Hyaluronan fragments synergize with interferon-gamma to induce the C-X-C chemokines mig and interferon-inducible protein-10 in mouse macrophages.

Hallmarks of chronic inflammation and tissue fibrosis are increased influx of activated inflammatory cells, mediator release, and increased turnover and production of the extracellular matrix (ECM). Recent evidence has suggested that fragments of the ECM component hyaluronan play a role in chronic inflammation by inducing macrophage expression of chemokines. Interferon-gamma (IFN-gamma), an important regulator of macrophage functions, has been shown to induce the C-X-C chemokines Mig and IP-10. These chemokines affect T-cell recruitment and inhibit angiogenesis. The purpose of this investigation was to determine the effect of hyaluronan (HA) on IFN-gamma-induced Mig and IP-10 expression in mouse macrophages. We found a marked synergy between HA and IFN-gamma on Mig and IP-10 mRNA and protein expression in mouse macrophages. This was most significant with Mig, which was not induced by HA alone. The synergy was specific for HA, was not dependent on new protein synthesis, was not mediated by tumor necrosis factor-alpha, was selective for Mig and IP-10, and occurred at the level of gene transcription. These data suggest that the ECM component HA may influence chronic inflammatory states by working in concert with IFN-gamma to alter macrophage chemokine expression.

Upregulation of alveolar epithelial fluid transport after subacute lung injury in rats from bleomycin.

Alveolar epithelial fluid transport was studied 10 days after subacute lung injury had been induced with intratracheal bleomycin (0.75 U). An isosmolar Ringer lactate solution with 5% bovine serum albumin and 125I-labeled albumin as the alveolar protein tracer was instilled into the right lung; the rats were then studied for either 1 or 4 h. Alveolar fluid clearance was increased in bleomycin-injured rats by 110% over 1 h and by 75% over 4 h compared with control rats (P < 0.05). The increase in alveolar fluid clearance was partially inhibited by amiloride (10(-3) M). Alveolar fluid clearance decreased toward normal levels in rats that were studied 60 days after bleomycin instillation. Remarkably, the measured increase in net alveolar fluid clearance occurred in the presence of a significant increase in alveolar epithelial permeability to protein. Moreover, the increase in alveolar epithelial fluid clearance occurred even though the mRNA for the alpha-subunit of the epithelial sodium channel was decreased in alveolar epithelial type II cells isolated from these rats. In addition, 22Na uptake by isolated alveolar epithelial type II cells from rats treated with bleomycin demonstrated a 52% decrease in uptake compared with type II cells from control rats. Morphological results demonstrated a significant hyperplasia of alveolar type II epithelial cells 10 days after bleomycin injury. Thus, these results provide evidence that proliferation of alveolar epithelial type II cells after acute lung injury may upregulate the transport capacity of the alveolar epithelium, even though the expression of epithelial sodium channels is reduced and the uptake of 22Na per cell is also reduced. These results may have clinical relevance for the resolution of alveolar edema in the subacute phase of lung injury.

Protein tyrosine phosphatases mediate cell readhesion in alveolar epithelial cells mechanically separated from in vitro matrix.

Alveolar epithelial type II cells are the progenitor cells for restoring the alveolar epithelial barrier after acute lung injury. During repair of lung injury, the alveolar epithelial type II cells reepithelialize denuded air spaces, a process that involves breaking and reforming cell adhesions. A novel technique of mechanical separation of cultured alveolar epithelial cells from in vitro matrix was used to examine the intracellular signals that result when alveolar epithelial cell adhesions are broken. The results show that the tyrosine phosphorylation levels of focal adhesion kinase, paxillin, and pp60(src) decreased immediately after mechanical separation of the cells. Levels returned to nearly normal by 24 h after mechanical separation. Paxillin and pp60(scr) coprecipitated with focal adhesion kinase regardless of their phosphorylation state. Interestingly, the tyrosine phosphorylation level of the mitogen-activated protein kinase, p42(erk2), increased 15 min after mechanical separation. Preincubation of cell monolayers with phenylarsine oxide, a protein tyrosine phosphatase inhibitor, blocked the decrease in tyrosine phosphorylation levels of focal adhesion kinase, paxillin and pp60(src). Phenylarsine oxide incubation also prevented readhesion of mechanically separated cells at 24 h, but genistein, a tyrosine kinase inhibitor, had no effect. We conclude that protein tyrosine phosphatases are activated immediately after cultured alveolar epithelial cells are mechanically separated from in vitro matrix, and their activation is required for alveolar epithelial cell readhesion.

Transforming growth factor-alpha and epidermal growth factor activate mitogen-activated protein kinase and its substrates in intestinal epithelial cells.

The signal transduction pathways of mitogenic stimuli in intestinal epithelial cells are not clearly understood. We report here a possible signaling pathway of two closely related agonists, transforming growth factor-alpha (TGF alpha) and epidermal growth factor (EGF). Both increase thymidine incorporation in the intestinal epithelial cell (IEC) line IEC-6. This increase is dose dependent and inhibited by the tyrosine kinase inhibitors genistein and tyrphostin. The addition of either TGF alpha or EGF to IEC-6 cells also stimulates the activities of the two forms of mitogen-activated protein kinase, p42erk2 MAPK and p44erk1 MAPK, as evidenced by increased incorporation of radiolabeled phosphate in myelin basic protein. The main difference between the MAPK activity levels induced by the two agonists is in the intensity of the response. Maximum TGF alpha-induced stimulation of p42erk2 MAPK activity is 9-fold at 2 ng/ml, while maximum EGF stimulation is only 4.5-fold at 25 ng/ml. These doses correlated closely with the dose required for maximum thymidine incorporation. The activity of the 90-kDa ribosomal S6 kinase, a downstream substrate for activated MAPK, is also enhanced as evidenced by increased incorporation of radiolabeled phosphate in the rsk kinase substrate peptide in IEC-6 cells following stimulation with either TGF alpha or EGF. This increase correlates closely with the stimulus-induced increase in MAPK activity with respect to dose, but the time of increased activity is more prolonged, especially after EGF stimulation. TGF alpha induced the synthesis of both c-Fos and c-Myc, two nuclear substrates for MAPK, and increased c-fos and c-myc message levels as well. However, c-Jun protein and c-jun mRNA were not induced. The increase in IEC-6 cell proliferation in response to TGF alpha and EGF stimulation may then be due, in part, to an increase in immediate early gene expression as a direct result of MAPK and RSK activation.

Transforming growth factor-alpha increases tyrosine phosphorylation of microtubule-associated protein kinase in a small intestinal crypt cell line (IEC-6).

The small intestinal crypt cell line (IEC-6) is an undifferentiated, untransformed, mitotically active cell used in this study to determine the effect of transforming growth factor-alpha (TGF-alpha) on tyrosine phosphorylation levels of cellular proteins. Thymidine incorporation increased maximally after addition of 2 ng/ml TGF-alpha for 24 h. At the same dose, TGF-alpha induced the tyrosine phosphorylation of proteins with approximate molecular masses of 42, 44, 52, 80, 150 and 175 kDa as shown by Western blots treated with anti-phosphotyrosine antibody. The most intense phosphorylation was seen in the 42 kDa (p42) and 44 kDa (p44) proteins, which were identified as two isoforms of microtubule-associated protein kinase (MAPK). This phosphorylation was seen as early as 5 min post stimulation and was dose dependent. Both p42 and p44 were found in the nucleus after stimulation, although a basal level of unphosphorylated protein was present before stimulation. The observed tyrosine phosphorylation of p42 and p44 was inhibited by genistein, a tyrosine kinase inhibitor, and tyrphostin 23, an epidermal growth factor receptor tyrosine kinase inhibitor. We conclude that MAPK is tyrosine phosphorylated in response to TGF-alpha stimulation of IEC-6 cells.

Superoxide anion production by rat neutrophils at various stages of bleomycin-induced lung injury.

This study investigated the level of activation of neutrophils isolated from rats at various stages of bleomycin-induced lung injury. Neutrophils were collected from blood and bronchoalveolar lavage (BAL) fluid and their superoxide anion (O2-)-generating capacity measured in response to phorbol myristate acetate (PMA) and opsonized zymosan (OZ) stimulation. When stimulated with PMA, BAL neutrophils isolated from animals 3 days after bleomycin treatment had a significantly greater capacity to produce O2- than BAL neutrophils from animals 7 days after bleomycin treatment. The O2- levels of 7 day BAL neutrophils more closely resembled the resting levels obtained with circulating neutrophils from both control and bleomycin-treated animals. There were no differences observed in any of the neutrophils when stimulated with OZ. Myeloperoxidase levels were measured in plasma and BAL and found to be elevated only in plasma at 7 days after bleomycin. These data demonstrate that neutrophil activation does occur in this model and that the activation appears to be transient, in response to specific stimuli and compartmentalized between the lung and blood.

A comparison of foreign body and hypersensitivity granuloma formation in the mouse: kinetic development, anergy, angiotensin converting enzyme levels, and response to captopril therapy.

Pulmonary granuloma size, anergy, serum and lung angiotensin converting enzyme (ACE) levels, and responsiveness to captopril therapy were measured in both pulmonary hypersensitivity granuloma (HSG) and foreign body granuloma (FBG) murine models. The kinetic development of the granuloma was similar in both models, peaking at 3 days and resolving by 14 days. The granuloma size of the FBG model was significantly larger than the HSG model at 1 and 3 days. The FBG model further differed from the HSG model in that anergy was observed at 3 days and lung ACE levels were increased at 1, 7, and 14 days. In the HSG model anergy was not observed and lung ACE levels were significantly elevated only at 1 and 7 days. Furthermore, the HSG model was shown to be responsive to captopril therapy whereas the FBG model was not. This study shows that two murine pulmonary granuloma models, although displaying similar growth and resolution kinetics, differ in terms of granuloma size, anergy, lung ACE activity, and responsiveness to captopril therapy, suggesting that the mechanism(s) involved in the inflammatory response of the two models may be different.

Superoxide production by rat neutrophils in the oleic acid model of lung injury.

The purpose of this study was to investigate the superoxide anion (O2-)-generating capacity of neutrophils isolated from rats at various stages of oleic acid(OA)-induced lung injury. Neutrophils were collected from blood, bronchoalveolar lavage (BAL), and peritoneal cavity (glycogen induced) after OA administration. Control neutrophils were collected from the blood of normal animals as a representative of nonprimed cells that produce low levels of O2-. A second control was the glycogen-elicited peritoneal neutrophil of normal animals which represented primed cells that produce enhanced levels of O2-. The ability of the neutrophils to produce O2- was evaluated by using both myristate acetate and opsonized zymosan as stimulants. Neutrophils isolated from blood and BAL from OA-injured lungs produced low levels of O2- and resembled closely the circulating, nonprimed neutrophil. Myeloperoxidase levels were measured in plasma and BAL and were found to be elevated in BAL of OA-injured animals. The inability of neutrophils to produce high levels of O2- and the elevation of myeloperoxidase suggest that neutrophils present in the lung may have degranulated in response to prior activation and are therefore incapable of further superoxide production.