Enhancement of innate and cell-mediated immunity by antimycobacterial antibodies.

We investigated the ability of human antibodies induced by Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination to protect against mycobacterial infections. Serum samples containing mycobacterium-specific antibodies were obtained from volunteers who had received two intradermal BCG vaccinations 6 months apart. Significant increases in lipoarabinomannan (LAM)-specific immunoglobulin G (IgG) were detected after both the primary and booster vaccinations. Effects of mycobacterium-specific antibodies on surface binding and internalization of BCG by neutrophils and monocytes/macrophages were studied, using green fluorescent protein (gfp)-expressing BCG. Surface-bound gfp-expressing BCG were distinguished from intracellular BCG by surface labeling with LAM-specific monoclonal antibody. Internalization of BCG by phagocytic cells was shown to be significantly enhanced in postvaccination serum samples. Furthermore, the inhibitory effects of neutrophils and monocytes/macrophages on mycobacterial growth were significantly enhanced by BCG-induced antibodies. The growth-inhibiting effects of postvaccination sera were reversed by preabsorption of IgG with Protein G. Finally, the helper effects of antimycobacterial antibodies for the induction of cell-mediated immune responses were investigated. BCG-induced antibodies significantly enhanced proliferation and gamma interferon production in mycobacterium-specific CD4(+) and CD8(+) T cells, as well as the proportion of proliferating and degranulating CD8(+) T cells. We conclude that mycobacterium-specific antibodies are capable of enhancing both innate and cell-mediated immune responses to mycobacteria.

C-reactive protein inhibits chemotactic peptide-induced p38 mitogen-activated protein kinase activity and human neutrophil movement.

Serum levels of the acute-phase reactant, C-reactive protein (CRP), increase dramatically during acute inflammatory episodes. CRP inhibits migration of neutrophils toward the chemoattractant, f-Met-Leu-Phe (fMLP) and therefore acts as an anti-inflammatory agent. Since tyrosine kinases are involved in neutrophil migration and CRP has been shown to decrease phosphorylation of some neutrophil proteins, we hypothesized that CRP inhibits neutrophil chemotaxis via inhibition of MAP kinase activity. The importance of p38 MAP kinase in neutrophil movement was determined by use of the specific p38 MAP kinase inhibitor, SB203580. CRP and SB203580 both blocked random and fMLP-directed neutrophil movement in a concentration-dependent manner. Additionally, extracellular signal-regulated MAP kinase (ERK) was not involved in fMLP-induced neutrophil movement as determined by use of the MEK-specific inhibitor, PD98059. Blockade of ERK with PD98059 did not inhibit chemotaxis nor did it alter the ability of CRP or SB203580 to inhibit fMLP-induced chemotaxis. More importantly, CRP inhibited fMLP-induced p38 MAP kinase activity in a concentration-dependent manner as measured by an in vitro kinase assay. Impressively, CRP-mediated inhibition of p38 MAP kinase activity correlated with CRP-mediated inhibition of fMLP-induced chemotaxis (r = -0.7144). These data show that signal transduction through p38 MAP kinase is necessary for neutrophil chemotaxis and that CRP intercedes through this pathway in inhibiting neutrophil movement.

Adhesion of bovine airway smooth muscle cells activates extracellular signal-regulated kinases.

Extracellular signal-regulated kinases (ERKs) phosphorylate and regulate cytoskeletal components of contractile cells and have been implicated in integrin-mediated adhesion. In this study, we examined the contributions of adherence, cell flattening, and cytoskeletal reorganization to adhesion-induced ERK activation in cultured bovine tracheal myocytes. We found, as evidenced by a reduction in electrophoretic mobility, that adhesion to fibronectin induced phosphorylation of both p44ERK1 and p42ERK2. In-gel kinase assays confirmed activation of both p44ERK1 and p42ERK2 in fibronectin-adherent cells, consistent with the notion that ligand-integrin binding is required for adhesion-induced ERK activation. However, ERK activation was maximal 2-4 h after plating, and adherence to either polystyrene or poly-L-lysine also caused ERK activation (fold increase 4 h after plating: fibronectin, 3.75 +/- 0.33; polystyrene, 3.95 +/- 0.78; poly-L-lysine, 2.14 +/- 0.36). Inspection of myocytes following passage onto fibronectin showed near 100% adhesion and cell spreading after 4 h, whereas cells plated onto poly-L-lysine demonstrated adherence but minimal spreading. To test whether the cytoskeletal reorganization accompanying cell spreading is required for adhesion-induced ERK activation, we assessed ERK activity following pretreatment with cytochalasin D, an inhibitor of actin polymerization. Cytochalasin inhibited both cell spreading and ERK activation following adhesion to fibronectin, but had no effect on growth factor-induced ERK activation in adherent cells. We conclude that adhesion-induced ERK activation in bovine tracheal myocytes may occur independently of ligand-integrin binding and is primarily related to the cell spreading that follows adhesion.

Role of C-reactive protein in acute lung injury.

Excessive inflammation caused by unregulated inflammatory processes can lead to disease. One example of this is seen in acute lung injury in which an individual is unable to regulate an inflammatory response in the lungs, with the net effect of self-induced tissue injury and loss of organ function. The acute-phase reactant, C-reactive protein, inhibits acute lung injury in animal models and, in this regard, acts as a major anti-inflammatory agent. Therefore, understanding the mechanism by which C-reactive protein elicits this inhibitory effect may provide important information about the design of therapeutic agents for the prevention or treatment of inflammation-mediated tissue injury and resultant loss of organ function.

Peptides derived from C-reactive protein inhibit neutrophil alveolitis.

C-reactive protein (CRP) is the classic acute phase reactant in humans, with serum levels elevated up to 1000-fold after the onset of inflammation. CRP inhibits chemotaxis of complement (C5a)-, LTB4-, IL-8-, and FMLP-stimulated neutrophils in vitro, and rabbits and transgenic mice with elevated serum CRP levels exhibit diminished neutrophil infiltration and vascular permeability in models of chemotactic factor-induced alveolitis. To evaluate the mechanism of CRP inhibition on chemoattractant-induced neutrophil inflammation in vivo, experiments were performed in mice infused with peptides of human CRP shown to inhibit C5a- and FMLP-stimulated neutrophil chemotaxis in vitro. After direct tracheal instillation of FMLP, mice previously injected via the retro-orbital plexus with CRP peptide 77-82 or 201-206 showed significant reductions (up to 90%) of neutrophils in the bronchoalveolar lavage fluid compared with vehicle-treated mice. Both CRP peptides also significantly (up to 55%) inhibited the increase in alveolar total protein levels. Control injections of native rabbit CRP (3 microM) inhibited neutrophil influx by 93% and protein leak by 55% in mice intratracheally instilled with FMLP. Despite similar levels of inhibition, approximately 10-fold more peptide by weight than native CRP was required. These data suggest that CRP degradation products at sites of tissue injury, in particular CRP peptides 77-82 and 201-206, are anti-inflammatory and can diminish lung injury by a reduction in neutrophil influx and protein leakage into alveoli following FMLP-induced inflammation.

Inhibition of C5a des Arg-induced neutrophil alveolitis in transgenic mice expressing C-reactive protein.

C-reactive protein (CRP) is the classic acute phase reactant in man with serum levels elevated up to 1,000-fold after the onset of inflammation. CRP inhibits chemotaxis of complement (C5a)-stimulated neutrophils in vitro and rabbits with elevated serum CRP levels exhibit diminished neutrophil infiltration and vascular permeability in a model of C5a-induced alveolitis. To specifically evaluate the effect of CRP on C5a-induced neutrophil inflammation in vivo, experiments were performed in transgenic mice capable of expressing rabbit CRP in an inducible fashion. After direct instillation of a known inflammatory agent (C5a des Arg) into the airways, transgenic mice with high plasma levels of CRP showed significantly diminished infiltration of neutrophils into bronchoalveolar lavage fluid (BALF) and a significant reduction of BALF total protein levels compared with normal mice. These data indicate that CRP can diminish lung injury by a reduction in neutrophil influx and protein leakage into alveoli following complement-induced inflammation.

Isolation and characterization of an NTP-dependent 3'-exoribonuclease from mitochondria of Saccharomyces cerevisiae.

RNA turnover in eukaryotes is thought to require 3'-exonuclease activity but so far no RNase with that specificity has been isolated from a eukaryote. We report here on the purification and characterization of a 3'-exoribonuclease isolated from the mitochondria of Saccharomyces cerevisiae. In vitro the purified enzyme displayed an absolute requirement of NTPs for activity. Each of the eight standard ribo- and deoxyribonucleotides supported activity with Km values ranging from 20 to 90 microM. The enzyme also displayed RNA-stimulated NTPase activity. The NTP-dependent enzyme cofractionated with three polypeptides of molecular masses 75,000, 90,000, and 110,000 daltons, although the native enzyme appears to have a molecular mass of 160,000 daltons predicted from the Stokes radius. The possible functions of this enzyme in vivo in the regulated decay of mitochondrial RNAs are discussed.

Rabbits with elevated serum C-reactive protein exhibit diminished neutrophil infiltration and vascular permeability in C5a-induced alveolitis.

In previous studies, we have shown that C-reactive protein (CRP) inhibits chemotaxis of neutrophils to complement fragments in vitro. To evaluate the effect of CRP on C5a-induced inflammation in vivo, a rabbit model of alveolitis was used. Rabbits pretreated with subcutaneous injections of croton oil had serum CRP increase from undetectable levels to 270 +/- 70 micrograms/ml 48 hours later. Rabbits were intubated and C5a des arg (10 micrograms/ml) instilled directly into the lungs via an endotracheal tube. Four to six hours later, the animals were killed and bronchoalveolar lavage performed. Rabbits pretreated with croton oil had significantly (P < 0.01) reduced C5a des arg-stimulated neutrophil infiltration (30 +/- 5%) into alveolar air spaces compared to untreated rabbits (64 +/- 9%). Increased numbers of total leukocytes in the alveolar washes coincided with increased neutrophil numbers whereas alveolar macrophages remained unchanged in all groups. Rabbits pretreated with croton oil also had a significant decrease (P < 0.05) in total protein (320 +/- 50 micrograms/ml) in lavage fluid after C5a instillation compared with untreated animals (850 +/- 140 micrograms/ml). In vitro, rabbit CRP (50 micrograms/ml) added to normal rabbit serum significantly (P < 0.05) inhibited chemotaxis of human neutrophils by 41%. Finally, direct intravenous pretreatment of rabbits with purified CRP also significantly reduced C5a-induced alveolitis. The CRP-C5a group had 33 +/- 10% neutrophil infiltration, a significant (P < 0.01) reduction from the C5a group (71 +/- 6%). The total protein content of the CRP-C5a rabbits was 986 +/- 165 micrograms/ml in the lavage fluid, which was significantly (P < 0.05) lower than the C5a group (1645 +/- 363 micrograms/ml). Therefore, CRP inhibits the development of neutrophil alveolitis and protein leakage in vivo and inhibits neutrophil chemotaxis in vitro. These data indicate that CRP offers a protective effect in neutrophil-mediated lung injury by reducing neutrophil influx and protein leak.