In vitro interaction between silicone gel and human monocyte-macrophages.

Controversy remains regarding the ability of silicone materials to induce a specific immune reaction versus a nonspecific inflammatory response. Histopathological analysis of the tissue around failed breast implants reveals chronic inflammation with silicone gel droplets either surrounded by giant cells or engulfed by macrophages, areas of fibrosis, and necrosis. Macrophages are the key cells engulfing or forming foreign body giant cells. To address the mechanisms of silicone-induced inflammation a model of human monocyte-derived macrophages (MDMs) was developed. After sonication of silicone gel, the silicone droplets were embedded in Type I collagen and used to coat glass coverslips; human MDMs were subsequently seeded on the coverslips and maintained in culture for up to 7 days. The advantage of the model was that human macrophages could be studied histologically, and cytochemically as they interacted with well-characterized silicone materials. Initial analysis of the human macrophages shows phagocytosis of the silicone gel within hours of exposure to the material. Analysis for pro-inflammatory cytokines reveals significant transient secretion of IL-1 (p < 0.01) over controls by human macrophages upon exposure to silicone gel at 24 h.

Changes in macrophage morphology and prolonged cell viability following exposure to polyethylene particulate in vitro.

The interaction of macrophages and ultra-high molecular weight polyethylene (PE) wear plays an important role in perpetuating chronic inflammation at the bone implant interface, leading to peri-implant osteolysis and mechanical failure of the implant. A model to study the interaction of human mature macrophages with orthopaedic biomaterial wear has been previously developed. With the use of the model, in this study, the mature human monocyte-derived macrophages (MDMs) were observed with light, fluorescent, and scanning electron microscopy (SEM), as well as transmission electron microscopy (TEM). The cell viability was investigated using calcein and ethidium staining. Following exposure to PE particulate, the morphology of the human MDMs was heterogeneous: rounded, flattened, and elongated. There was no morphological evidence of cytotoxicity or apoptosis. The MDM viability was not influenced by phagocytosis of PE particulate in a negative fashion. In fact, more prolonged cell viability was observed in the human MDMs exposed to PE particulate when compared to controls.

The role of T cells in polyethylene particulate induced inflammation.

OBJECTIVE: To investigate the role of T lymphocytes in ultra-high molecular weight polyethylene (UHMWPE) induced inflammation in joint arthroplasty. METHOD: We address the role of T cells in wear induced inflammation by injecting the knee joints of both immune competent rats and mice and severe combined immunodeficient (SCID) mice with UHMWPE. Histological and immunohistochemical analysis of the synovial tissues was compared. Interaction between human T cells and UHMWPE particles was examined in vitro using T cell activation assays. RESULTS: Histological and immunohistochemical analysis of the knees of the immune competent animals showed significant UHMWPE induced inflammation. In contrast, the tissue in the SCID mice knee joints showed very little inflammatory response to UHMWPE despite phagocytosis of the particulate. Since the SCID mice have no functional T or B lymphocytes, it is highly likely that the lack of inflammation in knee joints may be due to the absence of mouse T cells, as the infiltration of T cells into the joint tissue may enhance the inflammatory response to UHMWPE particles. T cell activation assays showed that T cells were not directly activated by UHMWPE particles and the nature of the interaction was not revealed from these experiments. CONCLUSIONS: Although T cells are not directly involved in UHMWPE particle induced inflammation, as shown by the T cell activation assays, the histological data from the mice studies clearly show differences in the amplitude of inflammation from animals with and without functional T cells. Our studies suggest that the T cells may enhance the inflammatory response due to a bystander effect. Since the macrophages upon ingestion of UHMWPE particles release several cytokines including tumor necrosis factor-alpha, interleukin 1, and IL-6, it is possible that T cells in the vicinity of these macrophages may become attracted to the knee joint and activated due to cytokine release.

Stereochemical requirements of alpha 2-adrenergic receptors for alpha-methyl substituted phenethylamines.

The alpha 1- and alpha 2-adrenergic effects of the stereoisomers of alpha-methyldopamine were evaluated in guinea pig aorta and field-stimulated guinea pig ileum, respectively, in order to establish the stereochemical requirements of these receptors for alpha-methyl substituted phenethylamines. The alpha 1-adrenergic receptor did not distinguish between the stereoisomers of alpha-methyldopamine which is in marked contrast to the alpha 2-adrenergic receptor where a dramatic stereochemical preference for the 2S(+)-isomer was observed. In addition, 2R(-)-alpha-methyldopamine displayed no alpha-receptor subtype specificity whereas 2S(+)-alpha-methyldopamine was highly selective (23 fold) for the alpha 2-adrenergic receptor. These results indicate that the alpha 2-adrenergic receptor can recognize and accept methyl substituents at the alpha-carbon atom of phenethylamines when correctly oriented, while the alpha 1-adrenergic receptor cannot. Thus, the alpha-carbon atom is a major determinant of the alpha 2-adrenergic effects of phenethylamines, and plays an important role in determining alpha-receptor subtype specificity. It is hypothesized that the alpha 2-adrenergic receptor (but not alpha 1) has an additional recognition site which will accommodate alpha-substituted phenethylamines.

Receptor interactions of imidazolines: alpha-adrenoceptors of rat and rabbit aortae differentiated by relative potencies, affinities and efficacies of imidazoline agonists.

1 Noradrenaline and a series of imidazolines were used to characterized and differentiate the postsynaptic alpha-adrenoceptors of rat and rabbit aortae. 2 Dose-response curves in each tissue revealed marked differences in the profile of agonist activity among the compounds. Based on the ED50 values for each compound, a rank order of potency of oxymetazoline greater than noradrenaline greater than tramazoline greater than tetrahydrozoline greater than clonidine was obtained in rabbit aorta and an order of noradrenaline greater than clonidine greater than tramazoline greater than oxymetazoline was obtained in rat aorta. Tetrahydrozoline had no agonist activity in rat aorta. 3 Dissociation constants were determined for each agonist in rat and rabbit aortae. Again, differences between the tissues were observed to the extent that the rank order of affinities for the imidazolines were exactly opposite for the two tissues. In rabbit aorta the order was, oxymetazoline greater than tramazoline greater than tetrahydrozoline greater than clonidine, whereas in rat aorta it was, clonidine greater than tetrahydrozoline greater then tramazoline greater than oxymetazoline. The extremes in tissue selectivity were observed with clonidine, which had approximately 125 fold higher affinity in rat aorta, and oxymetazoline, which had approximately 4 times higher affinity in rabbit aorta. 4 The absolute values of relative efficacies of the imidazolines studied, and their rank order, also differed between the two tissues. The relative efficacies of oxymetazoline and tramazoline were more than 15 fold greater in rabbit aorta than in rat aorta. Furthermore, tetrahydrozoline had a greater relative efficacy than clonidine in rabbit aorta while the converse was true in rat aorta. 5 Differences in the rank order of potency, affinity and relative efficacy of noradrenaline and a series of imidazolines in rat and rabbit aortae indicate that the postsynaptic alpha-adrenoceptors in these tissues are different. While the postsynaptic alpha-adrenoceptor of rabbit aorta is clearly of the alpha 1-subtype, the exact nature of the postsynaptic alpha-receptor of rat aorta is not clear. The unique alpha-receptor of rat aorta has properties of both alpha 1- and alpha 2-adrenoceptors.

Receptor interactions of imidazolines. IX. Cirazoline is an alpha-1 adrenergic agonist and an alpha-2 adrenergic antagonist.

The alpha-1 and alpha-2 adrenergic effects of cirazoline were evaluated in guinea-pig aorta and field-stimulated guinea-pig ileum, respectively. Cirazoline was found to be a full agonist at alpha-1 receptors having an ED50, dissociation constant (KA) and relative efficacy similar to that of (-)-norepinephrine. In contrast, cirazoline does not possess agonist activity at presynaptic alpha-2 receptors in the guinea-pig ileum. Thus, whereas norepinephrine and cirazoline both inhibited the twitch response of the field-stimulated ileum, only the response to norepinephrine was blocked by the selective alpha-2 antagonist, yohimbine. The nonadrenergic inhibition of the twitch response observed in the ileum with cirazoline resulted from weak anticholinergic activity (antimuscarinic) at the level of the postsynaptic effector organ and was observed only at high concentrations. At concentrations far below the level required to inhibit the twitch response, cirazoline was found to competitively antagonize the alpha-2-mediated inhibition of the twitch response elicited by norepinephrine. A Schild plot analysis indicated that cirazoline is a potent competitive alpha-2 receptor antagonist characterized by a pA2 value (i.e., -log KB) of 7.56. These results indicate that cirazoline is unique among imidazolines in that it is a potent alpha-1 adrenergic receptor agonist and an even more potent alpha-2 receptor antagonist. This unusual combination of activities could make cirazoline a particularly effective vasoconstricting agent.

Alpha and beta adrenergic effects of the stereoisomers of dobutamine.

Dobutamine and its stereoisomers were evaluated for alpha and beta adrenergic activities in vitro. The racemate and the (--)-isomer were found to be potent partial agonists of alpha adrenergic receptors in rat aorta. The (+)-isomer lacked alpha agonist activity. The affinities of (+/-)-, (+)- and (--)-dobutamine for the alpha adrenergic receptor were high (--log KB values of 7.01, 7.02 and 7.07, respectively) and not significantly different from one another. These data indicate that (+)- and (--)- dobutamine bind equally to the alpha adrenergic receptor, however, subsequent to binding, only the (--)-isomer is capable of activating the receptor and eliciting an alpha adrenergic response. The (+)-isomer, which has the same affinity as the (--)-isomer but which lacks agonist activity, is a potent competitive alpha blocker. Both stereoisomers of dobutamine were agonists of beta adrenergic receptors of cat papillary muscle and right atria. In contrast to the alpha adrenergic effects, the more potent isomer at the beta adrenergic receptor was (+)- dobutamine. The isomeric activity ratio for the stereoisomers of dobutamine was approximately 1 log unit in favor of the (+)-isomer with respect to both inotropic and chronotropic responses. Dose-response curves to the racemate were always situated between the stereoisomers, approximately 2-fold to the right of (+)-dobutamine. These results indicate that the stereochemical requirements of alpha and beta adrenergic receptors are opposite for the stereoisomers of dobutamine with the alpha receptor favoring the (--)-isomer and the beta receptor favoring the (+)-isomer.

Postsynaptic alpha adrenergic receptor subtypes differentiated by yohimbine in tissues from the rat. Existence of alpha-2 adrenergic receptors in rat aorta.

The selective alpha-2 adrenergic receptor antagonist, yohimbine, was used to differentiate postsynaptic alpha-adrenergic receptors in five peripheral tissues of the rat. Three distinct postsynaptic receptor subtypes were observed based on the affinity of the receptors for yohimbine. Receptors with high affinity for yohimbine were detected in the aorta, whereas low affinity receptors were observed in the vas deferens. The affinity for yohimbine in these two tissues differed by over 50-fold. Receptors with intermediate affinity for yohimbine were found in the portal vein, spleen and bladder. The Schild plot for yohimbine in the bladder suggests that blockade of alpha receptors in this tissue is not competitive or that there exists more than one type of alpha-adrenergic receptor present. Comparison of the dissociation constant of yohimbine in the aorta with dissociation constants obtained from the literature for this compound in a variety of tissues containing alpha-1 or alpha-2 adrenergic receptors indicates that the postsynaptic alpha adrenergic receptor in the aorta is of the alpha-2 type. Conversely, the postsynaptic alpha adrenergic receptor in the portal vein appears to be alpha-1. The differences in postsynaptic alpha appears to be alpha-1. The differences in postsynaptic alpha adrenergic receptors in these two vascular tissues may reflect the marked differences in adrenergic innervation and the possible relative lack of neuroeffector junctional alpha adrenergic receptors in the rat aorta.

Receptor interactrions of imidazolines. VI. Significance of carbon bridge separating phenyl and imidazoline rings of tolazoline-like alpha adrenergic imidazolines.

The pharmacological significance of the carbon bridge separating the imidazoline and phenyl rings of tolazoline-like alpha adrenergic imidazolines has been investigated. Extending the carbon bridge to two carbon atoms, or deleting the carbon bridge, lowers affinity of the imidazolines for the alpha receptor and markedly decreases or abolishes efficacy (i.e., agonist activity), suggesting that a single carbon atome optimallyu separates the phenyl and imidazoline rings. Although one carbon is optimal for alpha adrenergic activity, this particular atom does not appear to be essential since nitrogen may substitute for carbon with no marked or consistent changes observed in affinity or efficacy. Hydroxylation of the carbon bridge decreases affinity for the receptor approximately 10-fold but does not alter efficacy, whereas a similar substitution made in the norepinephrine-series of phenethylamines markedly increases affinity (Patil et al., 1974). With both the imidazolines and phenethylamines, this carbon atom may stereoselectively influence binding to the receptor. These results suggest that the carbon atom bridging the phenyl and imidazoline rings of tolazoline-like imidazolines serves only to provide optimal separation between these rings and does not contribute directly to the binding process. It is proposed that alpha adrenergic imidazolines interact differently with the alpha adrenergic receptor than the norepinephrine-like phenethylamines.

Receptor interactions of imidazolines. V. clonidine differentiates postsynaptic alpha adrenergic receptor subtypes in tissues from the rat.

The alpha-adrenergic effects of clonidine were studied in five tissues of the rat. Postsynaptic alpha adrenergic receptors in these tissues may be divided into three distinct classes based on affinity of the receptors for clonidine. High affinity receptors are located in the aorta and low affinity receptors in the vas deferens. Receptors with intermediate affinity are found in the portal vein, spleen and bladder. The bladder may also contain, to a small extent, a population of receptors similar to those found in the vas deferens which may mediate a different response. Available data suggest that the alpha adrenergic receptor of the rat aorta with high affinity for clonidine may be of the alpha-2 type but located postsynaptically in this tissue.

Receptor interactions of imidazolines. IV. Structural requirements for alpha adrenergic receptor occupation and receptor activation by clonidine and a series of structural analogs in rat aorta.

Clonidine and a series of nine halogen and alkyl-substituted structural analogs were evaluated for alpha adrenergic receptor agonist activity in rat aorta. Phenylephrine was included for comparison. All the imidazolines were partial agonists with respect to phenylephrine due to the fact that they only weakly activated the receptor. Whereas phenylephrine requires approximately 0.2% receptor occupancy to produce a response 20% of maximum, the imidazolines required from 25 to 100% receptor occupancy to produce the same response. Although clonidine and its structural analogs are from 125 to 500 times weaker activators of the alpha adrenergic receptor than phenylephrine, they have from 2 to 60 times higher affinity for the receptor than phenylephrine. The results support our previous conclusions that a dichotomy exists between the factors that direct receptor occupation and receptor activation since the structural requirements for each of these parameters differ.