Arthritis in Lewis rats induced by the non-immunogenic adjuvant CP20961: an immunohistochemical analysis of the developing disease.

OBJECTIVES: The role of lymphocytes and macrophages in developing adjuvant arthritis induced by an injection of CP20961 in inbred Lewis rats was studied over a 32 day period using a novel biotin-avidin immunoperoxidase histochemical technique. METHODS: Fresh frozen sections of hind paws and spleens, as well as lymph nodes draining the site of the injected adjuvant were immunostained using a panel of monoclonal antibodies specific for subsets of lymphocytes and macrophages and for MHC Class II antigen. RESULTS: An increase in the numbers of activated T-lymphocytes was detected early in the draining lymph nodes before hind paw swelling had begun. The presence of these cells in significant numbers was only observed in the vicinity of the joint after joint swelling and damage had begun. Macrophages were among the first cells to invade the swollen paws and later were found with T-lymphocytes and cells bearing the MHC class II antigen at the face of eroding and re-organising bone. CONCLUSIONS: The activity of T-lymphocytes in initiating arthritis appeared to occur early in lymph nodes. Joint destruction was more closely associated with the arrival of macrophages but later arrival of T-lymphocytes may have contributed to the maintenance of chronic inflammation.

Protective effect of clentiazem against epinephrine-induced cardiac injury in rats.

We investigated the effects of clentiazem, a 1,5-benzothiazepine calcium antagonist, on epinephrine-induced cardiomyopathy in rats. With 2-week chronic epinephrine infusion, 16 of 30 rats died within 4 days, and severe ischemic lesions and fibrosis of the left ventricles were observed. In epinephrine-treated rats, left atrial and left ventricular papillary muscle contractile responses to isoproterenol were reduced, but responses to calcium were normal or enhanced compared to controls. Left ventricular alpha and beta adrenoceptor densities were also reduced compared to controls. Treatment with clentiazem prevented epinephrine-induced death (P < .05), and attenuated the ventricular ischemic lesions and fibrosis, in a dose-dependent manner. Left atrial and left ventricular papillary muscle contractile responses to isoproterenol were reduced compared to controls in groups treated with clentiazem alone, but combined with epinephrine, clentiazem restored left atrial responses and enhanced left ventricular papillary responses to isoproterenol. On the other hand clentiazem did not prevent epinephrine-induced down-regulation of alpha and beta adrenoceptors. Interestingly, clentiazem, infused alone, resulted in decreased adrenergic receptor densities in the left ventricle. Clentiazem also did not prevent the enhanced responses to calcium seen in the epinephrine-treated animals, although the high dose of clentiazem partially attenuated the maximal response to calcium compared to epinephrine-treated animals. In conclusion, clentiazem attenuated epinephrine-induced cardiac injury, possibly through its effect on the adrenergic pathway.

Cysteine proteinase activity in the development of arthritis in an adjuvant model of the rat.

Cathepsin B and L activity was studied histochemically in arthritic rat ankle joints using specific synthetic substrates in a post coupling method on unfixed and undecalcified cryostat sections of rat ankle joints. Activity was strongly increased in chondrocytes and cells of the inflamed synovium with the development of arthritis induced by the synthetic adjuvant CP20961. Activity reached a maximum 20 days after induction of arthritis and decreased as the rats entered natural remission. Cathepsin B and L were at their highest level when macrophages were present in the joint space, as shown by using monoclonal antibody markers for rat macrophages (ED1 and ED2) in a biotin-avidin immunoperoxidase assay. This suggests that the macrophage infiltrate may have stimulated proteinase production in chondrocytes through cytokine release. The profile of appearance of cysteine proteinases suggests their involvement in the breakdown of cartilage and bone in the arthritic joint.

In vitro and in vivo effects of proteoglycan fractions in adjuvant treated rats.

Differential stimulatory effects by proteoglycan fractions: chondroitin (CS)- and keratan-sulphate regions; link protein and binding region were observed in cultures of spleen and lymph node lymphocytes taken from normal and adjuvant treated Lewis rats. In vivo, none of the fractions induced symptoms of arthritis but that pretreatment with the CS rich region produced an inhibition of Mycobacterium tuberculosis induced arthritis.

Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit.

The purpose of this study was to determine if chronic administration of L-arginine, the precursor of endothelium-derived relaxing factor (EDRF), normalizes endothelium-dependent relaxation and decreases atherosclerosis in hypercholesterolemic animals. Male rabbits were fed (a) normal rabbit chow; (b) 1% cholesterol diet; or (c) 1% cholesterol diet supplemented by 2.25% L-arginine HCl in drinking water. Arginine supplementation doubled plasma arginine levels without affecting serum cholesterol values. After 10 wk, the thoracic aorta was harvested for studies of vascular reactivity and histomorphometry. Endothelium-dependent relaxations (to acetylcholine and calcium ionophore A23187) were significantly impaired in thoracic aortae from animals fed a 1% cholesterol diet. By contrast, vessels from hypercholesterolemic animals receiving L-arginine supplementation exhibited significantly improved endothelium-dependent relaxations. Responses to norepinephrine or nitroglycerin were not affected by either dietary intervention. Histomorphometric analysis revealed a reduction in lesion surface area and intimal thickness in thoracic aortae from arginine-supplemented animals compared to those from untreated hypercholesterolemic rabbits. This is the first study to demonstrate that supplementation of dietary L-arginine, the EDRF precursor, improves endothelium-dependent vasorelaxation. More importantly, we have shown that this improvement in EDRF activity is associated with a reduction in atherogenesis.

Effects of fish oil on graft arteriosclerosis and MHC class II antigen expression in rat heterotopic cardiac allografts.

The effect of fish oil on accelerated graft coronary arteriosclerosis was assessed in Lewis to Brown-Norway rat heterotopic cardiac allografts. Twelve Brown-Norway rats were supplemented with 2 ml/kg/day of fish oil (68.3 mg eicosopentaenoic acid and 47.5 mg decosahexaenoic acid per milliliter). Eleven additional animals, receiving an isocaloric amount of safflower oil, served as control. All diets began 1 week before operation. Immunosuppression was obtained with low-dose cyclosporine (2 mg/kg/d). When killed (100 days), there were no significant differences in percentage weight gain, graft function, or histologic rejection score. Although lipid profiles were comparable, total cholesterol:high-density lipoprotein ratio was marginally higher in animals treated with fish oil (p = 0.069). Mean percentage luminal occlusion (before and after correcting for differences in size between coronary vessels analyzed) and average intimal thickness were similar between animals treated with fish oil and safflower oil as assessed by computer-assisted digitized, morphometric planimetry. In all allografts, donor interstitial dendritic cells were repopulated with recipient dendritic cells. The major histocompatibility complex class II cell density in the fish oil group did not differ significantly from rats supplemented with safflower oil (1.48 +/- 0.68 vs 1.48 +/- 0.65 cells per mm2, p = 0.995). In conclusion, fish oil did not exert any beneficial effect over safflower oil in terms of graft coronary arteriosclerosis, histologic rejection, or plasma lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose response of fish oil versus safflower oil on graft arteriosclerosis in rabbit heterotopic cardiac allografts.

With the advent of cyclosporin A, accelerated coronary arteriosclerosis has become the major impediment to the long-term survival of heart transplant recipients. Due to epidemiologic reports suggesting a salutary effect of fish oil, the dose response of fish oil on graft coronary arteriosclerosis in a rabbit heterotopic cardiac allograft model was assessed using safflower oil as a caloric control. Seven groups of New Zealand White rabbits (n = 10/group) received heterotropic heart transplants from Dutch-Belted donors and were immunosuppressed with low-dose cyclosporin A (7.5 mg/kg/day). Group 1 animals were fed a normal diet and served as control. Group 2, 3, and 4 animals received a daily supplement of low- (0.25 mL/kg/day), medium- (0.75 mL/kg/day), and high- (1.5 mL/kg/day) dose fish oil (116 mg n-3 polyunsaturated fatty acid/mL), respectively. Group 5, 6, and 7 animals were supplemented with equivalent dose of safflower oil (i.e., 0.25, 0.75, and 1.5 mL/kg/day). Oil-supplemented rabbits were pretreated for 3 weeks before transplantation and maintained on the same diet for 6 weeks after operation. The extent of graft coronary arteriosclerosis was quantified using computer-assisted, morphometric planimetry. When the animals were killed, cyclosporin A was associated with elevated plasma total cholesterol and triglyceride levels in the control group. While safflower oil prevented the increase in plasma lipids at all dosages, fish oil ameliorated the cyclosporin-induced increase in total cholesterol only with high doses. Compared to control animals, there was a trend for more graft vessel disease with increasing fish oil dose, as assessed by mean luminal occlusion and intimal thickness. A steeper trend was observed for increasing doses of safflower oil; compared to the high-dose safflower oil group, animals supplemented with low-dose safflower oil had less mean luminal occlusion (16.3% +/- 5.9% versus 41.4% +/- 7.6%, p less than 0.017) and intimal thickness (7.9 +/- 1.9 microns versus 34.0 +/- 13.0 microns, analysis of variance: p = 0.054). Low-dose safflower oil also had a slight, but nonsignificant, beneficial effect on graft vessel disease when compared to control rabbits. The same trends were observed in the degree of histologic rejection (0 = none to 3 = severe) in fish oil- and safflower oil-treated animals. Rejection score correlated weakly but significantly (p = 0.0001) with mean luminal occlusion (r = 0.52) and intimal thickness (r = 0.46). Therefore allograft coronary disease in this model appeared to exhibit an unfavorable, direct-dose response to fish oil and safflower oil, independent of effects on plasma lipids.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of accelerated cardiac allograft arteriosclerosis by fish oil.

Accelerated coronary arteriosclerosis remains the most important factor limiting long-term survival of heart transplant recipients, and dietary fish oil supplementation with omega-3 polyunsaturated fatty acids has been suggested to have a protective effect against coronary disease in epidemiologic studies and to inhibit arteriosclerosis in animal experiments. Therefore we tested the hypothesis that fish oil administration inhibits the development of allograft coronary arteriosclerosis by using a heterotopic heart transplant model. Three groups of Lewis rats (n = 10 each) received heterotopic heart transplants from Brown-Norway donors and were treated with cyclosporine intraperitoneally on a tapering schedule. Group 1 received fish oil daily by gavage (2 ml/kg/day; Emulsified Super MaxEpa, Twin Labs, Ronkonkona, N.Y.). Group 2 received an equal amount of safflower oil, as well as aspirin (1 mg/kg/day) and dipyridamole (3 mg/kg/day). Group 3 received safflower oil only. All rats were put to death 110 days later, at which time there was no statistically significant difference in graft function as assessed by palpation (scale 0 to 4, mean = 3.7 +/- 0.5 [+/- standard deviation]; analysis of variance: p = 0.72) or in microscopic grade of rejection (scale, 0 = none to 3 = severe, mean 2.1 +/- 0.6; analysis of variance: p = 0.68) between any of the groups. The coronary arteries were histologically scored for the degree of arteriosclerosis (scale, 0 = normal to 3 = occluded), and a mean grade of coronary disease was calculated for each heart. The fish oil-treated group had significantly less severe allograft coronary arteriosclerosis (analysis of variance: p = 0.005) than did groups 2 and 3 (mean grade 0.23 +/- 0.22 versus 1.04 +/- 0.75 and 0.96 +/- 0.55 (p less than 0.05, Scheffe F test), whereas groups 2 and 3 had similar degrees of coronary disease (p = no significant difference). These data demonstrate that fish oil supplementation inhibited accelerated coronary arteriosclerosis in this cyclosporine-treated heart allograft rat model, whereas antiplatelet agents in these doses were ineffective. Although the mechanism of this protective effect remains incompletely understood, it does not appear to involve enhanced immunosuppression. Fish oil and specific omega-3 polyunsaturated fatty acids should be further investigated as potentially useful agents to ameliorate accelerated allograft coronary arteriosclerosis in other animal species and perhaps eventually in man.