Early Accumulation Of Neutrophils Research Articles

Inhaled Nitric Oxide Therapy in the Preterm Infant Who Has Respiratory Distress Syndrome

After completing this article, readers should be able to: 1. Delineate the rationale for inhaled nitric oxide (iNO) use in respiratory distress syndrome. 2. List the toxicities of iNO. 3. Describe the primary findings of iNO therapy in major clinical trials. Respiratory distress syndrome (RDS) has been a major factor determining the limits of viability in the preterm infant. RDS is, in large part, the result of surfactant deficiency. Surfactant replacement and antenatal steroid use are the two major advances in the treatment of RDS to date and have resulted in significant decreases in mortality and chronic lung disease (CLD). Surfactant replacement became widely available in 1991, and antenatal steroids achieved widespread use following the National Institutes of Health Consensus Conference in 1994. (1) However, substantial mortality and CLD still are seen in the very low-birthweight infant (Fig. 1). Figure 1 National Institute of Child and Human Development (NICHD) Neonatal Research Network mortality and CLD rates for 2003 in preterm infants born at 23 to 30 weeks’ gestation. Following the identification of nitric oxide (NO) as the vascular endothelial relaxing factor, use of the agent has moved rapidly from bench to bedside. Inhaled nitric oxide (iNO) was found to improve oxygenation in adult and pediatric patients who had hypoxic respiratory failure. (2)(3)(4) Subsequent randomized, controlled trials in term and near-term infants who had hypoxic respiratory failure demonstrated an improvement in oxygenation as well as a significant decrease in death or the need for extracorporeal membrane oxygenation. (5)(6)(7) Following United States Food and Drug Administration review of these trials, iNO was approved for use in term and near-term infants who had hypoxic respiratory failure. The role of iNO in the preterm infant who has RDS has been an active area of investigation over the last decade. This …

Macrophage elastase (MMP-12): a pro-inflammatory mediator?

As many metalloproteinases (MMPs), macrophage elastase (MMP-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes. Studies using animal models of acute and chronic pulmonary inflammatory diseases, such as pulmonary fibrosis and chronic obstructive pulmonary disease (COPD), have given evidences that MMP-12 is an important mediator of the pathogenesis of these diseases. However, as very few data regarding the direct involvement of MMP-12 in inflammatory process in the airways were available, we have instilled a recombinant form of human MMP-12 (rhMMP-12) in mouse airways. Hence, we have demonstrated that this instillation induced a severe inflammatory cell recruitment characterized by an early accumulation of neutrophils correlated with an increase in proinflammatory cytokines and in gelatinases and then by a relatively stable recruitment of macrophages in the lungs over a period of ten days. Another recent study suggests that resident alveolar macrophages and recruited neutrophils are not involved in the delayed macrophage recruitment. However, epithelial cells could be one of the main targets of rhMMP-12 in our model. We have also reported that a corticoid, dexamethasone, phosphodiesterase 4 inhibitor, rolipram and a non-selective MMP inhibitor, marimastat could reverse some of these inflammatory events. These data indicate that our rhMMP-12 model could mimic some of the inflammatory features observed in COPD patients and could be used for the pharmacological evaluation of new anti-inflammatory treatment. In this review, data demonstrating the involvement of MMP-12 in the pathogenesis of pulmonary fibrosis and COPD as well as our data showing a pro-inflammatory role for MMP-12 in mouse airways will be summarized.

IP-10 Mediates Selective Mononuclear Cell Accumulation and Activation in Response to Intrapulmonary Transgenic Expression and During Adenovirus-Induced Pulmonary Inflammation

CXC chemokines that lack the glutamine-leucine-arginine (ELR) motif, including interferon (IFN)-inducible protein 10 (IP-10 or CXCL10), have been shown to mediate the generation of type 1 immune responses. In this study, we found that the intrapulmonary transient transgenic expression of murine IP-10 in mice using adenoviral gene transfer resulted in the early accumulation of neutrophils, natural killer (NK) cells, and NK T cells within the lung, followed by the delayed accumulation of CD4+ T cells. Adenovirus-mediated transgenic expression of IP-10 also resulted in selective activation of mononuclear cells, including gamma(delta)-T cells and NK cells, as manifest by CD69 expression or induction of cell-associated IFN-gamma. Importantly, the intratracheal (i.t.) administration of a control human type 5 adenovirus also caused significant accumulation of NK, NK T, and CD4+ T cells, which was maximal at 7 days post vector administration and was associated with the induction of IP-10. Neutralization of endogenous IP-10 in animals receiving control adenovirus resulted in decreases in the numbers of NK, CD4+, and CD8+ T cells. These results indicate that IP-10 can direct the accumulation and activation of neutrophils and selected mononuclear cells to the lung and that adenovirus-induced IP-10 contributes to lung inflammatory cell recruitment/activation observed in response to adenoviral vectors used for gene therapy.

Migration Defects of Leukocytes from Alpha4 or Beta2 Integrin-Deficient Mice during Aseptic Peritonitis.

Leukocyte migration to the inflammatory sites involves a dynamic and coordinated sequence of adhesion/ migration events, in which the α4 and β2 integrins in leukocytes play critical roles. In a model of aseptic peritonitis, normal mice respond with an initial influx of neutrophils followed by later recruitment of mononuclear cells. Previous studies used antibodies to inhibit α4 integrins in this model and provided only early observations (up to 24 hrs). Events at later stages were not addressed. To clarify the role of α4 integrins in this process, we induced aseptic peritonitis in α4-ablated (α4−/ −) mice and compared the results to similarly treated wild type (WT), and β2 integrin-deficient (β2−/ −) animals. A 4, 16 and 96 hrs post thioglycollate injection, leukocytes, harvested from the peritoneal cavity, were counted and evaluated by FACS and by differentials in smears. 3-5 animals per time point per genotype were analyzed. Early neutrophil accumulation in the peritoneum was similar in WT and α4−/ − mice (Table 1). β2−/ − mice were slow to accumulate neutrophils, but at 16 hrs the peaks were not different from α4−/ − or WT animals. However at later times in both α4−/ − and β2−/ − mice neutrophils persisted longer, suggesting either increased survival or prolonged recruitment. At 16 hrs large numbers of monocyte/ macrophages accumulated in the peritoneum and they were similar in all groups of mice up to 96 hours. The cumulative number of leukocytes in the peritoneum was compared to the concurrent circulating pool of leukocytes in each mouse group. At the peak time (16 hrs) the fraction recovered in the WT represented 394±148%, while in the α4−/ − mice it was 98±33% and in the β2−/ − mice it was 40±19%. Thus, although sufficient numbers of leukocytes accumulated in the peritoneum of both integrin-deficient mice, migration in the WT mice was significantly higher than that observed in the other two genetic models, suggesting impairment in animals with either integrin deficiency. Furthermore, lymphocyte accumulation was dramatically decreased at all time points in the α4−/ − mice (Table 2). Preliminary data in a model of combined, α4- and β2-integrin deficiency, generated in our laboratory, showed that only 3±2% of circulating leukocytes migrated into the peritoneum. Taken together, our results indicate that (1) for migration into peritoneum, neutrophils and monocyte/ macrophages can use α4 and β2 integrins interchangeably, (2) for optimal migration both integrins are needed, and (3) lymphocytes have an absolute requirement of α4 integrins for migration. These data extend previous knowledge on kinetic changes of leukocytes accumulating in the aseptic peritonitis model. Whether different kinetic changes are seen in other inflammatory models using our integrin-deficient animals requires further studies.Accumulation of myeloid cells in peritoneumNeutrophilsMonocyte/macrophages4 hrs16 hrs96 hrs4 hrs16 hrs96 hrsWT5.5±0.59.8±7.40.2±0.043.1±0.617.7±3.911.0±5.2β2−/ −2.5±0.810.5±5.33.7±1.64.7±1.312.8±5.211.0±4.0α4−/ −5.2±2.19.8±7.40.8±0.44.6±1.111.0±5.212.6±5.1Lymphocyte accumulation in peritoneum4 hrs16 hrs96 hrsWT2.7±1.22.5±0.33.1±0.5β2−/ −6.7±1.74.7±1.23.3±1.0α4−/ −0.5±0.10.4±0.10.6±0.1Cell number (x106) in peritoneum at various time points; in bold are the numbers that significantly differ from the WT for each time point (p<0.05)

Role of endothelin in the pathophysiology of renal ischemia-reperfusion in normal rabbits

The present study addressed the acute effects of endothelin-1 on renal function and neutrophils accumulation in the setting of in vivo severe (60 min) acute ischemia/reperfusion. Ischemia/reperfusion decreased renal functional parameters and increased renal neutrophil accumulation and medullary congestion. All these parameters markedly improved with the intrarenal administration of anti-endothelin-1 antiserum. Comparatively, the intrarenal infusion of endothelin-1 decreased renal function and increased neutrophil accumulation. Abnormalities in renal histology were, however, less pronounced than with ischemia/ reperfusion. In experiments using rabbit isolated perfused kidneys, endothelin-1 induced the accumulation of labeled neutrophils. This accumulation was similar to that observed in kidneys obtained after 60 minutes of ischemia plus 60 minutes of reperfusion. Both endothelin and ischemia/ reperfusion effects were counteracted by an anti-endothelin antibody. In further in vitro studies, we found that endothelin-1-induced the expression of the CD18 antigens on the neutrophil surface. In subsequent experiments based on this effect of ET-1 on CD18 antigens, a blockade of both ischemia/reperfusion-induced and endothelin-1-induced neutrophil accumulation was obtained by infusion an anti-CD18 antibody. In conclusion, our experiments disclosed the critical role of endothelin-1 as a major promoter of early neutrophil accumulation after ischemia/reperfusion, which occurred through an integrin-mediated mechanism.

Neutrophil elastase inhibitor (ONO-5046) decreases cytokine-induced neutrophil chemoattractant after reperfusion of pancreaticoduodenal transplantation in rats.

The protective effects of a neutrophil elastase inhibitor (ONO-5046) on reperfusion injury following pancreaticoduodenal transplantation in rats were studied by measuring serum concentrations of cytokine-induced neutrophil chemoattractant (CINC). Male Wistar rats were transplanted with syngeneic pancreaticoduodenal grafts. ONO-5046 was injected intravenously 5 min before vascular clamping and immediately after reperfusion at a dose of 10 mg/kg. No significant differences were observed in the peak serum concentrations of amylase between the groups treated with and treated without ONO-5046. The serum lipase concentrations in the untreated animals increased and peaked 3 hr after reperfusion. ONO-5046 significantly decreased the peak serum lipase concentration. The serum CINC concentrations, which were determined by enzyme-linked immunosorbent assay, increased and peaked 3 hr after reperfusion, decreasing gradually thereafter. However, pretreatment with ONO-5046 significantly inhibited the rise in serum CINC concentrations after reperfusion. Expression of CICN transcripts in the pancrease grafts was evaluated by Northern blot analysis and peaked 3 hr after reperfusion in untreated animals. Pretreatment with ONO-5046 also significantly inhibited the expression of CINC mRNA transcripts in the graft. ONO-5046 significantly decreased the number of neutrophils accumulated in the pancreas graft 24 hr after transplantation. In vitro CINC production by peritoneal macrophages was increased by neutrophil elastase in dose-dependent fashion. However, ONO-5046 decreased CINC production by peritoneal macrophages in response to neutrophil elastase. These results suggest that ONO-5046 prevents early neutrophil accumulation in the pancreas following ischemia/reperfusion of pancreaticoduodenal transplantation.

Flow cytometry analysis of human neutrophils labeled with rhodamine phalloidin: Effect of pentoxifylline

Pentoxifylline (PTX) has been reported to enhance the early accumulation of neutrophils at the site of Staphylococcus aureus subcutaneous infection in mice (1) and to stimulate in vitro PMN chemotaxis, particularly under dense agarose (2). Among the biochemical events contributing to chemotaxis are actin polymerization (3). The membrane cytoskeleton is believed to control the lateral mobility of integral membrane proteins as well as influencing cell shape and mobility. Thus, pharmacological modulations of neutrophil chemotaxis may be related to an effect of the pharmacological agents on the membrane cytoskeleton. The present study was designed to characterize the effect of PTX on actin polymerization of freely-suspended PMN before and after stimulation by the chemotactic factor f-MLP. We used flow cytometry to determine the proportion of actin in the filamentous form, and Rhodamine-Phalloidin as fluorescent probe (4). PTX decreased actin polymerization in response to stimulation by f-MLP. The reduction in F-actin by PTX was higher in the samples with higher activation ratios as compared with untreated PMN.