Larry D. Crouch

Role of CC Chemokines (Macrophage Inflammatory Protein-1β, Monocyte Chemoattractant Protein-1, RANTES) in Acute Lung Injury in Rats

The role of the CC chemokines, macrophage inflammatory protein-1β (MIP-1β), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1β, MCP-1, and RANTES were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1β and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response. Treatment of rats with anti-MIP-1β Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-α in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti-rat RANTES had no effect on the development of lung injury. In animals pretreated intratracheally with blocking Abs to MCP-1, RANTES, or MIP-1β, significant reductions in the bronchoalveolar lavage content of these chemokines occurred, suggesting that these Abs had reached their targets. Conversely, exogenously MIP-1β, but not RANTES or MCP-1, caused enhancement of the lung vascular leak. These data indicate that MIP-1β, but not MCP-1 or RANTES, plays an important role in intrapulmonary recruitment of neutrophils and development of lung injury in the model employed. The findings suggest that in chemokine-dependent inflammatory responses in lung CC chemokines do not necessarily demonstrate redundant function.

Role of IL-18 in Acute Lung Inflammation

We have examined the role of IL-18 after acute lung inflammation in rats caused by intrapulmonary deposition of IgG immune complexes. Constitutive IL-18 mRNA and protein expression (precursor form, 26 kDa) were found in normal rat lung, whereas in inflamed lungs, IL-18 mRNA was up-regulated; in bronchoalveolar (BAL) fluids, the 26-kDa protein form of IL-18 was increased at 2–4 h in inflamed lungs and remained elevated at 24 h, and the “mature” protein form of IL-18 (18 kDa) appeared in BAL fluids 1–8 h after onset of inflammation. ELISA studies confirmed induction of IL-18 in inflamed lungs (in lung homogenates and in BAL fluids). Prominent immunostaining for IL-18 was found in alveolar macrophages from inflamed lungs. When rat lung macrophages, fibroblasts, type II cells, and endothelial cells were cultured in vitro with LPS, only the first two produced IL-18. Intratracheal administration of rat recombinant IL-18 in the lung model caused significant increases in lung vascular permeability and in BAL content of neutrophils and in BAL content of TNF-α, IL-1β, and cytokine-induced neutrophil chemoattractant, whereas intratracheal instillation of anti-IL-18 greatly reduced these changes and prevented increases in BAL content of IFN-γ. Intratracheal administration of the natural antagonist of IL-18, IL-18 binding protein, resulted in suppressed lung vascular permeability and decreased BAL content of neutrophils, cytokines, and chemokines. These findings suggest that endogenous IL-18 functions as a proinflammatory cytokine in this model of acute lung inflammation, serving as an autocrine activator to bring about expression of other inflammatory mediators.

Neutrophil chemotactic activity and C5a following systemic activation of complement in rats

Using ELISA analysis, rat C5a was stimulated in serum from rats undergoing systemic activation of complement after intravenous infusion of purified cobra venom factor (CVF). Biological (neutrophil chemotactic) activity was also assessed. Serum levels of C5a were directly proportional to the amount of CVF infused. C5a and neutrophil chemotactic activity, peaked by 5 min, then plateaued. In vitro addition of anti-C5a to serum samples of CVF-infused rats totally abolished chemotactic activity, indicating that all biological activity could be ascribed to C5a. Blood neutrophils obtained from CVF-infused animals showed a significant upregulation of CD11b, the increase being reduced (38%) in animals pretreated with anti-C5a. These findings indicate that infusion of CVF into rats produces generation of C5a, all chemotactic activity in serum being related to C5a. The in vivo generation of C5a is, at least inpart, responsible for upregulation of CD11b on blood neutrophils.

REGULATORY EFFECTS OF INTERLEUKIN-11 DURING ACUTE LUNG INFLAMMATORY INJURY

The role of interleukin‐11 (IL‐11) was evaluated in the IgG immune complex model of acute lung injury in rats. IL‐11 mRNA and protein were both up‐regulated during the course of this inflammatory response. Exogenously administered IL‐11 substantially reduced, in a dose‐dependent manner, the intrapulmonary accumulation of neutrophils and the lung vascular leak of albumin. These in vivo anti‐inflammatory effects of IL‐11 were associated with reduced NF‐κB activation in lung, reduced levels of tumor necrosis factor α (TNF‐α) in bronchoalveolar lavage (BAL) fluids, and diminished up‐regulation of lung vascular ICAM‐1. It is interesting that IL‐11 did not affect BAL fluid content of the CXC chemokines, macrophage inflammatory protein‐2 (MIP‐2) and cytokine‐inducible neutrophil chemoattractant (CINC); the presence of IL‐11 did not affect these chemokines. However, BAL content of C5a was reduced by IL‐11. These data indicate that IL‐11 is a regulatory cytokine in the lung and that, like other members of this family, its anti‐inflammatory properties appear to be linked to its suppression of NF‐κB activation, diminished production of TNF‐α, and reduced up‐regulation of lung vascular ICAM‐1. J. Leukoc. Biol. 66: 151–157; 1999.

Dual blockade of P-selectin and β2-integrin in the liver inflammatory response after uncontrolled hemorrhagic shock

BACKGROUNDnNeutrophil infiltration is a characteristic feature of the hepatic injury associated with prolonged hypotension. Previous work has already stressed the important contribution of neutrophil-endothelial cell interactions in the organ injury seen after hemorrhagic shock. Single-blockade strategies using monoclonal antibodies (MAbs) against either selectin or integrin receptors have been demonstrated to be effective in limiting the tissue inflammatory response observed in this clinical disorder. One unexplored topic is the additive effect(s) and the potential antiinflammatory properties of the combined blocking of P-selectin plus beta2-integrin in the liver inflammatory response after uncontrolled hemorrhagic shock in rats.nnnSTUDY DESIGNnSprague-Dawley rats (n = 64) weighing 250-300 g were included in a three-phase model of uncontrolled hemorrhagic shock. A prehospital phase consisted of 90 minutes of fluid resuscitation with lactated Ringers solution to reach a mean arterial pressure (MAP) of 40 mmHg; a hospital phase consisted of 60 minutes of hemostasis and fluid resuscitation with lactated Ringers solution to reach a MAP of 80 mmHg; and the third phase was 3 days of observation. All rats had 3 mL/100 g of blood volume shed during the initial 15 minutes. At 30 minutes, 75% tail amputation produced uncontrolled hemorrhagic shock. Four groups were randomized (n = 16 per group), and treatment at the beginning of resuscitation included normal saline (group 1); anti-P-selectin MAb, RMP-1 (group 2); anti-beta2-integrin MAb, WT.3 (group 3); or anti-P-selectin plus anti-beta2-integrin MAbs (group 4). The following indices were evaluated: fluid requirements for resuscitation, liver injury tests, liver tissue myeloperoxidase, and liver histology.nnnRESULTSnDual blockade of P-selectin and beta2-integrin significantly reduced fluid requirements for resuscitation (p < 0.05). We also observed a statistically significant improvement (p < 0.05) in tests demonstrating hepatic injury, myeloperoxidase in hepatic tissue, and histology studies. Survival was increased from 40% in controls to 60% with the dual-blockade treatment.nnnCONCLUSIONSnThese results indicate that dual-blockade strategies aimed at P-selectin and beta-integrin provided a protective effect in the liver inflammatory response after uncontrolled hemorrhagic shock in rats. Although dual blockade was more effective than either individual blockade alone, questions remain about the possible redundancy in the inflammatory adhesion pathways after this clinical condition.

Plasma ammonia concentrations and the slow component of oxygen uptake kinetics during cycle ergometry.

Malek, MH, Housh, TJ, Crouch, LD, Johnson, GO, Hendrix, CR, Beck, TW, Mielke, M, Schmidt, RJ, and Housh, DJ. Plasma ammonia concentrations and the slow component of oxygen uptake kinetics during cycle ergometry. J Strength Cond Res 22(6): 2018-2026, 2008-The purposes of this study were to 1) compare the patterns of responses for plasma ammonia concentration ([NH3]) during moderate- vs. heavy-intensity cycle ergometry, and 2) examine the relationship between the &OV0312;o2 slow component (&OV0312;o2SC) and plasma [NH3]. Thirteen healthy, untrained men (mean ± SEM age = 24.8 ± 0.6 years) performed a total of eight constant power output exercises (7 minutes in duration) at two different intensities (moderate, 60% gas exchange threshold [GET] = 60% of the gas exchange threshold; and heavy, Δ50% = 50% of the difference between GET and &OV0312;o2max). Blood was collected from an antecubital vein before the exercise, during the last 3 minutes of the 6-minute warm-up, and during each minute of the 7-minute constant power output workbout. The time course of changes in plasma [NH3] and &OV0312;o2 during the two constant power output exercise intensities were assessed separately using 2 (intensity) × 7 (time) repeated-measures analyses of variance. For 60% GET, there were no significant differences in the mean normalized plasma [NH3] during the 7-minute workbout. For Δ50%, there was a significant increase in the mean normalized plasma [NH3] during the 7-minute workbout. These findings suggest a potential relationship between exercise-induced hyperammonemia and the &OV0312;o2SC during heavy-intensity exercise.