Biomedical subjects

Michael T Borchers

Publications and source records attributed to Michael T Borchers.

13 recordsLinked to original sources

PubMed2006

Lung-restricted macrophage activation in the pearl mouse model of Hermansky-Pudlak syndrome.

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring "pearl" HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1gamma) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-alpha, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-alpha, MIP1alpha, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-alpha responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-gamma/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-alpha at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-alpha secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.

Administration, Intranasal↗

PubMed2006

NKG2D ligands are expressed on stressed human airway epithelial cells.

Immune surveillance of the airways is critical to maintain the integrity and health of the lung. We have identified a family of ligands expressed on the surface of stressed airway epithelial cells whose function is to bind the NKG2D-activating receptor found on several pulmonary lymphocytes, including natural killer cells, gammadelta(+) T cells, and CD8(+) T cells. We employed real-time PCR and flow cytometry in normal and transformed airway epithelial cell to demonstrate that major histocompatibility complex class I chain-related (MIC) B and the UL-16 binding protein (ULBP) ligands (ULBP1-4) are ubiquitously expressed at the mRNA level in all cell lines. MICA/B surface expression was present on 70% of transformed cell lines but was undetectable on primary cells. We demonstrate that MICA/B and ULBP 1, 2, 3, and 4 expression is rare or absent on the cell surface of unstimulated normal human bronchial epithelial cells although transcripts and intracellular proteins are present. Normal human bronchial epithelial cells exposed to 0.3 mM hydrogen peroxide exhibit an induction of all ligands examined on the cell surface. Surface expression is independent of changes in transcript level or total cellular protein and is mediated by the ERK family of mitogen-activated protein kinases. The induction of NKG2D ligands on stressed airway epithelial cells represents a potentially important mechanism of immune cell activation in regulation of pulmonary health and disease.

Animals↗

PubMed2006

The NKG2D-activating receptor mediates pulmonary clearance of Pseudomonas aeruginosa.

The NKG2D-activating receptor is expressed on cytotoxic lymphocytes and interacts with ligands expressed on the surface of cells stressed by pathogenic and nonpathogenic stimuli. In this study, we investigated the physiologic importance of NKG2D receptor-ligand interactions in response to acute pulmonary Pseudomonas aeruginosa infection. P. aeruginosa infection increased the expression of mouse NKG2D ligands (Rae1) in airway epithelial cells and alveolar macrophages in vivo and also increased the cell surface expression of human NKG2D ligands (ULBP2) on airway epithelial cells in vitro. NKG2D receptor blockade with a specific monoclonal antibody inhibited the pulmonary clearance of P. aeruginosa. NKG2D receptor blockade also resulted in decreased production of Th1 cytokines and nitric oxide in the lungs of P. aeruginosa-infected mice. Additionally, NKG2D receptor blockade reduced the epithelial cell sloughing that accompanies P. aeruginosa infection. Macrophage phagocytosis and bronchoalveolar lavage cellularity were not different in P. aeruginosa-infected mice with and without NKG2D receptor blockade. These results demonstrate the importance of NKG2D-mediated immune activation in the clearance of acute bacterial infection and suggest that epithelial cell-lymphocyte interactions mediate pulmonary cytokine production, epithelial cell integrity, and bacterial clearance.

Animals↗

PubMed2005

The role of metallothionein in the pathogenesis of acute lung injury.

Often fatal, acute lung injury has a complicated etiology. Previous studies from our laboratory in mice have demonstrated that survival during acute lung injury is a complex trait governed by multiple loci. We also found that the increase in metallothionein (MT) is one of the greatest noted in transcriptome-wide analyses of gene expression. To assess the role of MT in nickel-induced acute lung injury, the survival of Mt-transgenic, Mt1/2(+/+), and Mt1/2(-/-) mice was compared. Pulmonary inflammation and global gene expression were compared in Mt1/2(+/+) and Mt1/2(-/-) mice. Gene-targeted Mt1/2(-/-) mice were more susceptible than Mt1/2(+/+) mice to nickel-induced inflammation, surfactant-associated protein B transcript loss, and lethality. Similarly, Mt-transgenic mice exhibited increased survival. MAPPFinder analyses also noted significant decreases in genes involved in protein processing (e.g., ubiquitination, folding), which were greater in Mt1/2(-/-) mice as compared with Mt1/2(+/+) mice early in the progression of acute lung injury, possibly due to a zinc-mediated transcript destabilization. In contrast, transcript levels of genes associated with the inflammatory response, extracellular matrix regulation, and coagulation/fibrinolysis were increased more in Mt1/2(-/-) mice as compared with Mt1/2(+/+) mice late in the development of acute lung injury. Thus, MT ultimately improves survival in the progression of acute lung injury in mice. Transcriptome-wide analysis suggests that this survival may be mediated through changes in the destabilization of transcripts associated with protein processing, the subsequent augmentation of transcripts controlling inflammation, extracellular matrix regulation, coagulation/fibrinolysis, and disruption of surfactant homeostasis.

Animals↗

PubMed2005

Gene expression changes during the development of acute lung injury: role of transforming growth factor beta.

RATIONALE: Acute lung injury can occur from multiple causes, resulting in high mortality. The pathophysiology of nickel-induced acute lung injury in mice is remarkably complex, and the molecular mechanisms are uncertain. OBJECTIVES: To integrate molecular pathways and investigate the role of transforming growth factor beta (TGF-beta) in acute lung injury in mice. METHODS: cDNA microarray analyses were used to identify lung gene expression changes after nickel exposure. MAPPFinder analysis of the microarray data was used to determine significantly altered molecular pathways. TGF-beta1 protein in bronchoalveolar lavage fluid, as well as the effect of inhibition of TGF-beta, was assessed in nickel-exposed mice. The effect of TGF-beta on surfactant-associated protein B (Sftpb) promoter activity was measured in mouse lung epithelial cells. MEASUREMENTS AND MAIN RESULTS: Genes that decreased the most after nickel exposure play important roles in lung fluid absorption or surfactant and phospholipid synthesis, and genes that increased the most were involved in TGF-beta signaling. MAPPFinder analysis further established TGF-beta signaling to be significantly altered. TGF-beta-inducible genes involved in the regulation of extracellular matrix function and fibrinolysis were significantly increased after nickel exposure, and TGF-beta1 protein was also increased in the lavage fluid. Pharmacologic inhibition of TGF-beta attenuated nickel-induced protein in bronchoalveolar lavage. In addition, treatment with TGF-beta1 dose-dependently repressed Sftpb promoter activity in vitro, and a novel TGF-beta-responsive region in the Sftpb promoter was identified. CONCLUSIONS: These data suggest that TGF-beta acts as a central mediator of acute lung injury through the alteration of several different molecular pathways.

Animals↗

PubMed2004

Metalloproteinases mediate mucin 5AC expression by epidermal growth factor receptor activation.

Chronic obstructive pulmonary disease is marked by alveolar enlargement and excess production of airway mucus. Acrolein, a component of cigarette smoke, increases mucin 5AC (MUC5AC), a prevalent airway mucin in NCI-H292 cells by transcriptional activation, but the signal transduction pathways involved in acrolein-induced MUC5AC expression are unknown. Acrolein depleted cellular glutathione at doses of 10 muM or greater, higher than those sufficient (0.03 muM) to increase MUC5AC mRNA, suggesting that MUC5AC expression was independent of oxidative stress. In contrast, acrolein increased MUC5AC mRNA levels by phosphorylating epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase 3/2, or MAPK 3/2(ERK1/2). Pretreating the cells with an EGFR-neutralizing antibody, or a metalloproteinase inhibitor, decreased the acrolein-induced MUC5AC mRNA increase. Small, interfering RNA directed against ADAM17 or MMP9 inhibited the acrolein-induced MUC5AC mRNA increase. Acrolein increased the release and subsequent activation of pro-MMP9. Acrolein increased MMP9 and decreased tissue inhibitor of metalloproteinase 3 (TIMP3), an endogenous inhibitor of ADAM17, transcripts. Together, these data suggest that acrolein induces MUC5AC expression via an initial ligand-dependent activation of EGFR mediated by ADAM17 and MMP9. In addition, a prolonged effect of acrolein may be mediated by altering MMP9 and TIMP3 transcription.

Acrolein↗

PubMed2003

A causative relationship exists between eosinophils and the development of allergic pulmonary pathologies in the mouse.

Asthma and mouse models of allergic respiratory inflammation are invariably associated with a pulmonary eosinophilia; however, this association has remained correlative. In this report, a causative relationship between eosinophils and allergen-provoked pathologies was established using eosinophil adoptive transfer. Eosinophils were transferred directly into the lungs of either naive or OVA-treated IL-5(-/-) mice. This strategy resulted in a pulmonary eosinophilia equivalent to that observed in OVA-treated wild-type animals. A concomitant consequence of this eosinophil transfer was an increase in Th2 bronchoalveolar lavage cytokine levels and the restoration of intracellular epithelial mucus in OVA-treated IL-5(-/-) mice equivalent to OVA-treated wild-type levels. Moreover, the transfer also resulted in the development of airway hyperresponsiveness. These pulmonary changes did not occur when eosinophils were transferred into naive IL-5(-/-) mice, eliminating nonspecific consequences of the eosinophil transfer as a possible explanation. Significantly, administration of OVA-treated IL-5(-/-) mice with GK1.5 (anti-CD4) Abs abolished the increases in mucus accumulation and airway hyperresponsiveness following adoptive transfer of eosinophils. Thus, CD4(+) T cell-mediated inflammatory signals as well as signals derived from eosinophils are each necessary, yet alone insufficient, for the development of allergic pulmonary pathology. These data support an expanded view of T cell and eosinophil activities and suggest that eosinophil effector functions impinge directly on lung function.

Adoptive Transfer↗

PubMed2003

Methacholine-induced airway hyperresponsiveness is dependent on Galphaq signaling.

Airway function in health and disease as well as in response to bronchospastic stimuli (i.e., irritants, allergens, and inflammatory mediators) is controlled, in part, by cholinergic muscarinic receptor regulation of smooth muscle. In particular, the dependence of airway smooth muscle contraction/relaxation on heterotrimeric G protein-coupled receptor signaling suggests that these events underlie the responses regulating airway function. Galphaq-containing G proteins are proposed to be a prominent signaling pathway, and the availability of knockout mice deficient of this subunit has allowed for an investigation of its potential role in airway function. Airway responses in Galphaq-deficient mice (activities assessed by both tracheal tension and in vivo lung function measurements) were attenuated relative to wild-type controls. Moreover, ovalbumin sensitization/aerosol challenge of Galphaq-deficient mice also failed to elicit an allergen-induced increase in airway reactivity to methacholine. These findings indicate that cholinergic receptor-mediated responses are dependent on Galphaq-mediated signaling events and identify Galphaq as a potential target of preventative/intervening therapies for lung dysfunction.

Airway Resistance↗

PubMed2002

Ablation of eosinophils leads to a reduction of allergen-induced pulmonary pathology.

A strategy to deplete eosinophils from the lungs of ovalbumin (OVA)-sensitized/challenged mice was developed using antibody-mediated depletion. Concurrent administration [viz. the peritoneal cavity (systemic) and as an aerosol to the lung (local)] of a rat anti-mouse CCR3 monoclonal antibody resulted in the abolition of eosinophils from the lung such that the airway lumen was essentially devoid of eosinophils. Moreover, perivascular/peribronchial eosinophil numbers were reduced to levels indistinguishable from saline-challenged animals. This antibody-mediated depletion was not accompanied by effects on any other leukocyte population, including, but not limited to, T cells and mast cells/basophils. In addition, no effects were observed on other underlying allergic inflammatory responses in OVA-treated mice, including OVA-specific immunoglobulin production as well as T cell-dependent elaboration of Th2 cytokines. The ablation of virtually all pulmonary eosinophils in OVA-treated mice (i.e., without concurrent effects on T cell activities) resulted in a significant decrease in mucus accumulation and abolished allergen-induced airway hyperresponsiveness. These data demonstrate a direct causative relationship between allergen-mediated pulmonary pathologies and eosinophils.

Allergens↗

PubMed2002

Gq signaling is required for allergen-induced pulmonary eosinophilia.

The complexity and magnitude of interactions leading to the selective infiltration of eosinophils in response to inhaled allergens are formidable obstacles to a larger understanding of the pulmonary pathology associated with allergic asthma. This study uses knockout mice to demonstrate a novel function for the heterotrimeric G protein, G(q), in the regulation of pulmonary eosinophil recruitment. In the absence of G(q) signaling, eosinophils failed to accumulate in the lungs following allergen challenge. These studies demonstrate that the inhibition of eosinophil accumulation in the airways is attributed to the failure of hemopoietically derived cells to elaborate GM-CSF in the airways. The data suggest that activation of a G(q)-coupled receptor(s) on resident leukocytes in the lung elicits expression of GM-CSF, which, in turn, is required for allergen-induced pulmonary eosinophilia, identifying a novel pathway of eosinophil-associated effector functions leading to pulmonary pathology in diseases such as asthma.

Aerosols↗

PubMed2002

A tale of two controversies: defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species.

Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide ((.)NO, nitrogen monoxide)-derived oxidant peroxynitrite (ONOO(-)). However, since the discovery that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) can generate nitrotyrosine via oxidation of nitrite (NO(2)(-)), several questions have arisen. First, the relative contribution of peroxidases to nitrotyrosine formation in vivo is unknown. Further, although evidence suggests that the one-electron oxidation product, nitrogen dioxide ((*)NO(2)), is the primary species formed, neither a direct demonstration that peroxidases form this gas nor studies designed to test for the possible concomitant formation of the two-electron oxidation product, ONOO(-), have been reported. Using multiple distinct models of acute inflammation with EPO- and MPO-knockout mice, we now demonstrate that leukocyte peroxidases participate in nitrotyrosine formation in vivo. In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, no contribution for either MPO or EPO to nitrotyrosine formation could be demonstrated. Head-space gas analysis of helium-swept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate (*)NO(2) formation using H(2)O(2) and NO(2)(-) as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO(2)(-)-dependent hydroxylation of targets under acidic conditions, a chemical reactivity shared with ONOO(-) but not (*)NO(2). Collectively, our results demonstrate that: 1) MPO and EPO contribute to tyrosine nitration in vivo; 2) the major reactive nitrogen species formed by leukocyte peroxidase-catalyzed oxidation of NO(2)(-) is the one-electron oxidation product, (*)NO(2); 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO(2)(-), producing a ONOO(-)-like product. We speculate that the latter reaction generates a labile Fe-ONOO complex, which may be released following protonation under acidic conditions such as might exist at sites of inflammation.

Animals↗

PubMed2002

Mucin apoprotein expression in COPD.

Mucins, which are complex glycoproteins that provide the viscoelastic properties of mucus that are essential for the protection of the airways, are characterized by a variable-number tandem repeats (VNTR) region that may undergo alternate splicing during transcription. Such transcripts may yield multiple proteins via diverse post-translational modifications involving glycosylation (within each VNTR). Fifteen distinct mucin genes have been identified, with several mapping to chromosomal clusters (ie, 7q22 and 11p15.5), possibly having evolved by gene duplication. The deduced protein sequences can be subdivided into both membrane-associated mucins and secreted mucins. Membrane-associated mucins consist of cytoplasmic, transmembrane, and extracellular domains. The membrane-associated mucins MUC1, MUC4, and MUC11 have been localized to the lung. In addition to VNTRs, secreted mucins possess repeated cysteine-rich D-domains (which are important in polymerization). Secreted mucins that are localized to the lung include MUC2 (in cells with and without secretory granules), MUC5AC (in surface and submucosal mucous cells), MUC5B and MUC8 (in submucosal mucous cells), and MUC7 (in submucosal serous cells). Currently, little is known about the regulation of mucins in COPD patients. Recent studies with acrolein and cigarette smoke have suggested that MUC5AC is inducible (accompanied by epidermal growth factor [EGF] ligand formation and the activation of EGF receptor-dependent pathways), whereas MUC5B is constitutively expressed (increasing through gland enlargement). Similarly, little is known about the genetic determinants that control mucus hypersecretion, but preliminary findings in animal models suggest that intrastrain differences in acrolein-induced mucin formation are amenable to genetic analysis. As our understanding of the functional genomics of mucin biology increases, further clinical targets and therapeutic strategies are likely to emerge.

Acrolein↗

PubMed2002

In vitro assessment of chemokine receptor-ligand interactions mediating mouse eosinophil migration.

Eosinophil migration from circulation is controlled, in part, by chemokines through a family of G-protein-coupled chemokine receptors (CCR). Studies of human eosinophils have demonstrated that signaling through CCR3 receptors is a prominent pathway leading to chemotaxis, although several other receptor-ligand interactions also appear to mediate eosinophil recruitment. The availability of genetically unique strains of mice permits a reductionist approach to assess the signaling pathways in experimental models of human disease. However, despite similarities in these pathways between mice and humans, significant species differences exist, complicating the translation of results from animal models to humans. Purified mouse eosinophils were used in this study to investigate the chemokine receptor expression and the activities of 18 chemokines. Mouse eosinophils isolated from IL-5 transgenic mice expressed transcripts encoding the chemokine receptors CCR1, CCR2, CCR3, CCR5, CCR8, CXCR2, and CXCR4, but not CCR4. Mouse eosinophils also migrated in response to human and mouse eotaxin-1 and -2, but not human eotaxin-3. In addition, the induced migration of mouse eosinophils by TARC, MIP-1beta, and KC suggests that unidentified receptor-ligand interactions contribute to eosinophil recruitment. It is interesting that the potent chemoattractant of human eosinophils, RANTES, was unable to mediate mouse eosinophil migration. Furthermore, despite the ability of MIP-1alpha to bind receptors on purified mouse eosinophils, it was only able to induce significant eosinophil migration in a mixed splenocyte population and was unable to induce migration of highly purified eosinophils. Collectively, these observations reveal physiologically relevant distinctions in mechanisms mediating human and mouse eosinophil migration that potentially reflect evolutionary disparities between these species.

Amino Acid Sequence↗