Interleukin-2-induced lymphocyte infiltration of multiple organs is differentially suppressed by soluble tumor necrosis factor receptor.

Interleukin-2 (IL-2) mediates the regression of metastatic cancer, but clinical application is restricted by associated toxicities. Previous studies implicate tumor necrosis factor (TNF) as an important mediator of certain IL-2-induced toxicities. We hypothesized that soluble TNF receptor (sTNFr), a TNF antagonist, would alter lymphocyte trafficking into normal tissues and ameliorate IL-2-induced toxicity. Four groups of C57BL/6 mice were treated for 4 days with intraperitoneal injections of 100,000 IU IL-2 alone, 100,000 IU IL-2 and 30 micrograms sTNFr combined, 30 micrograms sTNFr alone, or equal volumes of saline. Animal activity was graded and blood obtained for SGPT and SGOT. At necropsy, organs were harvested for wet:dry ratios as a measurement of organ edema. The lung, liver, and thymus were examined histologically for lymphocytic infiltration and graded on a scale of 1 to 5. IL-2-treated groups had a statistically significant increase in organ edema, lymphocytic infiltration into the lung and liver, liver enzyme elevation, and pancytopenia when compared with controls. Soluble TNFr significantly suppressed IL-2-induced pulmonary lymphocytic infiltration and associated serum lymphopenia without significant alteration of other IL-2-induced effects. These data implicate TNF as a mediator of the pulmonary lymphocytic infiltration and of lymphopenia that accompanies IL-2 therapy and further suggest that alternative mechanisms are involved in other IL-2-induced deleterious effects.

Medium pH modulates matrix, mineral, and energy metabolism in cultured chick bones and osteoblast-like cells.

The effects of medium pH were tested on calvariae, tibiae, and osteoblast-like cells from chick embryos. Bones and isolated cells were incubated for 5 h or 2 days in Hepes-buffered medium at pH values ranging from 6.8 to 8.2. Osteoblast function was evaluated by lactate production, oxygen consumption, alkaline phosphatase activity (AlPase), Ca and inorganic phosphate (Pi) flux, proline hydroxylation, DNA content, and thymidine incorporation. As medium pH was increased, glycolysis, collagen synthesis, and AlPase increased, while Ca efflux decreased. No effect of pH was seen on mitochondrial activity, Pi efflux, or cell number or proliferation. The importance of glycolysis as an endogenous pH regulator was demonstrated by inhibition with iodoacetic acid or glucose restriction and by adding lactate to the medium. The results suggest that the pH of bone interstitial fluid may be regulated by glycolysis and that changes in pH of this compartment may have marked effects on osteoblast function.

Smokeless tobacco contains an inhibitor of prolyl hydroxylase activity.

The effects of smokeless tobacco extract (STE) and various constituents of STE on prolyl hydroxylase activity were determined using enzyme extracted from chick embryos. STE inhibited prolyl hydroxylase activity in a concentration-dependent manner, but nicotine and anabasine had essentially no effect. Enzymatic activity was inhibited by zinc, but not by the other inorganic elements in STE; however, the zinc concentration in STE was not high enough to produce the observed inhibition. The inhibition by STE was diminished by increasing concentrations of 2-oxoglutarate, but not by increasing concentrations of other cofactors. Thus, STE contains an inhibitor of prolyl hydroxylase which may be competitive with 2-oxoglutarate.

Inhibition of cell metabolism by a smokeless tobacco extract: tissue and species specificity.

Smokeless tobacco contains a nonnicotine inhibitor of posttranslational modification of collagen (hydroxylation of [3H]proline) by cultured chick embryo tibias and osteoblasts. This study was undertaken to determine whether a methanol extract of smokeless tobacco (STE) containing the inhibitor has similar effects on collagen-producing cells and tissues other than bone. Its effects on DNA synthesis and cell proliferation (incorporation of [3H]thymidine) were also determined. Frontal bone, aorta, and cartilage were incubated for 2 days in medium containing STE. Glycolysis (lactate production) was stimulated by 80% in cartilage, but was not affected in the other tissues; medium alkaline phosphatase activity was unaffected. In frontal bone and cartilage, [3H] hydroxyproline content was decreased 88% and 57%, respectively, and [3H]proline content was decreased 68% and 37%, respectively; neither was affected in the aorta. Confluent cultures of collagen-producing mouse fibroblasts or primary osteoblasts obtained from chick embryo calvarias were incubated for 2 days in medium containing increasing concentrations of STE. Glycolysis and DNA synthesis were not affected. Cell proliferation was unaffected in fibroblasts, but was inhibited (34%) at the highest STE concentration in osteoblasts. AIPase activity was not detectable in fibroblast medium, but was decreased up to 72% in osteoblast medium. Inhibition of collagen synthesis by STE was concentration related in both cell types. At the highest concentration, [3H] hydroxyproline and [3H]proline contents in the cell layers were decreased to the following respective values: fibroblasts 56% and 45% and osteoblasts 50% and 29%, respectively. When incubation with STE was discontinued for 1 day, recovery did not occur. These findings suggest that inhibition of collagen synthesis by STE is not specific for bone, that collagen-producing cells are directly affected, and that recovery is not immediate. This inhibitor could contribute to the periodontal disease often seen in users of smokeless tobacco. Its identification and removal would produce a safer product.

A serum substitute promotes osteoblast-like phenotypic expression in cultured cells from chick calvariae.

The effects of medium supplements were tested on embryonic chick calvarial cells in culture. Isolates were divided among four treatment groups: Nu-Serum, chicken serum, fetal bovine serum, or calf serum. Expression of the osteoblastic phenotype was assessed by cell morphology, DNA content, [3H]thymidine incorporation, lactate production, cellular and medium alkaline phosphatase activities, and collagen synthesis. Cells grown in Nu-Serum demonstrated increased alkaline phosphatase activity and a six-fold higher rate of collagen synthesis compared to chicken serum. These cells displayed a polygonal profile, abundant rough endoplasmic reticulum, Golgi apparati, and elaborated an extensive matrix of banded collagen which was well mineralized by day 10 of culture. Although highly mitogenic, chicken serum promoted a more fibroblastoid morphology. Compared to the sera tested, Nu-Serum preferentially promoted the osteoblast-like phenotype in chick calvarial cells in culture.

Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells.

Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of [3H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [3H]hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of [3H]thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.

Comparison of the effects of smokeless tobacco extract with the effects of prolyl hydroxylase inhibitors on collagenous and noncollagenous protein synthesis by osteoblasts.

The effects of smokeless tobacco extract (STE) and prolyl hydroxylase inhibitors on protein synthesis by isolated osteoblast-like cells were compared. STE and 2,2'dipyridyl markedly inhibited alkaline phosphatase (Alpase) and [3H]proline hydroxylation without affecting glycolysis (lactate production). However, pyridine 2,5-dicarboxylate (2,5-PDC) did not inhibit [3H] proline hydroxylation, Alpase activity, or glycolysis at moderate concentrations. The [3H]hydroxyproline to [3H]proline ratio in the cell layers demonstrated a concentration-dependent decrease with increasing STE and inhibitor concentrations. In the cell layers, the collagenous protein (CP) content was decreased after exposure to STE, 2,2'dipyridyl, and 2,5-PDC and the noncollagenous protein (NCP) content was decreased after exposure to STE and 2,5-PDC. However, the effects on CP were at least twofold greater than on NCP. Similar results were observed regarding protein released to the culture medium. These data demonstrate that STE, like 2,2'dipyridyl, inhibits the hydroxylation of proline and the synthesis of collagenase-digestible protein.

Smokeless tobacco contains a nonnicotine inhibitor of bone metabolism.

The effects of smokeless tobacco on bone were investigated using tibiae from chick embryos. The bones were cultured in nicotine (15-1500 micrograms/ml) or in smokeless tobacco extract (STE, 15 mg tobacco/ml culture medium) and the effects on bone glucose metabolism (oxygen consumption and lactate production) and collagen synthesis ([3H]proline hydroxylation) were tested in vitro. Only the highest concentration of nicotine tested produced substantial effects as demonstrated by decreased oxygen consumption and [3H]hydroxyproline content (82 and 90%, respectively) and by a 15% increase in lactate production. Results with STE, which contained nicotine concentrations ranging from 104 to 125 micrograms/ml, showed similarly reduced [3H]hydroxyproline content (90%), but oxygen consumption was only reduced 36%, while lactate production was elevated 60%. The ability of the bones to recover from treatment with STE was demonstrated by increased oxygen consumption and [3H]hydroxyproline content (18 and 78%, respectively) and decreased lactate production (47%) compared to the STE-treated bones. These findings suggest the following conclusions: (1) both nicotine and STE at concentrations found in the saliva of smokeless tobacco users stimulate glycolysis and markedly inhibit bone collagen synthesis and mitochondrial activity; (2) effects of STE on bone are not due to nicotine; and (3) under the conditions studied, bone partially recovers from the effects of STE.