Release of TNF-alpha from in vitro-stimulated monocytes is negatively associated with serum levels of apolipoprotein B in patients with type 2 diabetes.

Impaired course of inflammation is a likely mechanism behind a number of diabetic complications. The present study was undertaken to investigate lipopolysaccharide-induced production of tumour necrosis factor (TNF)-alpha in monocytes from patients with type 2 diabetes and to assess its relationship with diabetes-associated metabolic abnormalities. Monocytic TNF-alpha mRNA production was lower in the diabetic participants compared to their corresponding controls. Diabetic subjects who had been receiving simvastatin treatment had TNF-alpha mRNA production similar to that of the healthy participants. The release of TNF-alpha from diabetic cells correlated negatively with serum levels of apolipoprotein B (apoB) (R = -0.755, P = 0.001), total plasma cholesterol (R = - 0.702, P = 0.002) and the presence of retinopathy (R = -0.572, P = 0.021). No such associations were found in the control subjects. In a multiple linear regression model, only the level of apoB and diabetes duration demonstrated significant effects on the release of TNF-alpha, with apoB alone accounting for 57% of the variation. We conclude that production of TNF-alpha mRNA in response to the bacterial stimulant is compromised in poorly controlled type 2 diabetes. Lipid abnormalities are associated with the observed defect. Impaired cytokine production represents a significant defect in the functioning of the immune system and may contribute to aberrations in the course of inflammation in the diabetic state.

Critical role of mast cells in morphine-mediated impairment of zymosan-induced peritonitis in mice.

OBJECTIVE AND DESIGN: Zymosan-induced peritoneal inflammation is significantly inhibited in mice injected with an irritant supplemented with morphine. The aim of the present study was to examine the putative mast cell involvement in this inhibition. SUBJECTS: Peritonitis was induced in WBB6FI mice (genetically mast cell-deficient W/Wv and their control littermaters +/+) and in Balb/c mice, with normal mast cells (MC) and mast cell-depleted (MCx) by pretreatment with compound 48/80. Bone marrow leukocytes from intact Balb/c mice were tested for their sensitivity to chemoattractants after in vitro incubation with morphine (10(-6) M), with or without preincubation with naltrexone (10(-8) M). Control cells were incubated in medium only. TREATMENT: Peritonitis was induced by i.p. injection of either zymosan only (Z, 2 mg/ml) or zymosan supplemented with morphine (ZM, M: 20 mg/kg), without or with pretreatment with naltrexone (NZM, N: 5 mg/kg). METHODS: Thirty minutes after induction of peritonitis, the histamine levels (ELISA) and vascular permeability (Evans blue leakage) were measured. At 6 h, the number of exudatory leukocytes (haemocytometer) and chemotaxis/chemoattractant level (48-well chemotactic chamber) were estimated. RESULTS: (1) At 6 h of peritonitis, the number of exudatory leukocytes and levels of plasma chemoattractants were significantly lower in animals injected with zymosan supplemented with morphine (ZM) than in Z and NZM groups of WBB6F1 and Balb/c mice, but only in those with normal mast cells, and not in their mast cell-deficient/depleted counterparts. (2) In contrast, at 30 minutes, vascular permeability and histamine levels were higher in ZM than in Z group of mice with normal mast cells (MC), but not in those depleted of mast cells (MCx). (3) In vitro preincubation of leukocytes with morphine inhibited their migratory activity only towards peritoneal fluid from zymosan-treated MC mice but not from their MCx counterparts. CONCLUSIONS: Mast cell-derived factors are involved in morphine-mediated impairment of zymosan-induced peritonitis in mice.

Strain differences in some immune parameters can be obscured by circadian variations and laboratory routines: studies of male C57BL/6J, Balb/c and CB6 F1 mice.

This is a report on the potential influence of circadian changes and laboratory routines on some immune parameters (thymic and splenic weights, the numbers of bone marrow, peripheral blood, and peritoneal leukocytes) in: (1) males of C57BL/6J, Balb/c, and CB6 F1 mice kept under identical laboratory conditions; (2) males of CB6 mice kept under the same laboratory conditions, except for opposite light/dark regimes, either light/dark (LD) or dark/light (DL). All the animals were purchased from the same supplier and adapted for 4-5 weeks to strictly controlled housing conditions. Some parameters were similar at certain time points but statistically significantly different at others due to strain-specific daily variations. In order to make the interstrain comparisons more reliable, the data collected around the day/night cycle were pooled for calculations of mean values. Several immune parameters of CB6 mice kept under DL conditions were significantly different than those in mice under the conventional LD conditions. In conclusion, the extrapolation of results (especially in the field of neuroimmunology) to other strains (or species) should be done with great caution; and all interstrain (interspecific) comparisons, especially those from various laboratories, should always be related to specific time points and laboratory conditions.

A permissive role for tumor necrosis factor in vascular endothelial growth factor-induced vascular permeability.

Vascular endothelial growth factor (VEGF) induces both angiogenesis and an increase in vascular permeability, 2 processes that are considered to be important for both tumor growth and the delivery of drugs to the site of tumors. This study demonstrates that transmembrane expression of tumor necrosis factor (tmTNF) is up-regulated in the endothelium of a murine methylcholanthrene (meth A)-induced sarcoma in comparison to the adjacent normal dermal vasculature and is also present on cultivated human endothelial cells. It is further shown that tmTNF is required for VEGF-mediated endothelial hyperpermeability in vitro and in vivo. This permissive activity of TNF appears to be selective, because anti-TNF antibodies ablated the VEGF-induced permeability but not proliferation of cultivated human endothelial cells. Furthermore, tnf gene-deficient mice show no obvious defects in vascularization and develop normally but failed to respond to administration of VEGF with an increase in vascular permeability. Subsequent studies indicated that the tmTNF and VEGF signaling pathways converge at the level of a secondary messenger, the "stress-activated protein kinase-2" (SAPK-2)/p38: (1) up-regulated endothelial expression of tmTNF resulted in the continuous activation of SAPK-2/p38 in vitro, and (2) an inhibitor of SAPK-2/p38 activation abolished the vascular permeability activity of VEGF in vivo. In conclusion, the study's finding that continuous autocrine signaling by tmTNF sensitizes endothelial cells to respond to VEGF by increasing their vascular permeability provides new therapeutic concepts for manipulating vascular hyperpermeability.

Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice.

Macrophage dysfunction is a likely mechanism underlying common diabetic complications such as increased susceptibility to infection, accelerated atherosclerosis, and disturbed wound healing. There are no available studies on the function of tissue macrophages in diabetes in humans. We have therefore studied peritoneal macrophages from diabetic type 2-like db/db mice. We found that the release of tumor necrosis factor-alpha and interleukin-1beta from lipopolysaccharide plus interferon-gamma-stimulated macrophages and vascular endothelial growth factor from both stimulated and nonstimulated macrophages was significantly reduced in diabetic animals compared with nondiabetic controls. Nitric oxide production from the stimulated db/db macrophages was significantly higher than that in the db/+ cultures, whereas there was no difference in their ability to generate reactive oxygen species. When studied both at light and electron microscopic levels, macrophages in diabetic animals had an altered morphological appearance compared with those of normal controls. We conclude that the function and morphology of the macrophages are disturbed in db/db mice and that this disturbance is related to the mechanisms underlying common inflammatory and degenerative manifestations in diabetes.

Early events of hepatic metastasis formation in mice: role of Kupffer and NK-cells in natural and interferon-gamma-stimulated defense.

Surgical manipulation of a tumor may result in increased influx of tumor cells into the systemic and portal circulation and give rise to formation of metastases. In addition, major surgery has been reported to cause profound immunosuppression. In an attempt to increase the host-antitumor immune mechanisms following surgery we have studied the effect of preoperative administration of interferon-gamma, related to the antimetastatic effects of Kupffer cells (KC) and natural killer cells (NK-cells) in the early phase of liver metastasis formation. Colon carcinoma cells were injected into the superior mesenteric vein of syngeneic mice and after 17 days metastases were quantified by weight, number, and uptake of [125I]iododeoxyuridine. Unstimulated control mice developed 10.5 surface nodules per liver 17 days following injection of colon carcinoma cells into the superior mesenteric vein of syngeneic mice. This figure was only 2.6 in mice stimulated with a single dose of 1000 IU IFN-gamma 4 h prior to inoculation of tumor cells. Administration of GdCl3, which is reported to deplete and block the function of Kupffer cells, 24 h prior to tumor cell inoculation resulted in a 5-fold tumor mass increase relative to control. Injection of anti-asiolo-GM1 antiserum, which eliminates the hepatic NK-cells, induced a 10-fold increase in tumor mass. These results indicate an important early antimetastatic function of hepatic NK-cells and KC and that presurgical administration of IFN-gamma may be important for eliminating circulating tumor cells and inhibiting development of residual tumors.

Tumor stroma.

The accumulated evidence indicates that tumor stroma with its cells and cell products plays a much more active and important role than previously believed. Growth factors and cytokines produced by macrophages and other cells are crucial for stroma formation and angiogenesis. Lytic enzymes provided by stromal cells may be essential for invasion. TNF and other inflammatory mediators may be operative in the systemic effects of tumors, e.g. cachexia. All these effects may come about through the action of soluble substances produced by tumor cells or by more intimate interactions. There is no evidence that stromal cells are directly involved in carcinogenesis--i.e. the cellular transformation to produce the malignant cell. On the other hand, stromal cells and other components of the interstitia are instrumental in tumorigenesis--i.e. the development of a real malignant tumor from its start on the cellular or subcellular level. In one way of looking at it, the stromal cells, e.g. macrophages may be considered as "slaves", kept to carry out certain functions, synthesize essential substances e.g. growth factors that the tumor cells do not have the capacity or the degree of finely tuned machinery to produce. The objective of immunomodulation should then be to create a "slave uprising", to make the macrophages and other cells turn against their masters, stop producing growth factors and start producing harmful factors that would lead to the elimination of the malignant growth. The first target of immunomodulation in tumor disease should probably be local malignancies where no effective treatment exists today- and selected cases of metastatic prevention (181, 182).

Conjugates of Tremella polysaccharides with microbeads and their TNF-stimulating activity.

A mannan (A) and a heteroglycan (D) were prepared by partial acidic hydrolysis of T3, a major polysaccharide fraction of the fungus Tremella fuciformis Berk. Methylation analysis of A and D showed that they contained a 1-->3 linked mannosyl main chain, to which were linked different side chains at position 2, 4, or 6 of the mannosyl residues. A and D were conjugated to albumin microbeads (AM) by reductive amination. The conjugates showed significant cytokine-stimulating activity in vitro whereas unconjugated AM had no activity. Not conjugated A and D showed cytokine-stimulating activity only in about 100 times higher concentrations.

Inhibition of establishment and growth of mouse liver metastases after treatment with interferon gamma and beta-1,3-D-glucan.

The purpose of this study was to investigate the combined antitumor effect of aminated beta-1,3-D-glucan (AG) and interferon-gamma (IFN-gamma) in an experimental liver metastasis model. Liver metastases were established by inoculation of C-26 colon carcinoma cells into the superior mesenteric vein of syngeneic mice. Treatment of mice started 24 hours after inoculation of tumor cells by daily intravenous injections of either AG, IFN-gamma, or a combination of both for a duration of 6 days. The resultant liver metastases were then quantified after an additional period of 11 days. Combination of IFN-gamma and AG inhibited the growth of liver metastases almost entirely. IFN-gamma was also very efficient, while AG alone did not exert any significant antitumor effect. These results, along with histological studies from mice receiving AG and IFN-gamma, indicated that activation and recruitment of liver macrophages may be a part of the mechanism responsible for the inhibition of metastatic growth observed in this study.

Characterization and cytokine-stimulating activities of acidic heteroglycans from Tremella fuciformis.

Four acidic heteroglycans, T2a-T2d, were isolated from the body of Tremella fuciformis Berk. They contained 1.9%-2.9% of acetyl groups and were composed of mannose (Man), glucuronic acid (GlcA), and small amounts of xylose (Xyl), glucose (Glc), and fucose (Fuc). According to methylation analysis they had a mannan backbone consisting of 3-linked Man, and side chains containing glucosyl, mannosyl, fucosyl, xylosyl, and glucuronic acid residues. The side chains were attached through O-2, O-4, or O-6 in about 40 percent of backbone mannosyl residues. Molecular masses of the four polysaccharides were 410, 250, 34, and 20 kDa, respectively. T2a-T2d induced human monocytes to produce interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in vitro. The products of Smith degradation (T2a-S) and lithium degradation (T2a-L) of T2a and the product of deacetylation (T2b-D) of T2b also induced monocytes to secret IL-1 as efficiently as the original polysaccharides, indicating that xylosyl and glucuronic acid residues as well as acetyl groups were not important to promote the cytokine-stimulating activity.

Cytotoxic effect of cytokines on murine colon carcinoma cells involves TNF-mediated apoptosis.

We have studied the cytotoxic effect of TNF-alpha on C-26 murine colon carcinoma cells in vitro. Treatment with TNF-alpha alone did not result in any demonstrable cytotoxicity. However, when combined with IFN-gamma, the cytotoxic effect of TNF-alpha was enhanced in a dose-dependent manner. An agonistic TNF-R1 specific antibody and recombinant human TNF-alpha both exerted a cytotoxic effect when combined with IFN-gamma, suggesting that the cytotoxicity was mediated through the TNF-R1. The cytotoxicity was associated with production of nitric oxide without any direct involvement in the cytotoxic effect. At the ultrastructural level, treated cells displayed a typical apoptotic morphology which was not accompanied by internucleosomal cleavage of DNA as shown by conventional electrophoresis.

Rhythms of immunity.

Rhythms of daily activity are found in all vertebrate species, some of them being diurnal (like humans, dogs, pigeons), others--nocturnal (like mice, rats and bats). Some species undergo very pronounced seasonal changes, as they hibernate in the winter or mate only at the specific seasons. The main regulator (a clock and a calendar) for daily and seasonal rhythms is the periodicity of the external light-darkness, reflected by the periodicity of melatonin secretion from the pineal gland, which is inhibited by light and induced during the darkness. In contrast to melatonin which peaks during the night both in diurnal and noctural species, the cyclicity of other hormones and several immune parameters correlates with the pattern of the animal locomotor activity-resting. The immune parameter that peaks at one time of day for a diurnal species peaks about 12 h later for a nocturnal one. Various immune parameters peak at various time points, anticipating an encounter with pathogens during the period of activity while energetically expensive resolution of the immune response during the resting. Daily and seasonal cyclicity of the immune functions are temporally integrated with other physiologic and behavioral processes and all of them are regulated and coordinated with daily and seasonal changes of an external environment by the neuroendocrine homeostatic system.

Characterization and cytokine stimulating activities of heteroglycans from Tremella fuciformis.

Three heteroglycans, T1a, T1b, and T1c, have been isolated from the body of Tremella fuciformis Berk. They are composed of mannose (Man), xylose (Xyl), glucose (Glc), fucose (Fuc), and glucuronic acid (GlcA). According to methylation analysis and partial acidic hydrolysis the main chains of T1a, T1b, and T1c consisted of (1-->3)-linked Man, which was branched at the 2, 4, or 6 positions. The branching points were linked with nonreducing terminal GIcA-residues or (1-->6)-linked glucan-chains. Molecular weights of the three heteroglycans are 53,000, 18,000, and 12,000 D respectively, but they undergo self-aggregation in water. T1a-T1c induce human monocytes to produce interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in vitro. Acidic hydrolysate fractions of T1a (T1a-1, 2, 3, 4, 5) with molecular weight from 53,000 to 1,000 D, also induce human monocytes to produce IL-6 as efficient as T1a.

Characterisation of acidic heteroglycans from Tremella fuciformis Berk with cytokine stimulating activity.

Four kinds of acidic heteroglycan, T3a-T3d, were isolated from the body of the fungus Tremella fuciformis Berk. The molecular weights of T3a-T3d were 550, 420, 55, and 48 kDa, respectively. Glycosidic linkage analysis showed that they had a mannan backbone consisting of 3-linked Man p, and side chains containing glucosyl, mannosyl, fucosyl, xylosyl, and glucuronic acid residues attached through O-2, O-4, or O-6 of about half of the backbone mannosyl residues. A partial acidic hydrolysate of T3a could be divided into a low-branching region (T3a-1, 2, 3, 4) mainly branched at the 2-position of 3-linked Man p in the mannan backbone, and a high-branching region (T3a-5A) branched at the 2,4- or 2,4,6-positions of 3-linked Man p in the backbone. The low-branching region, which is predominant in the backbone, was substituted with non-reducing terminal Glc pA, Fuc p and short side chains consisting of (1-->6)-linked Glc p and (1-->2)-linked Man p linked through C-2 of the mannan backbone. The high-branching region, which is a minor component of the backbone, was linked with long side chains of (1-->6)-linked Glc p and (1-->4)-linked Glc pA in their branching points. T3a-T3d were able to induce human monocytes to produce interleukin-1, interleukin-6, and tumor necrosis factor in vitro. The different fragments of the acidic hydrolysate of T3a (T3a-1, 2, 3, 4, 5A) also induced monocytes to secret interleukin-6 with high potency, indicating that the activity may be caused by a common structure, (1-->3)-mannan in the four heteroglycans and their fragments. The change of molecular weight had no obvious influence on the activity of the heteroglycans.

Macrophage cytotoxicity against murine meth A sarcoma involves nitric oxide-mediated apoptosis.

We have studied the cytotoxic effect of stimulated macrophages on Meth A tumor cells in vitro. When stimulated with interferon-gamma and soluble beta-1,3-D-glucan, macrophages exerted cytotoxicity towards syngeneic Meth A tumor cells. This cytotoxicity was associated with a high level of nitric oxide production. Both cell death and nitric oxide production were significantly inhibited by the addition of aminoguanidine, a specific inhibitor of inducible nitric oxide synthase (iNOS), to the culture medium. The cytotoxic effect was accompanied by internucleosomal cleavage of DNA as shown by electrophoresis and DNA fragmentation assay.

IFN-gamma inhibits internalization of soluble aminated beta-1,3-D-glucan by macrophages and thereby down-regulates the glucan induced release of TNF-alpha and IL-1 beta.

We have previously shown that soluble animated beta-1,3-D-glucan (AG) and glucan-derivatized microbeads (GDM) bind to the specific beta-glucan receptor on mouse peritoneal macrophages. Phagocytosis of GDM by macrophages is mediated through the beta-glucan receptor. IFN-gamma which increases macrophage phagocytic capacity, also increased the phagocytosis of GDM. In the present study we show that IFN-gamma inhibits internalization of AG in macrophages in a dose- and time-dependent manner. The inhibitory effect of IFN-gamma was neutralized by treatment of the macrophages with cycloheximide. These results were confirmed by confocal laser scanning microscopy which showed that IFN-gamma treated cells incorporated less fluorescein-labelled AG than did untreated cells. IFN-gamma did not change the macrophage-binding capacity for AG showing that the inhibitory effect of IFN-gamma is not caused by decreased number of beta-glucan receptors on the cells. The stimulatory effect of AG on IL-1 beta and TNF-alpha release from macrophages was reduced by pretreatment of the cells with IFN-gamma. We conclude that the uptake of AG and GDM in macrophages, both mediated through the beta-glucan receptor, are differently regulated by IFN-gamma. The reduced internalization of AG after IFN-gamma treatment of macrophages, is probably responsible for the down-regulation of IL-1 and TNF-alpha secretion.

Aminated beta-1,3-D-polyglucose activates salmon pronephros macrophages in vitro.

Salmon pronephros macrophages were stimulated in vitro using a novel immunomodulator, aminated beta-1,3-D-polyglucose. Activation of these cells was judged by the measurement of superoxide anion formation, pinocytotic activity, acid phosphatase activity and morphological criteria. Stimulated macrophages showed increased superoxide anion formation compared with unstimulated control cells as measured by reduction of Nitroblue tetrazoleum, and an increased level of acid phosphatase activity. Furthermore, stimulated macrophages showed increased pinocytotic activity and a significant increase in cell diameter and spreading.