A lack of evidence for down-modulation of CD3 zeta expression in colorectal carcinoma and pregnancy using multiple detection methods.

Loss of the T cell receptor-associated CD3 zeta chain has been proposed as a possible mechanism of the acquired immunosuppression in both tumour-bearing hosts, and in symptomatic patients with HIV infection. However, other reports suggest that the zeta-chain loss may in part be caused by protease activity of contaminating phagocytes ex vivo. Using flow cytometry and Western blot analysis on highly purified T cells, and ensuring adequate addition of protease inhibitors, we have studied the expression of CD3zeta on peripheral blood T cells from patients with colorectal carcinoma, and compared these with normal controls, and pregnant donors, as a further example of an immunocompromised state. Immunohistochemistry was performed on tumour sections from patients with colorectal carcinoma to measure CD3zeta expression in tumour infiltrating T cells, and compared with normal mucosa and tonsil. Using these three approaches, our data provide no evidence for downregulation of CD3zeta chain expression either in colorectal carcinoma or pregnancy and suggest that this explanation is unlikely to fully account for the reduced T cell function associated with these conditions in all patients.

The modulation of IL-6 and TNF-alpha release by nitric oxide following stimulation of J774 cells with LPS and IFN-gamma.

Lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) induced nitric oxide synthase (NOS) activity, tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and prostaglandin (PG)E2 release in J774 cells, a murine macrophage cell line. The role of endogenous NO in modulating TNF-alpha and IL-6 release was investigated using N-iminoethyl-L-ornithine (L-NIO), a specific inhibitor of NOS. L-NIO (10-1000 microM) produced a concentration-dependent potentiation of LPS and IFN-gamma induced IL-6 release. Time-course studies demonstrated a significant potentiation of IL-6 release at 12 h with a maximum effect at 48 h. By contrast to its effects on IL-6, L-NIO significantly attenuated TNF-alpha release, and at 48 h reduced PGE2 release. The NO-donor S-nitroso-N-acetyl-penicillamine (SNAP,300 microM), significantly inhibited LPS and IFN-gamma induced IL-6 release, but potentiated TNF-alpha release. In addition, SNAP prevented the potentiation of IL-6 and the inhibition of TNF-alpha release by L-NIO. Stimulation of J774 cells with a combination of LPS and IFN-gamma for 24 h or 48 h reduced cell viability which was prevented by L-NIO. Furthermore, SNAP also reduced cell viability determined after 24 h incubation. These results indicate that NO can differentially modulate LPS and IFN-gamma-induced cytokine release from J774 cells, up-regulating TNF-alpha but down-regulating IL-6, and that NO is cytotoxic to these cells.

Role of oxygen radicals and arachidonic acid metabolites in the reverse passive Arthus reaction and carrageenin paw oedema in the rat.

1. The role of arachidonic acid metabolites and oxygen radicals in carrageenin-induced rat paw oedema and dermal reverse passive Arthus reaction (RPA) have been investigated. 2. Indomethacin (10 mg kg-1, p.o.) inhibited carrageenin paw oedema when administered 30 min before, but not 2 h after carrageenin. BWB70C (10 mg kg-1, p.o.), a selective inhibitor of 5-lipoxygenase, had no effect whether administered before or after carrageenin. Administration of both indomethacin and BWB70C had no greater anti-inflammatory effect than indomethacin alone. 3. BW755C (20 mg kg-1, p.o.), which inhibits the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism, or superoxide dismutase-polyethylene glycol conjugate (SOD-PEG, 3000 u, i.v.) inhibited carrageenin paw oedema whether administered either 30 min before, or 2 h after carrageenin. 4. Pretreatment with dexamethasone (0.1 mg kg-1) or colchicine (2 mg kg-1), likewise suppressed carrageenin paw oedema. 5. BW755C (25-100 mg kg-1, p.o.) dose-dependently reduced plasma leakage in the RPA, whereas indomethacin (5 mg kg-1, p.o.) or BWB70C either alone or in combination, did not. 6. SOD-PEG (300-3000 u, i.v.) dose-dependently inhibited plasma leakage in the RPA. In addition, the iron chelator and peroxyl radical scavenger, desferrioxamine (200 mg kg-1, s.c.) also inhibited plasma leakage. 7. Pretreatment with dexamethasone (0.1 mg kg-1) or colchicine (1 mg kg-1) reduced the plasma leakage in RPA, whereas MK-886 (10 mg kg-1) had no effect. 8. These results indicate an important role for oxygen radicals but not arachidonic acid metabolites in the maintenance of carrageenin paw oedema and the plasma leakage in RPA. Furthermore, the results suggest that the anti-inflammatory actions of BW755C can be dissociated from its effects on arachidonic acid metabolism and are attributed to its anti-oxidant activity.

Actions of nitric oxide on the acute gastrointestinal damage induced by PAF in the rat.

The actions of nitric oxide (NO) on the acute gastrointestinal damage induced by platelet-activating factor (PAF) have been investigated in the rat. S-nitroso-N-acetyl penicillamine, which spontaneously generates NO, dose-dependently inhibited PAF-induced gastrointestinal plasma leakage, a measure of the initiation of vascular damage. The inhibitor of NO synthase, NG-monomethyl-L-arginine substantially potentiated gastrointestinal damage and plasma leakage induced by E. coli endotoxin, but had no effect on that induced by intravenous infusion of PAF. Endogenous NO may thus have a protective role in the gastrointestinal vascular that can be mimicked by generators of NO. The protection afforded by endogenous NO may, however, be dependent on the nature of the inflammatory stimulus used to induce gastrointestinal damage.

Protective effect of S-nitroso-N-acetyl-penicillamine in endotoxin-induced acute intestinal damage in the rat.

Macroscopic jejunal damage and plasma leakage induced within 15 min by E. coli lipopolysaccharide (LPS 50 mg kg-1 i.v.) in the rat was enhanced by the inhibitor of nitric oxide (NO) formation, NG-monomethyl-L-arginine (L-NMMA 50 mg kg-1 i.v.). The nitro-vasodilator, S-nitroso-N-acetyl-penicillamine (SNAP; 10 micrograms kg-1 min-1 i.v.), which generates NO, attenuated both LPS-induced intestinal damage and the enhancement of such damage and plasma leakage produced by L-NMMA. Endogenous NO may thus have a protective role in the intestinal vasculature that can be mimicked by generators of NO.