagenase synthesis.     

Stimulation of prostaglandin E2 and thromboxane B2 production by human monocytes in response to interleukin-2

Interleukin 2 (IL-2) is a potent lymphokine involved in the regulation of immune responses and is classically regarded as a stimulus for the activation and growth of T-cells. Recent reports have demonstrated the IL-2 dependent activation of human peripheral blood lymphocytes into lymphokine activated killer cells capable of lysing tumor cells both in vitro and in vivo. In this study we report data which clearly show IL-2 may also act to down-regulate the immune response by inducing the synthesis of arachidonic acid metabolites with known immunosuppressive actions. Stimulation of peripheral human blood monocytes with IL-2 caused an increased production of prostaglandin E2 (PGE2) and thromboxane (TXB2) in a dose-dependent manner. Kinetic analysis showed no increase above controls after 6 hours and maximal levels by 10 hours; elevated levels were maintained after 45 hours of incubation. After 20 hours of stimulation with 2000 U/ml IL-2, the level of PGE2 and TXB2 were greater than three-fold above controls, 0.7 and 19 ng/10(6) cells, respectively. The stimulation was relatively specific in that neither prostacyclin nor leukotrienes were produced in response to IL-2. These data demonstrate that IL-2 acts on human monocytes to induce the secretion of PGE2 and TXB2.     

Regulation of interleukin-1 production in murine macrophages and human monocytes by a normal physiological constituent

The in vitro production of large quantities of interleukin-1 (IL-1) in mouse peritoneal exudate macrophages and human peripheral blood monocytes is possible through the use of the proteolytic enzyme pepsin and its zymogen pepsinogen. Equal amounts of IL-1 are generated by pepsin in the absence or presence of polymixin B. The addition of pepsin or pepsinogen had no effect on the proliferation of C3H/HeJ thymocytes to the plant mitogen phytohemagglutinin. Pepsin and pepsinogen are present in significant quantities in immune cells and the plasma. Although little is known concerning the physiological role of pepsin and pepsinogen outside of the gastrointestinal system, it may be proposed that the in vivo production of IL-1 may in part be regulated by the cellular and plasma concentrations of pepsin and pepsinogen.     

Hyperthermia induced NFκB mediated apoptosis in normal human monocytes

Conceptual approaches of heat-induced cytotoxic effects against tumor cells must address factors affecting therapeutic index, i.e., the relative toxicity for neoplastic versus normal tissues. Accordingly, we investigated the effect of hyperthermia treatment (HT) on the induction of DNA fragmentation, apoptosis, cell-cycle distribution, NFκB mRNA expression, DNA-binding activity, and phosphorylation of IκBα in the normal human Mono Mac 6 (MM6) cells. For HT, cells were exposed to 43°C. FACS analysis showed a 48.5% increase in apoptosis, increased S-phase fraction, and reduced G2 phase fraction after 43°C treatments. EMSA analysis showed a dose-dependent inhibition of NFκB DNA-binding activity after HT. This HT-mediated inhibition of NFκB was persistent even after 48 h. Immunoblotting analysis revealed dose-dependent inhibition of IκBα phosphorylation. Similarly, RPA analysis showed that HT persistently inhibits NFκB mRNA. These results demonstrate that apoptosis upon HT exposure of MM6 cells is regulated by IκBα phosphorylation mediated suppression of NFκB.     

Leukotrienes augment interleukin 1 production by human monocytes

The effects of leukotrienes (LT) on production of interleukin 1 (IL 1) by human peripheral blood monocytes were examined. LTB4 enhanced IL 1 production by lipopolysaccharide (LPS)-stimulated monocytes twofold to threefold, and the most efficient concentrations of LTB4 were 10(-8) to 10(-7) M. LTD4 also enhanced IL 1 production, but to a lesser extent than LTB4. Adherence-purified, but otherwise unstimulated, human monocytes could also be induced to produce IL 1 in response to LTB4. Similarly, IL 1 production by monocytes stimulated with the known IL 1 inducers muramyl dipeptide, silica, or zymosan was also enhanced by LTB4. Inhibition of cyclooxygenase with use of indomethacin during IL 1 production by LPS-treated monocytes enhanced thymocyte response to IL 1, but LTB4 further enhanced IL 1 production when added to indomethacin-treated monocyte cultures. Neither LTB4 nor indomethacin had any direct effect on thymocyte proliferation. Optimal enhancement of IL 1 production occurred when LPS and LTB4 were present together at the initiation of the 24-hr monocyte culture. Significant enhancement was also observed, however, when monocyte cultures were either preincubated with LTB4 before addition of LPS or cultured with LPS alone for 3 hr before addition of LTB4. These results indicate that leukotrienes can modulate IL 1 production by human monocytes and suggest that they may play a role in IL 1-mediated functions of monocytes in inflammatory and immune reactions.     

27-hydroxycholesterol: production rates in normal human subjects.

We attempted to quantitate production of bile acid via the 27-hydroxylation pathway in six human subjects. After bolus intravenous injection of known amounts of [24-14C]cholic acid and [24-14C]chenodeoxycholic acid, each subject underwent a constant intravenous infusion of a mixture of [22, 23-3H]-27-hydroxycholesterol and [2H]-27-hydroxycholesterol for 6;-10 h. Production rate of 27-hydroxycholesterol was calculated from the infusion rate of [2H]-27-hydroxycholesterol and the serum ratio of deuterated/protium 27-hydroxycholesterol, which reached a plateau level by 4 h of infusion. Conversion of 27-hydroxycholesterol to cholic and chenodeoxycholic acids was determined from the 3H/14C ratio of these two bile acids in bile samples obtained the day after infusion. In five of the six subjects, independent measurement of bile acid synthesis by fecal acidic sterol output was available from previous studies. Endogenous production of 27-hydroxycholesterol averaged 17.6 mg/day and ranged from 5.0 to 28.2 mg/day, which amounted to 8.7% (range 3.0;-17.9%) of total bile acid synthesis. On average 66% of infused 27-hydroxycholesterol was converted to bile acid, of which 72.6% was chenodeoxycholic acid.These data suggest that relatively little bile acid synthesis takes place via the 27-hydroxylation pathway in healthy humans. Nevertheless, even this amount, occurring predominantly in vascular endothelium and macrophages, could represent an important means for removal of cholesterol deposited in endothelium.     

Calcitonin enhances production of prostaglandins by stimulated human monocytes

Stimulated monocytes produce prostaglandins that may play a role in bone resorption. We studied the effects of salmon calcitonin (sCT) on human monocyte production of various prostaglandin metabolites. Latex particle-stimulated human monocyte production of prostaglandin E2, thromboxane A2 measured as thromboxane B2, and prostacyclin measured as 6-keto-PGF1 alpha was each increased in the presence of sCT. This effect required surface stimulation, was blocked by indomethacin, was less marked with equivalent concentrations of human calcitonin, and was not seen with parathyroid hormone. Calcitonin specifically affects prostaglandin pathways in stimulated human monocytes.     

5''-Methylthioadenosine in urine from normal subjects and cancer patients

A new type of device can prepare liposomes continuously, in large quantities and with excellent aqueous space capture efficiency. At initial lipid concentration of 300 mumol/ml these liposomes capture approx. 75% of cytosine arabinoside used as an aqueous space marker. Liposome size can be reduced by increasing the number of times the preparations are recycled through the microemulsifier. Liposomes less than 0.1 micron in diameter, as shown by electron microscopy, can be made easily. Liposomes prepared at 300 mumol/ml, composed of phosphatidylglycerol/phosphatidylcholine/cholesterol in a 0.1:0.4:0.5 molar ratio leaked less than 1% of entrapped cytosine arabinoside (Ara-C) at 4 degrees C, and less than 10% Ara-C at 37 degrees C plus serum, over a 48 h period. These liposomes could be useful for a number of applications including diagnostics, therapeutics and model membrane studies.     

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained in vitro were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E2. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E2 release on the concentration of isoproterenol were similar, each response was maximal at 10(-6) M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E2 from amnion discs. Although prostaglandin E2, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, may be regulators of prostaglandin production by the amnion.     

Tumour-cell-induced production of tumour necrosis factor by monocytes of gastric cancer patients receiving BCG immunotherapy

Summary Human peripheral blood monocytes cocultured with tumour cells were used as an in vitro model of in situ interactions between tumour-infiltrating macrophages and the tumour. Tumour cells stimulated de novo expression of the human tumour necrosis factor (TNF) gene in monocytes and caused the release of TNF into the culture supernatant. A group of 14 patients with stage IVA gastric cancer receiving adjuvant chemotherapy (5-FU, Adriamycin, mitomycin C: FAM) or immunochemotherapy (BCG+FAM) was investigated for the ability of monocytes to produce TNF in vitro upon stimulation with tumour cells or purified protein derivative of tuberculin (PPD). Patients were followed at biweekly intervals, i.e. before each instillation of BCG epicutaneously over a period of 10 weeks. It was found that monocytes of some patients receiving BCG at the end of the observation period had an enhanced ability to produce TNF following stimulation with tumour cells. In contrast, such production was not substantially altered during the study period in patients on chemotherapy. PPD-induced TNF production was much weaker and was not significantly changed during this observation time. We infer that BCG immunotherapy may induce the subtle changes in some cancer patients that lead to an increased interaction between monocytes and tumour cells and result in enhanced production of cytokine(s) with antitumour properties.     

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E₂. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E₂ release on the concentration of isoproterenol were similar, each response was maximal at 10⁻⁶ M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E₂ from amnion discs. Although prostaglandin E₂, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, my be regulators of prostaglandin production by the amnion.     

Platelet-activating factor induces the production of leukotrienes by human monocytes

The aim of this study was to evaluate the role of platelet-activating factor (PAF) as a stimulator of leukotriene production by human monocytes. The production of leukotrienes was time- and concentration-dependent. Release of leukotrienes was half-maximal after 2 min and reached a maximum after 10 min. At a concentration of 10(-8) M, PAF induced the production of 0.14 +/- 0.01 ng LTB4/10(6) cells (mean +/- S.E., n = 8). At concentrations of 10(-6) M, PAF induced the production of 1.0 +/- 0.04 ng LTB4 and 0.22 +/- 0.03 ng peptidoleukotrienes (mean +/- S.E., n = 16). There was no metabolism of LTB4 as judged from stability of [3H]LTB4 added to the incubations. LTC4 was slowly metabolized by human monocytes to LTD4 and LTE4. The two specific PAF-receptor antagonists BN 52021 and WEB 2086 in concentrations of 10(-4) and 10(-6) M, respectively, inhibited the PAF (10(-6) M) stimulated LTB4 production completely. In this study, we demonstrate that nanomolar concentrations of PAF can stimulate the production of LTB4 and peptidoleukotrienes in human monocytes by a receptor-mediated mechanism.     

Influence of surgery on in-vitro cytokine production by human monocytes

Surgery leads to significant modulation of the immune system, in which cytokines play a major role. Circulating interleukin 6 (IL-6) and IL-1 have been reported following surgery whereas tumor necrosis factor alpha (TNF-alpha) is only found in gut ischemia-associated surgery. We have investigated the consequences of surgery on in-vitro cytokine production by human monocytes stimulated by lipopolysaccharide (LPS) and staphylococcal toxic shock syndrome toxin-1 (TSST-1). Comparisons were made between the responsiveness of cells obtained the day before (D-1), during (D0) and after (D1, D2, D3) surgery. Patients undergoing abdominal aortic surgery (N = 9), carotid surgery (N = 4) and spinal surgery (N = 4) have been studied. A significant decrease of TNF-alpha, IL-1 beta and IL-1 alpha production by monocytes prepared from blood samples taken during the surgery was noticed, whereas IL-6 production was not significantly modified. On D2 a significant increase of monocyte responsiveness was observed and levels of cytokine productions rose back to initial values by the end of the follow up. The diminished in-vitro cytokine production observed during surgery might be the consequence of the effects of anaesthetic drugs, whereas the enhancement observed on D2 might reflect the surgical stress, leading to in-vivo priming of circulating monocytes.     

Tumor cytotoxicity and interleukin 1 production of blood monocytes of lung cancer patients

Summary The effects of lung cancer on the abilities of blood monocytes to produce interleukin-1 and to mediate antitumor activity were examined. The functional integrity of blood monocytes was determined by their capacity to respond in vitro to a variety of activating agents and become tumoricidal, as assessed by a radioactive release assay and ability to produce interleukin-1 in vitro. The results show that the presence of lung cancer significantly increased the number of harvested blood monocytes and that the spontaneous tumoricidal activity of these monocytes was slightly high as compared to monocytes obtained from healthy donors. The production of interleukin-1 by monocytes of healthy donors and lung cancer patients was similar. Blood monocytes obtained from lung cancer patients were less cytotoxic against allogeneic A375 melanoma cells as compared with those of healthy donors subsequent to incubation with a soluble muramyl dipeptide analog or lipopolysaccharide, but were as tumoricidal as those from healthy donors when activated with lipophilic muramyl tripeptide (MTP-PE) entrapped in multilamellar liposomes. The finding that monocytes of patients with lung cancer can respond to MTP-PE encapsulated in liposomes, recommends the use of these liposomes in therapy of human lung cancer.