Plasminogen activator and thromboplastin activity from sheep alveolar macrophages.

Alveolar lavage cells from normal sheep were found to be composed of over 95% macrophages. When the cells were cultured, fibrinolytic and thromboplastin-like activities could be detected within 2-4 hours of incubation. As the number of cultured cells was increased the two activities in the conditioned medium increased proportionately. The cells were separated into two distinct subpopulations by means of a sedimentation velocity cell fractionation technique. The macrophage subpopulations were examined for differences in size, morphology, esterase staining and ability to release plasminogen activator and procoagulant activity respectively. These activities were confined to the large cell subpopulation. The fibrinolytic activity was shown to be plasminogen-dependent and could be inhibited by DFP. On the basis of this the fibrinolytic activity has been designated as plasminogen activator. The procoagulant activity was shown to be thromboplastin in nature because it was Factor VII dependent, inactivated by phospholipase C and not inhibited by DFP. The procoagulant activity has been designated as macrophage thromboplastin. The two activities could be distinguished on the basis of DFP inhibition.

Surface activation of factor XII (Hageman factor)--critical role of high molecular weight kininogen and another potentiator).

When factor XII was adsorbed to kaolin it slowly became activated and converted prekallikrein to kallikrein. In the presence of HMW-kininogen the rate of activation of factor XII and consequently that of prekallikrein was markedly enhanced. The enhancing effect of HMW-kininogen was a dose-dependent phenomenon. In order to enhance the activation of factor XII on a surface the HMW-kininogen molecule had to be intact. Cleavage of HMW-kininogen by kallikrein decreased the enhancing effect of HMW-kininogen, there being an inverse relation between the bradykinin-generated and the capacity to enhance factor XII activation. Another 'potentiator' of factor XII activation was isolated from proteins adsorbed to aluminum hydroxide. This potentiator further increased the activation of factor XII, also in a dose-dependent fashion. It was postulated that factor XII is slowly converted into its active form by exposure to negatively charged surfaces; that this process is enhanced by kallikrein and further accelerated by HMW-kininogen and the 'potentiator'; and that these enhancing substances probably act by opening active sites on the factor XII molecule.

Acute inflammation and a Shwartzman-like reaction induced by interleukin-1 and tumor necrosis factor. Synergistic action of the cytokines in the induction of inflammation and microvascular injury.

A Shwartzman-like reaction was elicited in rabbits by preparing the skin with intradermal injections of recombinant human tumor necrosis factor alpha (TNF alpha) and recombinant human interleukin-1 (IL-1 alpha or beta). The animals were challenged intravenously with endotoxin or by intravascular activation of complement with immune complexes or zymosan 18 hours later and were sacrificed after another 2 hours. Animals challenged with saline did not develop Shwartzman-like reactions. The sites prepared with endotoxin or with either form of IL-1 plus TNF alpha developed visible hemorrhage, whereas sites injected with either IL-1 or TNF alpha alone did not. Hemorrhage and microthrombosis were quantitated with 59Fe-labeled erythrocytes and 111In-platelets for 2 hours after the intravenous challenge, and the findings confirmed the observations made on gross inspection. Dermal sites prepared with the cytokines and challenged intravenously with endotoxin, immune complexes, or zymosan exhibited some diffuse hemorrhage and an intense erythrocyte extravasation around distended vessels, along skin appendages, and the panniculus carnosus muscle. The lumens of many large and postcapillary venules contained aggregates of platelets and leukocytes. These changes were superimposed on those seen at prepared sites (leukocyte infiltration). By electron microscopy fibrin was demonstrable in association with the formed elements of the blood. Histologic examination of the 18-hour-old preparative lesions or 20-hour-old lesions of saline-"challenged" animals revealed accumulation of leukocytes in the dermis, predominantly neutrophils. This accumulation was sparse at sites treated with only IL-1 or TNF alpha, but very intense at sites treated with both IL-1 and TNF alpha or with endotoxin. These observations were confirmed quantitatively by measuring the accumulation of 51Cr-labeled neutrophils for 2 hours after injection. The potency of IL-1 alpha was comparable to that in our earlier report, and TNF alpha was about three log times less potent. Sites treated with both IL-1 alpha and TNF alpha resulted in 69% greater neutrophil emigration than the additive response elicited by each cytokine. The reported findings implicate a synergism between IL-1 and TNF alpha in the induction of both the inflammatory reaction (preceding the Shwartzman reaction) and the thrombohemorrhagic component of the Shwartzman reaction proper.

The in vivo effect of leukotriene B4 on polymorphonuclear leukocytes and the microcirculation. Comparison with activated complement (C5a des Arg) and enhancement by prostaglandin E2.

The effect of synthetic leukotriene B4 (LTB4) on chemotaxis in vivo (51Cr-polymorphonuclear leukocyte [PMN] accumulation) was examined and its potency compared with that of C5a des Arg-containing zymosan-activated plasma (ZAP). On a molar basis the amount of C5a des Arg calculated to be in our preparation of ZAP was found to be up to approximately 80 times more potent than LTB4, although in vitro the two chemotaxins have been reported to be about equipotent. ZAP is more representative of what may happen in vivo than its principal constituent C5a des Arg, but for a more precise comparison the purified and isolated peptide will have to be compared with synthetic LTB4. Whereas ZAP induced severe PMN-dependent microvascular injury (increase in vessel permeability [125I-albumin] and hemorrhage [59Fe-erythrocytes]), LTB4 only induced an increase in vascular permeability, and this occurred only in the presence of simultaneously injected prostaglandin E2 (PGE2). PGE2 also enhanced substantially the number of PMNs and the amount of exuded plasma at injection sites of the chemotaxins. However, unlike in two other reports, LTB4 did not cause an immediate transient increase in vessel permeability, nor did it enhance the permeability-increasing effect of bradykinin. Furthermore, unlike PGE2 LTB4 did not induce an increase in blood flow, but a decrease (57Co-microspheres). It is concluded that LTB4 may act as a host-derived chemoattractant in vivo, but, compared with that of ZAP (primarily activated complement), its role in acute inflammation is probably less significant than that of the complement-derived chemotaxin(s).