Patrick G. Arndt

Role of p38 Mitogen-Activated Protein Kinase in a Murine Model of Pulmonary Inflammation

Early inflammatory events include cytokine release, activation, and rapid accumulation of neutrophils, with subsequent recruitment of mononuclear cells. The p38 mitogen-activated protein kinase (MAPK) intracellular signaling pathway plays a central role in regulating a wide range of inflammatory responses in many different cells. A murine model of mild LPS-induced lung inflammation was developed to investigate the role of the p38 MAPK pathway in the initiation of pulmonary inflammation. A novel p38 MAPK inhibitor, M39, was used to determine the functional consequences of p38 MAPK activation. In vitro exposure to M39 inhibited p38 MAPK activity in LPS-stimulated murine and human neutrophils and macrophages, blocked TNF-α and macrophage inflammatory protein-2 (MIP-2) release, and eliminated migration of murine neutrophils toward the chemokines MIP-2 and KC. In contrast, alveolar macrophages required a 1000-fold greater concentration of M39 to block release of TNF-α and MIP-2. Systemic inhibition of p38 MAPK resulted in significant decreases in the release of TNF-α and neutrophil accumulation in the airspaces following intratracheal administration of LPS. Recovery of MIP-2 and KC from the airspaces was not affected by inhibition of p38 MAPK, and accumulation of mononuclear cells was not significantly reduced. When KC was instilled as a proinflammatory stimulus, neutrophil accumulation was significantly decreased by p38 MAPK inhibition independent of TNF-α or LPS. Together, these results demonstrate a much greater dependence on the p38 MAPK cascade in the neutrophil when compared with other leukocytes, and suggest a means of selectively studying and potentially modulating early inflammation in the lung.

Selective Suppression of Neutrophil Accumulation in Ongoing Pulmonary Inflammation by Systemic Inhibition of p38 Mitogen-Activated Protein Kinase

The p38 mitogen-activated protein kinase (MAPK) signaling pathway regulates a wide range of inflammatory responses in many different cells. Inhibition of p38 MAPK before exposing a cell to stress stimuli has profound anti-inflammatory effects, but little is known about the effects of p38 MAPK inhibition on ongoing inflammatory responses. LPS-induced activation of p38 MAPK in human neutrophils was inhibited by poststimulation exposure to a p38 MAPK inhibitor (M39). Release of TNF-α, macrophage-inflammatory protein (MIP)-2 (MIP-1β), and IL-8 by LPS-stimulated neutrophils was also reduced by poststimulation p38 MAPK inhibition. In contrast, release of monocyte chemoattractant protein-1 was found to be p38 MAPK independent. Ongoing chemotaxis toward IL-8 was eliminated by p38 MAPK inhibition, although the rate of nondirectional movement was not reduced. A murine model of acute LPS-induced lung inflammation was used to study the effect of p38 MAPK inhibition in ongoing pulmonary inflammation. Initial pulmonary cell responses occur within 4 h of stimulation in this model, so M39 was administered 4 h or 12 h after exposure of the animals to aerosolized LPS to avoid inhibition of cytokine release. Quantities of TNF-α, MIP-2, KC, or monocyte chemoattractant protein-1 recovered from bronchial alveolar lavage or serum were not changed. Recruitment of neutrophils, but not other leukocytes, to the airspaces was significantly reduced. Together, these data demonstrate the selective reduction of LPS-induced neutrophil recruitment to the airspaces, independent of suppression of other inflammatory responses. These findings support the feasibility of p38 MAPK inhibition as a selective intervention to reduce neutrophilic inflammation.

Toll/IL-1R Domain-Containing Adaptor Protein (TIRAP) Is a Critical Mediator of Antibacterial Defense in the Lung against Klebsiella pneumoniae but Not Pseudomonas aeruginosa

Bacterial pneumonia is a leading cause of mortality and is associated with extensive neutrophil accumulation. Major pathogens associated with this disease include nonflagellated Klebsiella pneumoniae (Kp) and flagellated Pseudomonas aeruginosa (Pa). TLRs are essential for innate immune defense. TIRAP (Toll/IL-1R domain-containing adaptor protein) is an adaptor in TLR1, TLR2, TLR4, and TLR6 signaling, whereas MyD88 is an adaptor for all TLRs. However, the importance of TIRAP in pulmonary defense against Kp or Pa has not been examined. To demonstrate the role of TIRAP, TIRAP-deficient and wild-type littermates were intratracheally inoculated with Kp or Pa. We found that TIRAP−/− mice had substantial mortality, higher bacterial burden in the lungs, and enhanced dissemination following Kp challenge. Furthermore, Kp-induced neutrophil sequestration, histopathology, and MIP-2, TNF-α, IL-6, and LIX (lipopolysaccharide-induced CXC chemokine) production were attenuated in the lungs of TIRAP−/− mice. In contrast, TIRAP is not required for Pa-induced mortality, pulmonary bacterial burden, bacterial dissemination, neutrophil accumulation, or histopathology, yet it is necessary for MIP-2, TNF-α, and IL-6 production, but not LIX production. However, both Kp- and Pa-induced neutrophil influxes are MyD88 dependent. To determine the mechanisms associated with Pa-induced neutrophil accumulation, we inoculated mice with a flagellin C mutant of Pa (PaΔfliC) or purified flagellin, a TLR5 agonist. PaΔfliC-induced neutrophil sequestration and LIX expression are dependent on TIRAP, whereas flagellin-induced neutrophil influx and LIX expression are independent of TIRAP. These novel findings illustrate a pathogen-specific role for TIRAP in pulmonary defense and suggest that TLR5 plays an essential role for Pa-induced neutrophil influx via LIX production.

Regulation of Lipopolysaccharide-Induced Lung Inflammation by Plasminogen Activator Inhibitor-1 through a JNK-Mediated Pathway

The neutrophil is of undoubted importance in lung inflammation after exposure to LPS. We have shown recently that systemic inhibition of JNK decreased neutrophil recruitment to the lung after exposure to LPS, although the mechanisms underlying this inhibition are incompletely understood. As plasminogen activator inhibitor-1 (PAI-1) accentuates cell migration, with JNK activation recently shown to up-regulate PAI-1 expression, this suggested that systemic JNK inhibition may down-regulate LPS-induced pulmonary neutrophil recruitment through a decrease in PAI-1 expression. We show in this study that exposure of mice to aerosolized LPS increased PAI-1 expression in the lung and alveolar compartment, which was decreased by pretreatment with the JNK inhibitor SP600125. Exogenous, intratracheally administered PAI-1 prevented the inhibition of pulmonary neutrophil recruitment in the setting of systemic JNK inhibition, thereby suggesting a role for PAI-1 in the JNK-mediated pathway regulating LPS-induced neutrophil recruitment. In addition, PAI-1−/− mice had a decrease in neutrophil recruitment to the alveolar compartment after exposure to LPS, compared with wild-type controls, further suggesting a role for PAI-1 in LPS-induced lung inflammation. An increase in the intravascular level of KC is a likely mechanism for the inhibition of pulmonary neutrophil recruitment after LPS exposure in the setting of decreased PAI-1 expression, as systemic KC levels after exposure to LPS were increased in PAI-1-deficient mice and in mice pretreated with SP600125, with augmentation of intravascular KC levels inhibiting neutrophil recruitment to the lung after exposure to LPS.

Mycoplasma pneumoniae-Associated Bronchiolitis Causing Severe Restrictive Lung Disease in Adults: Report of Three Cases and Literature Review

Study objectives To characterize adult Mycoplasma pneumoniae-induced bronchiolitis requiring hospitalization. Design We encountered an adult patient with severe bronchiolitis in the absence of pneumonia due to M pneumoniae. To determine the relative frequency of such a condition, we retrospectively reviewed the medical records of adults over a 4-year period with a hospital discharge diagnosis of “bronchiolitis” from a university hospital. Setting University Hospital of the University of Colorado Health Sciences Center, Denver, CO. Study subjects From 1994 to 1998, 10 adult inpatients were identified with a diagnosis of bronchiolitis. There were two with respiratory bronchiolitis, one with panbronchiolitis, one patient with bronchiolitis obliterans organizing pneumonia (BOOP), and six with acute inflammatory bronchiolitis. Including the initial patient, three had a definitive clinical diagnosis of Mycoplasma-associated bronchiolitis. Results The three adult patients with bronchiolitis due to M pneumoniae are unusual because they occurred in the absence of radiographic features of a lobar or patchy alveolar pneumonia. Hospital admission was occasioned by the severity of symptoms and gas exchange abnormalities. One patient had bronchiolitis as well as organizing pneumonia (BOOP) that responded favorably to corticosteroid treatment. The other two had high-resolution CT findings diagnostic of an acute inflammatory bronchiolitis. One of the patients with inflammatory bronchiolitis had an unusual pattern of marked ventilation and perfusion defects localized predominantly to the left lung. All three had restrictive ventilatory impairment on physiologic testing. Conclusions In adults, Mycoplasma-associated bronchiolitis without pneumonia is rarely reported, but in hospitalized patients, it may be more common than expected and may be associated with severe physiologic disturbances.

Leukocyte ADAM17 regulates acute pulmonary inflammation.

The transmembrane protease ADAM17 regulates the release and density of various leukocyte cell surface proteins that modulate inflammation, including L-selectin, TNF-α, and IL-6R. At this time, its in vivo substrates and role in pulmonary inflammation have not been directly examined. Using conditional ADAM17 knock-out mice, we investigated leukocyte ADAM17 in acute lung inflammation. Alveolar TNF-α levels were significantly reduced (>95%) in ADAM17-null mice following LPS administration, as was the shedding of L-selectin, a neutrophil-expressed adhesion molecule. Alveolar IL-6R levels, however, were reduced by only ≈25% in ADAM17-null mice, indicating that ADAM17 is not its primary sheddase in our model. Neutrophil infiltration into the alveolar compartment is a key event in the pathophysiology of acute airway inflammation. Following LPS inhalation, alveolar neutrophil levels and lung inflammation in ADAM17-null mice were overall reduced when compared to control mice. Interestingly, however, neutrophil recruitment to the alveolar compartment occurred earlier in ADAM17-null mice after exposure to LPS. This decrease in alveolar neutrophil recruitment in ADAM17-null mice was accompanied by significantly diminished alveolar levels of the neutrophil-tropic chemokines CXCL1 and CXCL5. Altogether, our study suggests that leukocyte ADAM17 promotes inflammation in the lung, and thus this sheddase may be a potential target in the design of pharmacologic therapies for acute lung injury.

Systemic Inhibition of the Angiotensin-Converting Enzyme Limits Lipopolysaccharide-Induced Lung Neutrophil Recruitment through Both Bradykinin and Angiotensin II-Regulated Pathways

Recruitment of neutrophils to the lung is a sentinel event in acute lung inflammation. Identifying mechanisms that regulate neutrophil recruitment to the lung may result in strategies to limit lung damage and improve clinical outcomes. Recently, the renin angiotensin system (RAS) has been shown to regulate neutrophil influx in acute inflammatory models of cardiac, neurologic, and gastrointestinal disease. As a role for the RAS in LPS-induced acute lung inflammation has not been described, we undertook this study to examine the possibility that the RAS regulates neutrophil recruitment to the lung after LPS exposure. Pretreatment of mice with the angiotensin-converting enzyme (ACE) inhibitor enalapril, but not the anti-hypertensive hydralazine, decreased pulmonary neutrophil recruitment after exposure to LPS. We hypothesize that inhibition of LPS-induced neutrophil accumulation to the lung with enalapril occurred through both an increase in bradykinin, and a decrease in angiotensin II (ATII), mediated signaling. Bradykinin receptor blockade reversed the inhibitory effect of enalapril on neutrophil recruitment. Similarly, pretreatment with bradykinin receptor agonists inhibited IL-8-induced neutrophil chemotaxis and LPS-induced neutrophil recruitment to the lung. Inhibition of ATII-mediated signaling, with the ATII receptor 1a inhibitor losartan, decreased LPS-induced pulmonary neutrophil recruitment, and this was suggested to occur through decreased PAI-1 levels. LPS-induced PAI-1 levels were diminished in animals pretreated with losartan and in those deficient for the ATII receptor 1a. Taken together, these results suggest that ACE regulates LPS-induced pulmonary neutrophil recruitment via modulation of both bradykinin- and ATII-mediated pathways, each regulating neutrophil recruitment by separate, but distinct, mechanisms.

Tec kinases regulate actin assembly and cytokine expression in LPS-stimulated human neutrophils via JNK activation

The acute inflammatory response involves neutrophils wherein recognition of bacterial products, such as lipopolysaccharide (LPS), activates intracellular signaling pathways. We have shown that the mitogen-activated protein kinase (MAPK) c-Jun NH(2) terminal kinase (JNK) is activated by LPS in neutrophils and plays a critical role in monocyte chemoattractant protein (MCP)-1 expression and actin assembly. As the Tec family kinases are expressed in neutrophils and regulate activation of the MAPKs in other cell systems, we hypothesized that the Tec kinases are an upstream component of the signaling pathway leading to LPS-induced MAPKs activation in neutrophils. Herein, we show that the Tec kinases are activated in LPS-stimulated human neutrophils and that inhibition of the Tec kinases, with leflunomide metabolite analog (LFM-A13), decreased LPS-induced JNK, but not p38, activity. Furthermore, LPS-induced actin polymerization as well as MCP-1, tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta expression are dependent on Tec kinase activity.

c-Jun NH2-TERMINAL KINASE REGULATES LIPOPOLYSACCHARIDE-INDUCED PULMONARY MONONUCLEAR CELL RECRUITMENT VIA CCL2

Subsequent to the initial recruitment of neutrophils, monocytes are recruited to the lung after an injurious insult. Previously the authors have shown that inhibition of either p38 or c-Jun NH2-terminal kinase (JNK) decreased pulmonary neutrophil recruitment in mice exposed to lipopolysaccharide (LPS). As the signaling pathways regulating the influx of mononuclear cells to the lung are poorly understood, the authors undertook the present study to examine the roles of p38 and JNK. In a model of LPS-induced lung inflammation, systemic inhibition of JNK, but not p38, decreased the recruitment of mononuclear cells to the lung. Levels of CCL2 (monocyte chemoattractant protein 1 [MCP-1]) were decreased in the setting of JNK inhibition, with LPS-induced pulmonary mononuclear cell recruitment in CCL2-deficient mice similar to that found with JNK inhibition. The decrease in LPS-induced CCL2 levels in the lung seen with JNK inhibition, however, was independent of neutrophil recruitment, as systemic depletion of neutrophils had no effect on pulmonary CCL2 levels after LPS exposure. In sum, these results suggest that JNK, but not p38, regulates LPS-induced mononuclear cell recruitment to the lung, that this occurs through a CCL2-dependent pathway, and that LPS-induced pulmonary CCL2 expression is dependent on JNK but independent of pulmonary neutrophil recruitment.

Spontaneous complete resolution of pneumomediastinum and pneumatosis intestinalis caused by acute GVHD

A 31-year-old male with relapsed Hodgkin lymphoma (nodular sclerosing) after autologous haemopoietic stem cell transplantation (HCT) received allogeneic (allo) HCT from an human leukocyte antigen (HLA) identical sibling donor for consolidation in second complete remission. He presented with acute diarrhea at day 134, and was diagnosed with gut grade II acute graft-versus-host disease (aGVHD) proven by histopathological examination of a rectal biopsy. Steroids initially controlled the diarrhea, however, gut aGVHD flared at day 1100 while steroids were Image 1. CT images show air tracking in the anterior mediastinum, along the bowel wall and sub diaphragmatic region. Follow up CT images demonstrate complete resolution of the findings.