Hyaluronan mediates airway hyperresponsiveness in oxidative lung injury.

Chlorine (Cl2) inhalation induces severe oxidative lung injury and airway hyperresponsiveness (AHR) that lead to asthmalike symptoms. When inhaled, Cl2 reacts with epithelial lining fluid, forming by-products that damage hyaluronan, a constituent of the extracellular matrix, causing the release of low-molecular-weight fragments (L-HA, <300 kDa), which initiate a series of proinflammatory events. Cl2 (400 ppm, 30 min) exposure to mice caused an increase of L-HA and its binding partner, inter-α-trypsin-inhibitor (IαI), in the bronchoalveolar lavage fluid. Airway resistance following methacholine challenge was increased 24 h post-Cl2 exposure. Intratracheal administration of high-molecular-weight hyaluronan (H-HA) or an antibody against IαI post-Cl2 exposure decreased AHR. Exposure of human airway smooth muscle (HASM) cells to Cl2 (100 ppm, 10 min) or incubation with Cl2-exposed H-HA (which fragments it to L-HA) increased membrane potential depolarization, intracellular Ca(2+), and RhoA activation. Inhibition of RhoA, chelation of intracellular Ca(2+), blockade of cation channels, as well as postexposure addition of H-HA, reversed membrane depolarization in HASM cells. We propose a paradigm in which oxidative lung injury generates reactive species and L-HA that activates RhoA and Ca(2+) channels of airway smooth muscle cells, increasing their contractility and thus causing AHR.

The lipocalin alpha1-microglobulin protects erythroid K562 cells against oxidative damage induced by heme and reactive oxygen species.

Alpha(1)-microglobulin is a 26 kDa plasma and tissue glycoprotein that belongs to the lipocalin protein superfamily. Recent reports show that it is a reductase and radical scavenger and that it binds heme and has heme-degrading properties. This study has investigated the protective effects of alpha(1)-microglobulin against oxidation by heme and reactive oxygen species in the human erythroid cell line, K562. The results show that alpha(1)-microglobulin prevents intracellular oxidation and up-regulation of heme oxygenase-1 induced by heme, hydrogen peroxide and Fenton reaction-generated hydroxyl radicals in the culture medium. It also reduces the cytosol of non-oxidized cells. Endogeneous expression of alpha(1)-microglobulin was up-regulated by these oxidants and silencing of the alpha(1)-microglobulin expression increased the cytosol oxidation. alpha(1)-microglobulin also inhibited cell death caused by heme and cleared cells from bound heme. Binding of heme to alpha(1)-microglobulin increased the radical reductase activity of the protein as compared to the apo-protein. Finally, alpha(1)-microglobulin was localized mainly at the cell surface both when administered exogeneously and in non-treated cells. The results suggest that alpha(1)-microglobulin is involved in the defence against oxidative cellular injury caused by haemoglobin and heme and that the protein may employ both heme-scavenging and one-electron reduction of radicals to achieve this.

Inhibitory and anti-inhibitory factors of rat serum active on the G1-S transition of hepatocyte cell cycle.

Rat hepatocytes are responsive to a serum factor inhibiting their progression through the cell cycle from the late G1 phase to the S phase. After fractionation of normal adult rat serum by two chromatographic steps on DEAE cellulose and sephadex gel filtration, the inhibitory activity was linked to proteins having a high electronegative charge and of apparent high molecular weight. Polyacrylamide gel electrophoretic analysis of active fraction showed that the alpha1 macroglobulin was its main component. Male and female baby rats were sensitive to the inhibitory factor from normal rats. Contrary to the normal adult rat serum the whole hepatectomized adult rat serum did not exhibit any ingibitory activity on the G1-S transition. However, two components having antagonist activities: an alpha1 globulin and a gamma globulin, were separated by chromatographic procedures from hepatectomized rat serum. (a) The alpha1 globulin showed an inhibitory activity. It had an apparent molecular weight lower than that found in normal rats. Its activity was sex related: only male baby rats were responsive. (b) The factor present in the gamma globulin fraction was found to be antagonistic to the alpha1 globulin factor. Its occurrence after hepatectomy explains the absence of inhibitory activity in the serum of hepatectomized rats.

Studies of the inhibition of C56-initiated lysis (reactive lysis). IV. Antagonism of the inhibitory activity c567-INH by poly-L-lysine.

The stable intermediate complex C56 can initiate the lysis (reactive lysis) of unsensitized erythrocytes (E) by the membrane attack machanism of complement. Certain serum constituents designated C567-INH inhibit reactive lysis by preventing the C567 complex, once formed, from attaching to a membrane surface. It is shown here that microgram quantities of poly-L-lysine (PLL), a synthetic polycation of molecular weight 180,000, can reverse the effests of C567-INH, and thereby potentiate formation of EC567 by erythrocytes, C56 and C7 in whole serum. Erythrocytes exposed to PLL in a preincubation step did not show either increased susceptibility to C567 or resistance to C567-INH, and reversal of C567-IHN by given amounts of PLL was not diminished as cell concentrations were greatly increased, indicating that the effect of PLL was predominantly directed against fluid phase rather than against erythrocyte membrane substrates. The effects of PLL and C567-INH were quantitatively reciprocal. Thus, PLL-induced potentiation of C56-induced lysis is a solute effect which seems to involve direct neutralization of naturally occurring serum inhibitors of the C567 trimolecular complex of complement. The use of PLL thus provides a suitable antagonist for C567-INH in reaction mixtures, and allows evaluation of the role of C567 and C567-INH in a variety of situations involving C-mediated lysis.

Inhibitors of kallikrein in human plasma.

Human plasma was fractionated by ammonium sulfate precipitation, DEAE-cellulose chromatography, and Sephadex G-200 gel filtration to determine which method would give the greatest number of clearly separable kallikrein inhibitory peaks. With G-200 gel filtration three peaks could be separated which were demonstrated to contain alpha(2)-macroglobulin, C1 inactivator, and alpha(1)-antitrypsin. No other kallikrein inhibitors could be identified. The fractions containing C1 inactivator and alpha(2)-macroglobulin appeared to be more effective against kallikrein than that containing alpha(1)-antitrypsin. A patient with hereditary angioneurotic edema was shown to have an abnormal C1 inactivator protein capable of interfering with kallikrein's biologic, but not its esterolytic activity. Heat-treated human plasma, a commonly used source of kininogen for experiments with kallikrein, was shown to have kallikrein inhibitory activity.