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Biomedical subjects

G W Hunninghake

Publications and source records attributed to G W Hunninghake.

At least 19 recordsLinked to original sources
PubMed2001

Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-alpha secretion through IKK regulation of NF-kappa B.

Reactive oxygen species (ROS) are important second messengers generated in response to many types of environmental stress. In this setting, changes in intracellular ROS can activate signal transduction pathways that influence how cells react to their environment. In sepsis, a dynamic proinflammatory cellular response to bacterial toxins (e.g. lipopolysaccharide or LPS) leads to widespread organ damage and death. The present study demonstrates for the first time that the activation of Rac1 (a GTP-binding protein), and the subsequent production of ROS, constitutes a major pathway involved in NFkappaB-mediated tumor necrosis factor-alpha (TNFalpha) secretion following LPS challenge in macrophages. Expression of a dominant negative mutant of Rac1 (N17Rac1) reduced Rac1 activation, ROS formation, NFkappaB activation, and TNFalpha secretion following LPS stimulation. In contrast, expression of a dominant active form of Rac1 (V12Rac1) mimicked these effects in the absence of LPS stimulation. IKKalpha and IKKbeta were both required downstream modulators of LPS-activated Rac1, since the expression of either of the IKK dominant mutants (IKKalphaKM or IKKbetaKA) drastically reduced NFkappaB-dependent TNFalpha secretion. Moreover, studies using CD14 blocking antibodies suggest that Rac1 induces TNFalpha secretion through a pathway independent of CD14. However, a maximum therapeutic inhibition of LPS-induced TNFalpha secretion occurred when both CD14 and Rac1 pathways were inhibited. Our results suggest that targeting both Rac1- and CD14-dependent pathways could be a useful therapeutic strategy for attenuating the proinflammatory cytokine response during the course of sepsis.

Adenoviridae↗
PubMed2001

Lipopolysaccharide activates Akt in human alveolar macrophages resulting in nuclear accumulation and transcriptional activity of beta-catenin.

Exposure of human alveolar macrophages to bacterial LPS results in activation of a number of signal transduction pathways. An early event after the alveolar macrophage comes in contact with LPS is activation of the phosphatidylinositol 3 kinase (PI 3-kinase). This study evaluates the downstream effects of that activation. We observed that LPS exposure results in phosphorylation of Akt (serine 473). We found this using both phosphorylation-specific Abs and also by in vivo phosphorylation with (32)P-loaded cells. AKT activation resulted in the phosphorylation-dependent inactivation of glycogen synthase kinase (GSK-3) (serine 21/9). We found that both of these events were linked to PI 3-kinase because the PI 3-kinase inhibitors, wortmannin and LY294002, inhibited LPS-induced phosphorylation of both AKT and GSK-3. Inactivation of GSK-3 has been shown to reduce the ubiquitination of beta-catenin, resulting in nuclear accumulation and transcriptional activity of beta-catenin. Consistent with this, we found that LPS caused an increase in the amounts of PI 3-kinase-dependent nuclear beta-catenin in human alveolar macrophages and expression of genes that require nuclear beta-catenin for their activation. This is the first demonstration that LPS exposure activates AKT, inactivates GSK-3, and causes accumulation and transcriptional activity of beta-catenin in the nucleus of any cell, including alveolar macrophages.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2000

Protein kinase C zeta plays a central role in activation of the p42/44 mitogen-activated protein kinase by endotoxin in alveolar macrophages.

Human alveolar macrophages respond to endotoxin (LPS) by activation of a number of mitogen-activated protein kinase pathways, including the p42/44 (extracellular signal-related kinase (ERK)) kinase pathway. In this study, we evaluated the role of the atypical protein kinase C (PKC) isoform, PKC zeta, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC zeta, mitogen-activated protein/ERK kinase (MEK, the upstream activator of ERK), and ERK. LPS did not activate Raf-1, the classic activator of MEK. Pseudosubstrate-specific peptides with attached myristic acid are cell permeable and can be used to block the activity of specific PKC isoforms in vivo. We found that a peptide specific for PKC zeta partially blocked activation of both MEK and ERK by LPS. We also found that this peptide blocked in vivo phosphorylation of MEK after LPS treatment. In addition, we found that LPS caused PKC zeta to bind to MEK in vivo. These observations suggest that MEK is an LPS-directed target of PKC zeta. PKC zeta has been shown in other systems to be phosphorylated by phosphatidylinositol (PI) 3-kinase-dependent kinase. We found that LPS activates PI 3-kinase and causes the formation of a PKC zeta/PI 3-kinase-dependent kinase complex. These data implicate the PI 3-kinase pathway as an integral part of the LPS-induced PKC zeta activation. Taken as a whole, these studies suggest that LPS activates ERK kinase, in part, through activation of an atypical PKC isoform, PKC zeta.

Amino Acid Sequence↗
PubMed2000

A constitutive active MEK --> ERK pathway negatively regulates NF-kappa B-dependent gene expression by modulating TATA-binding protein phosphorylation.

Endotoxin-induced cytokine gene expression is regulated, in part, by NF-kappaB. We have shown that both the ERK and p38 mitogen-activated protein (MAP) kinases are necessary for cytokine gene transcription and that the p38 MAP kinase is required for NF-kappaB-driven transcription, so we hypothesized that the MEK --> ERK pathway regulated NF-kappaB-driven transcription as well. We found that a constitutive active MEK --> ERK pathway inhibited NF-kappaB-driven transcription. In addition, both PD 98059 and a dominant negative ERK2 augmented NF-kappaB-driven transcription; however, neither PD 98059 nor MEK1 altered NF-kappaB activation at any level. The constitutive active MEK --> ERK pathway inhibited the phosphorylation of TBP, which is necessary for both interaction with RelA and binding to the TATA box. Due to the fact that we have shown that the p38 MAP kinase modulates TBP activation, we evaluated the effect of the constitutive active MEK --> ERK pathway on p38 MAP kinase activity. We found that the MEK --> ERK pathway negatively regulates NF-kappaB-driven transcription, in part, by inhibiting p38 MAP kinase activity. Thus, the ERK and p38 MAP kinases have differential effects on NF-kappaB-driven transcription.

Cell Line↗
PubMed2000

Effects of ragweed and Th-2 cytokines on the secretion of IL-8 in human airway epithelial cells.

Ragweed-induced asthma is a very common pulmonary disease. An important part of asthma is a large increase in inflammatory cells, including neutrophils and eosinophils, in ragweed-sensitilized subjects. In this study, we determined if ragweed treatment could induce the release of interleukin-8 (IL-8) by human airway epithelial cells. We found that ragweed induces a substantial increase of the secretion of IL-8 from A549 cells in a dose- and time-dependent manner. Th-2 cytokines, IL-4 and IL-13, partially inhibited the ragweed-induced secretion of IL-8. Our results suggest that airway epithelial cells may be one of the cell sources that provide IL-8 during ragweed-induced asthma. The results also indicate that IL-4 and IL-13 may exert inhibitory effects on IL-8 secretion by airway epithelium.

Allergens↗
PubMed1999

The p38 mitogen-activated protein kinase is required for NF-kappaB-dependent gene expression. The role of TATA-binding protein (TBP).

Endotoxin-induced cytokine gene transcription in monocytes and macrophages is regulated in part by NF-kappaB. We have previously shown that the p38 mitogen-activated protein (MAP) kinase is necessary for endotoxin-induced cytokine gene transcription. Due to the fact that most cytokine promoter sequences have active NF-kappaB sites, we hypothesized that the p38 MAP kinase was necessary for NF-kappaB-dependent gene expression. We found that NF-kappaB-dependent gene expression was reduced to near control levels with either SB 203580 or a dominant-negative p38 MAP kinase expression vector. Inhibition of the p38 MAP kinase did not alter NF-kappaB activation at any level, but it significantly reduced the DNA binding of TATA-binding protein (TBP) to the TATA box. The dominant-negative p38 MAP kinase expression vector interfered with the direct interaction of native TFIID (TBP) with a co-transfected p65 fusion protein. Likewise, this dominant-negative plasmid also interfered with the direct interaction of a co-transfected TBP fusion protein with the native p65 subunit. The p38 kinase also phosphorylated TFIID (TBP) in vitro, and SB 203580 inhibited phosphorylation of TFIID (TBP) in vivo. Thus, the p38 MAP kinase regulates NF-kappaB-dependent gene transcription, in part, by modulating activation of TFIID (TBP).

Cytokines↗
PubMed1999

Human alveolar macrophages are markedly deficient in REF-1 and AP-1 DNA binding activity.

Although many functions of human alveolar macrophages are altered compared with their precursor cell, the blood monocyte (monocyte), the reason(s) for these functional changes have not been determined. We recently reported that human alveolar macrophages do not express AP-1 DNA binding activity (Monick, M. M., Carter, A. B., Gudmundsson, G., Geist, L. J., and Hunninghake, G. W. (1998) Am. J. Physiol. 275, L389-L397). To determine why alveolar macrophages do not express AP-1 DNA binding activity, we first showed that there was not a decrease in expression of the FOS and JUN proteins that make up the AP-1 complex. There was, however, a significant difference in the amounts of the nuclear protein, REF-1 (which regulates AP-1 DNA binding by altering the redox status of FOS and JUN proteins), in alveolar macrophages compared with monocytes. In addition, in vitro differentiation of monocytes to a macrophage-like cell resulted in decreased amounts of REF-1. Finally, addition of REF-1 from activated monocytes to alveolar macrophage nuclear proteins resulted in a marked increase in AP-1 DNA binding. These studies strongly suggest that the process of differentiation of monocytes into alveolar macrophages is associated with a loss of REF-1 and AP-1 activity. This observation may explain, in part, some of the functional differences observed for alveolar macrophages compared with monocytes.

Carbon-Oxygen Lyases↗
PubMed1999

Viral infection modulates expression of hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is a granulomatous, inflammatory lung disease caused by inhalation of organic Ags, most commonly thermophilic actinomycetes that cause farmer's lung disease. The early response to Ag is an increase in neutrophils in the lung, whereas the late response is a typical Th1-type granulomatous disease. Many patients who develop disease report a recent viral respiratory infection. These studies were undertaken to determine whether viruses can augment the inflammatory responses in HP. C57BL/6 mice were exposed to the thermophilic bacteria Saccharopolyspora rectivirgula (SR) for 3 consecutive days per wk for 3 wk. Some mice were exposed to SR at 2 wk after infection with respiratory syncytial virus (RSV), whereas others were exposed to SR after exposure to saline alone or to heat-inactivated RSV. SR-treated mice developed a typical, early neutrophil response and a late granulomatous inflammatory response. Up-regulation of IFN-gamma and IL-2 gene expression was also found during the late response. These responses were augmented by recent RSV infection but not by heat-inactivated RSV. Mice with a previous RSV infection also had a greater early neutrophil response to SR, with increased macrophage inflammatory protein-2 (MIP-2, murine equivalent of IL-8) release in bronchoalveolar lavage fluid. These studies suggest that viral infection can augment both the early and late inflammatory responses in HP.

Alveolitis, Extrinsic Allergic↗
PubMed1999

A phosphatidylcholine-specific phospholipase C regulates activation of p42/44 mitogen-activated protein kinases in lipopolysaccharide-stimulated human alveolar macrophages.

This study uses human alveolar macrophages to determine whether activation of a phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) is linked to activation of the p42/44 (ERK) kinases by LPS. LPS-induced ERK kinase activation was inhibited by tricyclodecan-9-yl xanthogenate (D609), a relatively specific inhibitor of PC-PLC. LPS also increased amounts of diacylglycerol (DAG), and this increase in DAG was inhibited by D609. LPS induction of DAG was, at least in part, derived from PC hydrolysis. Ceramide was also increased in LPS-treated alveolar macrophages, and this increase in ceramide was inhibited by D609. Addition of exogenous C2 ceramide or bacterial-derived sphingomyelinase to alveolar macrophages increased ERK kinase activity. LPS also activated PKC zeta, and this activation was inhibited by D609. LPS-activated PKC zeta phosphorylated MAP kinase kinase, the kinase directly upstream of the ERK kinases. LPS-induced cytokine production (RNA and protein) was also inhibited by D609. As an aggregate, these studies support the hypothesis that one way by which LPS activates the ERK kinases is via activation of PC-PLC and that activation of a PC-PLC is an important component of macrophage activation by LPS.

Bridged-Ring Compounds↗
PubMed1999

Interstitial lung disease: A quantitative study using the adaptive multiple feature method.

We have previously described an adaptive multiple feature method (AMFM) for the objective assessment of global and regional changes in pulmonary parenchyma to detect emphysema. This computerized method uses a combination of statistical and fractal texture features for characterization of lung tissues based upon high resolution computed tomography (HRCT) scans. This present study was a substantial extension of the AMFM to simultaneously discriminate between multiple pulmonary disease processes. Normal subjects and those with emphysema, idiopathic pulmonary fibrosis (IPF), or sarcoidosis were studied. The AMFM was compared with two currently utilized computer-based methods: mean lung density (MLD) and the histogram analysis (HIST). Globally, when comparing two-subject groups the AMFM overall accuracy was 2 to 18% better than the overall accuracy of MLD and as much as 36% better than the accuracy of the HIST methods. In three-subject group discrimination tasks, the AMFM performed 7 to 27% better than the MLD and 4 to 36% better than the HIST methods. Finally, in discriminating all four subject groups at a time, the AMFM overall accuracy was 81%, which was 21% better than the MLD and 25% better than the HIST method. In most three-subject group comparisons and in the four-subject group comparison, the AMFM was significantly (p < 0.01) better than the MLD and HIST methods. Next, the AMFM was applied to local discrimination between normal and each disease group individually. The normal versus emphysema, normal versus IPF, and normal versus sarcoidosis samples were discriminated with an accuracy of 95, 86, and 77%, respectively. The AMFM is an objective quantitative method that can be adapted for successful discrimination of multiple parenchymal lung diseases.

Diagnosis, Computer-Assisted↗
PubMed1999

Variable airway responsiveness to inhaled lipopolysaccharide.

Individuals exposed to inhaled endotoxin (lipopolysaccharide [LPS]) can develop airway symptomatology and exacerbations of asthma. Moreover, among those occupationally exposed to organic dusts, the progression of airflow obstruction is related to the endotoxin concentration in the bioaerosol. Not everyone exposed to high concentrations of LPS develops these problems. To determine whether individuals express a differential response to inhaled LPS, we challenged 72 healthy volunteers with increasing doses of LPS. Airflow was assessed after each dose and the protocol was terminated for decline in FEV1 >/= 20%. Marked differences in the response to inhaled LPS were observed: eight "sensitive" subjects had at least 20% decline in their FEV1 after inhaling 6.5 micrograms or less of LPS, whereas 11 "hyporesponsive" subjects maintained an FEV1 >/= 90% of their baseline even after inhaling 41.5 micrograms of LPS. Serial testing demonstrated that the response to inhaled LPS is reproducible. Sensitive subjects were more commonly female and hyporesponsive subjects were more often male (p = 0.016). Peripheral blood monocytes from hyporesponsive subjects, compared with sensitive subjects, released less interleukin (IL)-6 and IL-8. These findings demonstrate that an LPS phenotype can be reproducibly elicited in humans, which creates an opportunity to identify genes involved in this response to inhaled LPS.

Administration, Inhalation↗
PubMed1999

Computer recognition of regional lung disease patterns.

We have developed an objective, reproducible, and automated means for the regional evaluation of the pulmonary parenchyma from computed tomography (CT) scans. This method, known as the Adaptive Multiple Feature Method (AMFM) assesses as many as 22 independent texture features in order to classify a tissue pattern. In this study, the six tissue patterns characterized were: honeycombing, ground glass, bronchovascular, nodular, emphysemalike, and normal. The lung slices were evaluated regionally using 31 x 31 pixel regions of interest. In each region of interest, an optimal subset of texture features was evaluated to determine which of the six patterns the region could be characterized as. The computer output was validated against experienced observers in three settings. In the first two readings, when the observers were blinded to the primary diagnosis of the subject, the average computer versus observer agreement was 44.4 +/- 8.7% and 47.3 +/- 9.0%, respectively. The average interobserver agreement for the same two readings were 48.8 +/- 9.1% and 52.2 +/- 10.0%, respectively. In the third reading, when the observers were provided the primary diagnosis, the average computer versus observer agreement was 51.7 +/- 2.9% where as the average interobserver agreement was 53.9 +/- 6.2%. The kappa statistic of agreement between the regions, for which the majority of the observers agreed on a pattern type, versus the computer was found to be 0.62. For regional tissue characterization, the AMFM is 100% reproducible and performs as well as experienced human observers who have been told the patient diagnosis.

Diagnosis, Computer-Assisted↗
PubMed1999

Both Erk and p38 kinases are necessary for cytokine gene transcription.

A critical feature of sepsis-induced acute lung injury is the release of cytokines from endotoxin (LPS)- stimulated alveolar macrophages (AM). LPS is also known to activate various members of the mitogen- activated protein kinase (MAPK) family in other types of cells. In this study, we evaluated whether multiple members of the MAPK family regulate cytokine gene expression in LPS-stimulated AM. We found that LPS activates both the extracellular signal-regulated kinase (Erk) and p38 kinases, and that this activation is augmented when the cells are cultured in serum. Inhibition of either the Erk (with PD98059) or p38 (with SB203580) kinase pathway resulted in only a partial reduction in cytokine (interleukin-6 and tumor necrosis factor) messenger RNA accumulation and cytokine release, whereas inhibition of both pathways simultaneously resulted in a decrease in cytokine gene expression to near-control levels. Nuclear run-on assays showed that the effect of these MAPK pathways on LPS-induced expression of the cytokine genes was attributable, at least in part, to regulation of gene transcription. These findings suggest that activation of both the Erk and p38 kinase pathways is necessary for optimal cytokine gene expression in LPS-stimulated human AM, and that the MAPK pathways play a critical role in the inflammatory response that occurs in sepsis-induced acute lung injury.

Adult↗
PubMed1998

IL-12 modulates expression of hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is a granulomatous, inflammatory lung disease caused by inhalation of organic Ags, most commonly thermophilic actinomycetes. Only a minority of individuals exposed to these Ags develops disease, suggesting that host factors are important for the expression of HP. We compared the expression of HP in a sensitive strain of mice, C57BL/6, and in a resistant strain of mice, DBA/2. They were exposed to the thermophilic bacteria Saccharopolyspora rectivirgula (SR) or to saline alone for 3 consecutive days/week for 3 wk. After exposure to Ag, C57BL/6 mice, but not DBA/2 mice, developed granulomatous inflammation with an increase in lung index (lung weight). Both strains had similar amounts of Ag delivered to the lungs after intranasal installation, as determined with 14C-labeled Ag. Both also had similar increases in total bronchoalveolar cells after Ag exposure, but the C57BL/6 mice had more lymphocytes. Compared with the resistant strain, the sensitive strain had a significantly greater Ag-induced increase in IL-12 and IFN-gamma gene expression. DBA/2 mice resembled sensitive, C57BL/6 mice if they received IL-12 augmentation therapy at the time of Ag exposure. These findings were not limited to lung, since both unstimulated and SR-stimulated spleen cells from C57BL/6 mice released significantly more IL-12 than cells from DBA/2 mice. However, spleen cells from DBA/2 mice made more IFN-gamma when exposed to IL-12, than cells from C57BL/6 mice. These results suggest that the IL-12 response to Ag may modulate in part the expression of HP.

Adjuvants, Immunologic↗