H S Shin

Stimulation of rat astrocytes in vitro by calcium ionophore A23187 and/or lipopolysaccharide results in the generation of a cytotoxic factor that is functionally similar to the previously described macrophage-derived cytotoxic factor, tumor necrosis factor. Like the macrophage product, the astrocyte cytotoxic factor kills murine L 929 cell targets. In addition, it kills rat oligodendrocytes, the myelin-producing cells of the central nervous system. Human recombinant tumor necrosis factor also has cytotoxic activity directed against rat oligodendrocytes.

Macrophages play an important role in the acute tissue inflammatory response through the release of cytokines and growth factors in response to stimuli such as lipopolysaccharide (LPS). Macrophage inflammatory effector functions are also influenced by interactions with the extracellular matrix (ECM). Such macrophage-ECM interactions may be important in regulating chronic inflammatory responses. Recent evidence has suggested that hyaluronan (HA), a glycosaminoglycan (GAG) component of ECM can induce inflammatory gene expression in murine macrophages. HA exists in its native form as a large polymer, but is found as smaller fragments under inflammatory conditions. The NF-kappa B/I-kappa B transcriptional regulatory system has been shown to be a critical component of the host inflammatory response. We examined the effects of high molecular weight HA and lower molecular weight HA fragments on NF-kappa B activation in mouse macrophages. Only the smaller HA fragments were found to activate NF-kappa B DNA binding activity. After HA stimulation, I-kappa B alpha mRNA was induced and I-kappa B alpha protein levels, which initially decreased, were restored. The induction of I-kappa Balpha expression was not observed for other GAGs. The time course of I-kappa B alpha protein regeneration in response to HA fragments was consistent with an autoregulatory mechanism. In support of this mechanism, in vitro translated murine I-kappa B alpha inhibited HA fragment-induced NF-kappa B DNA binding activity. The NF-kappa B DNA binding complex in HA-stimulated extracts was found to contain p50 and p65 subunits. Activation of the NF-kappa B/I-kappa B system in macrophages by ECM fragments may be an important mechanism for propagating the tissue inflammatory response.

Large amounts of each C'3, C'5, C'6, C'7, C'8, and C'9 were consumed when guinea pig serum was incubated with endotoxic lipopolysaccharide, zymosan, or preformed immune complexes. Since these C' components subserve several of the biological activities which follow the injection of endotoxins into experimental animals, these experiments support the hypothesis that certain biological effects induced by endotoxins may be mediated via the C' system, and may account for some of the known similarity in the reactivities evoked by endotoxins and immune complexes in vivo.

We have examined the kinetics of 1,2-diacylglycerol production in quiescent IIC9 fibroblasts. alpha-Thrombin and epidermal growth factor (EGF) both stimulate an increase in the mass of cellular 1,2-diacylglycerol. The generation of 1,2-diacylglycerol is biphasic when stimulated by a high concentration of alpha-thrombin (500 ng/ml), with an early phase peaking at 15 s and a late phase peaking at 5 min. Production of 1,2-diacylglycerol is monophasic when stimulated by: (a) a low concentration of alpha-thrombin (100 pg/ml); (b) a high concentration of alpha-thrombin added to cultures which had been pretreated with chymotrypsin; or (c) EGF. In all cases the stimulation of 1,2-diacylglycerol was sustained for at least 30 min. In a previous report (Raben, D. M., Yasuda, K., and Cunningham, D. D. (1987) Biochemistry 26, 2759-2765), it was demonstrated that alpha-thrombin stimulates lipid metabolism in fibroblasts via two coupling mechanisms designated R1 and R2. We now present evidence that the early phase of alpha-thrombin-stimulated 1,2-diacylglycerol production is related to R1, which is characterized by: 1) increased release of arachidonic acid, 2) hydrolysis of polyphosphoinositides, and 3) inhibition by pretreating cultures with chymotrypsin. The late phase is related to R2 which is characterized by 1,2-diacylglycerol production in the absence of stimulated phosphoinositide hydrolysis and arachidonic acid release. In addition, EGF activates an R2-like mechanism in that it does not stimulate the release of arachidonic acid or hydrolysis of polyphosphoinositides but does stimulate a 2-fold increase in 1,2-diacylglycerol mass.

Upon activation of the complement system in guinea pig serum by endotoxin, a factor chemotactic for polymorphonuclear leukocytes is generated. This chemotactic factor has an approximate molecular weight (M.W.) of 15,000 and is independent of three minor chemotactic factors (M.W. τ;150,000, ∼68,000 and <4000) present in normal, heat-inactivated or EDTA-treated guinea pig serum. Purified, radiolabeled guinea pig C′3 and C′5 were used to determine whether the 15,000 M.W. chemotactic factor generated in serum was derived from either of these components. Endotoxin was incubated with guinea pig serum to which had been added either 125I C′3 or 125I C′5. Gel filtration elution profiles of the reaction mixtures revealed that both C′3 and C′5 were cleaved in endotoxin-treated serum. Moreover, the 15,000 M.W. chemotactic factor was identified with a cleavage product of C′5 but not of C′3. In addition, rabbit anti-guinea pig C′5, but not anti-C′3, significantly inhibited the activity of the 15,000 M. W. chemotactic factor. We conclude that the majority of chemotactic activity found in whole guinea pig serum after interaction with endotoxin is a product cleaved from C′5.