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W Conrad Liles

Publications and source records attributed to W Conrad Liles.

35 records · Page 2Linked to original sources
PubMed2004

Optimal timing to repopulation of resident alveolar macrophages with donor cells following total body irradiation and bone marrow transplantation in mice.

To determine the time required to repopulate mouse lungs with donor alveolar macrophages following total body irradiation (TBI) and bone marrow transplantation (BMT), C57Bl/6 mice were subjected to TBI with 900 cGy, followed by transplantation of bone marrow cells from mice expressing green fluorescent protein (GFP) in their somatic cells. The mice were euthanized at either 30 (n=5), 60 (n=5) or 90 (n=5) days following BMT. Thirty days following transplantation, 87.8 +/- 3.9% (mean +/- S.E.M.) circulating leukocytes in recipient mice were derived from the donor, as determined by fluorescence activated cell sorting (FACS) analysis for GFP. However, only 46.9 +/- 7.4% of the resident alveolar cells expressed GFP, indicating incomplete repopulation. By day 60 post-transplantation, the percentage of bronchoalveolar lavage fluid (BALF) cells expressing GFP reached 74.5 +/- 2.4%, remaining stable 90 days after transplantation (80.4 +/- 1.9%). We conclude that 60 days after TBI with 900 cGy and bone marrow transplantation, the majority of the lung resident alveolar macrophages is of donor origin. This study provides useful information regarding the time of reconstitution with donor alveolar macrophages in the pulmonary airspaces of recipient mice following marrow transplantation.

Adoptive Transfer↗
PubMed2004

Differential response of human lung epithelial cells to fas-induced apoptosis.

The Fas (CD95)/Fas ligand (CD178) system plays an important role in epithelial damage during the acute respiratory distress syndrome. The goal of this study was to determine whether proximal and distal human lung epithelial cells differ in their sensitivity to Fas ligand (rh-sFasL), and whether the response of lung epithelium to Fas ligation is modulated by proinflammatory cytokines. Although the expression of both Fas message and protein was similar in proximal and distal lung epithelial cells, only distal cells became apoptotic when exposed to serial dilutions of rh-sFasL. Stimulation with tumor necrosis factor-alpha, interleukin-1beta, or interferon-gamma significantly increased the sensitivity of proximal cells to rh-sFasL, and exposure to either tumor necrosis factor-alpha or interferon-gamma enhanced the sensitivity of distal cells to Fas ligation. These findings suggest that in normal human lungs, the responses of the epithelium to Fas ligation become more pronounced from proximal to distal locations. Furthermore, proinflammatory cytokines sensitize lung epithelium to Fas-induced death. These findings are relevant for understanding the role of the Fas/FasL system in acute lung injury, in which epithelial damage occurs primarily in distal airway and alveolar epithelium, whereas sFasL is present throughout the airspaces.

Antigens, CD↗
PubMed2004

Sustained lipopolysaccharide-induced lung inflammation in mice is attenuated by functional deficiency of the Fas/Fas ligand system.

To determine whether the Fas/Fas ligand (FasL) (CD95/CD178) system contributes to the development of an inflammatory response in vivo, 2.5 microg of bacterial lipopolysaccharide (LPS; endotoxin) per g was administered intranasally to healthy mice (C57BL/6) and mutant mice deficient in either Fas (lpr mice) or FasL (gld mice). Sustained LPS-induced neutrophilic inflammation in the lungs was attenuated in both lpr and gld mice. These observations provide further evidence of a proinflammatory role for the Fas/FasL system in the lungs.

Administration, Intranasal↗
PubMed2003

Fas and Fas-associated death domain protein regulate monocyte chemoattractant protein-1 expression by human smooth muscle cells through caspase- and calpain-dependent release of interleukin-1alpha.

We previously reported that treatment of human vascular smooth muscle cells (SMCs) with proapoptotic stimuli, including Fas ligand plus cycloheximide (FasL/Chx), or overexpression of Fas-associated death domain protein (FADD) result in increased expression of monocyte chemoattractant protein-1 (MCP-1) and other proinflammatory genes. In this study, we demonstrate that Fas/FADD-induced MCP-1 upregulation is driven by an autocrine/paracrine signaling loop in which interleukin (IL)-1alpha synthesis and release are activated through caspase- and calpain-dependent processes. Untreated SMCs contain very little IL-1alpha protein or transcript. Both were increased greatly in response to Fas/FADD activation, primarily through an autocrine/paracrine pathway in which secreted IL-1alpha stimulated additional IL-1alpha synthesis and release. Caspase 8 (Csp8) activity increased in response to FasL/Chx treatment, and Csp8 inhibitors markedly reduced IL-1alpha release and MCP-1 upregulation. In contrast, Csp8 activity was not significantly increased in response to FADD overexpression and caspase inhibitors did not effect FADD-induced MCP-1 upregulation. Both FasL/Chx treatment and FADD overexpression increased the activity of calpains. Calpain inhibitors reduced IL-1alpha release and MCP-1 upregulation in both FADD-overexpressing SMCs and FasL/Chx-treated SMCs without blocking Csp8 activity. This indicates that calpains are not required for activation of caspases and that caspase activation is not sufficient for IL-1alpha release and MCP-1 upregulation. These data suggest that calpains play a dominant role in Fas/FADD-induced IL-1alpha release and MCP-1 upregulation and that caspase activation may function to amplify the effects of calpain activation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Mobilization of hematopoietic progenitor cells in healthy volunteers by AMD3100, a CXCR4 antagonist.

Stromal cell-derived factor 1 (SDF1/CXCL12) and its cognate receptor, CXCR4, play key regulatory roles in CD34+ cell trafficking. We investigated whether AMD3100, a selective CXCR4 antagonist, could mobilize hematopoietic progenitor cells from marrow to peripheral blood in healthy human volunteers. Initially, 10 persons each received a single dose of AMD3100 (80 microsubcutaneously), which induced rapid, generalized leukocytosis associated with an increase in peripheral blood CD34+ cells, representing pluripotent hematopoietic progenitors by in vitro colony-forming unit assays, from 3.8 +/- 0.5/microL to 20.7 +/- 3.5/microL at 6 hours. Subsequent dose-response studies showed a maximum increase in circulating CD34+ cells from 2.6 +/- 0.3/microL to 40.4 +/- 3.4/microL at 9 hours after 240 micro/kg AMD3100. Serial administration of AMD3100 (80 microg/kg/d for 3 days) resulted in consistent, reversible increases in peripheral blood CD34+ cells. AMD3100 was well tolerated and caused only mild, transient toxicity. These findings suggest potential clinical application of AMD3100 for CD34+ cell mobilization and collection for hematopoietic stem cell transplantation.

Adult↗
PubMed2003

Fas (CD95) induces proinflammatory cytokine responses by human monocytes and monocyte-derived macrophages.

Fas (CD95, APO-1) is regarded as the prototypical cell death receptor of the TNFR superfamily. Fas-induced apoptosis is generally considered to be a noninflammatory process, contributing to the silent resolution of immune and inflammatory responses. However, accumulating evidence indicates that Fas may also induce cellular activation signals. We hypothesized that Fas could activate proinflammatory cytokine responses by normal human monocytes and macrophages. Monocytes were isolated by negative immunoselection from the PBMC fraction of venous blood from healthy volunteers, and monocyte-derived macrophages were cultivated in vitro. Both monocytes and monocyte-derived macrophages released TNF-alpha and IL-8 following Fas ligation, and conditioned medium from Fas-activated monocytes and macrophages induced the directed migration of neutrophils in a chemotaxis assay. Fas-induced monocyte cytokine responses were associated with monocyte apoptosis, nuclear translocation of NF-kappaB, and cytokine gene expression and were blocked by caspase inhibition but not by inhibition of IL-1beta signaling. In contrast, Fas-induced macrophage cytokine responses occurred in the absence of apoptosis and were caspase independent, indicating maturation-dependent differences in the Fas signaling pathways that lead to proinflammatory cytokine induction. Rather than contributing to the resolution of inflammation, Fas ligation on circulating monocytes and tissue macrophages may induce proinflammatory cytokine responses that can initiate acute inflammatory responses and tissue injury.

Apoptosis↗
PubMed2003

The p17 cleaved form of caspase-3 is present within viable macrophages in vitro and in atherosclerotic plaque.

OBJECTIVE: In vitro studies of macrophage death in response to oxidized LDL (oxLDL) were undertaken as a model for the formation of the necrotic core of atherosclerotic plaque. METHODS AND RESULTS: Thioglycollate-elicited mouse peritoneal macrophages avidly incorporated both oxLDL and acetylated LDL (acLDL) to become foam cells. oxLDL-treated macrophages, but not acLDL-treated macrophages, showed nearly 100% death, with characteristics consistent with apoptosis, including cell surface phosphatidylserine exposure, intracellular caspase-3 activity, cleavage of caspase-3 substrates, and DNA fragmentation, as shown by TUNEL assay. The activated form of caspase-3 (p17 cleaved form) was present in attached, viable macrophages before exposure to oxLDL. This p17 form was also found in apparently viable as well as in TUNEL-positive cells within atherosclerotic lesions of chow-fed apolipoprotein E-deficient (ApoE-/-) mice. The amount of p17 caspase-3 was reduced by in vitro blockade of FasL with an FasL-blocking antibody and was absent in macrophages from lpr/lpr mice, which lack functional Fas. Moreover, lpr/lpr macrophages resisted oxLDL cytotoxicity. CONCLUSIONS: The naturally occurring Fas-FasL induction of caspase-3 cleavage after macrophage attachment may represent an important physiologic mechanism that primes for cytotoxicity by oxLDL and possibly, other death-inducing molecules.

Amino Acid Chloromethyl Ketones↗
PubMed2003

Cellular and molecular abnormalities in severe congenital neutropenia predisposing to leukemia.

Severe congenital neutropenia (SCN) is a rare hematological disease characterized by a selective decrease in the level of circulating neutrophils in peripheral blood, maturation arrest at the promyelocyte stage of differentiation in the bone marrow, recurrent severe infections, and evolution to acute myelogenous leukemia (AML). Cellular and molecular studies of 12 SCN patients, including 5 patients that evolved to develop AML, revealed impaired proliferative characteristics and accelerated apoptosis of bone marrow progenitor cells in SCN compared with 11 healthy controls as demonstrated by flow cytometry analysis. Sequencing analysis revealed heterozygous deletion or substitution mutations in the neutrophil elastase (NE) gene in 9 of 12 patients but not in healthy controls. Expression of various NE mutants, but not normal NE, resulted in accelerated apoptosis of human promyelocytic HL-60 progenitor cells, similar to impaired survival observed in patients' cells. Bone marrow-derived primitive CD34(+) and CD33(+)/CD34(-) progenitor cells from SCN patients evolving to AML, all with mutations in the granulocyte colony-stimulating factor receptor (G-CSFR) gene, demonstrated normal cell survival, whereas more differentiated CD15(+)/CD33(-)/CD34(-) cells negative for mutant G-CSFR gene, continue to exhibit accelerated apoptosis. These data demonstrate that impaired survival of bone marrow myeloid progenitor cells, probably driven by expression of mutant NE, is the cellular mechanism responsible for neutropenia in SCN. Furthermore, our results suggest that acquired G-CSFR mutations may initiate signaling events that override the pro-apoptotic effect of mutant NE in primitive progenitor cells, resulting in an expansion of the abnormal AML clone.

Antigens, CD34↗
PubMed2003

Differential role of MyD88 in macrophage-mediated responses to opportunistic fungal pathogens.

Toll-like receptors mediate macrophage recognition of microbial ligands, inducing expression of microbicidal molecules and cytokines via the adapter protein MyD88. We investigated the role of MyD88 in regulating murine macrophage responses to a pathogenic yeast (Candida albicans) and mold (Aspergillus fumigatus). Macrophages derived from bone marrow of MyD88-deficient mice (MyD88(-/-)) demonstrated impaired phagocytosis and intracellular killing of C. albicans compared to wild-type (MyD88(+/+)) macrophages. In contrast, ingestion and killing of A. fumigatus conidia was MyD88 independent. Cytokine production by MyD88(-/-) macrophages in response to C. albicans yeasts and hyphae was substantially decreased, but responses to A. fumigatus hyphae were preserved. These results provide evidence that MyD88 signaling is involved in phagocytosis and killing of live C. albicans, but not A. fumigatus. The differential role of MyD88 may represent one mechanism by which macrophages regulate innate responses specific to different pathogenic fungi.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Broad-spectrum caspase inhibition paradoxically augments cell death in TNF-alpha -stimulated neutrophils.

It is increasingly clear that there are caspase-dependent and -independent mechanisms for the execution of cell death and that the utilization of these mechanisms is stimulus- and cell type-dependent. Intriguingly, broad-spectrum caspase inhibition enhances death receptor agonist-induced cell death in a few transformed cell lines. Endogenously produced oxidants are causally linked to necroticlike cell death in these instances. We report here that broad-spectrum caspase inhibitors effectively attenuated apoptosis induced in human neutrophils by incubation with agonistic anti-Fas antibody or by coincubation with tumor necrosis factor-alpha (TNF-alpha) and cycloheximide ex vivo. In contrast, the same caspase inhibitors could augment cell death upon stimulation by TNF-alpha alone during the 6-hour time course examined. Caspase inhibitor-sensitized, TNF-alpha-stimulated, dying neutrophils exhibit apoptoticlike and necroticlike features. This occurred without apparent alteration in nuclear factor-kappaB (NF-kappaB) activation. Nevertheless, intracellular oxidant production was enhanced and sustained in caspase inhibitor-sensitized, TNF-alpha-stimulated neutrophils obtained from healthy subjects. However, despite reduced or absent intracellular oxidant production following TNF-alpha stimulation, cell death was also augmented in neutrophils isolated from patients with chronic granulomatous disease incubated with a caspase inhibitor and TNF-alpha. These results demonstrate that, in human neutrophils, TNF-alpha induces a caspase-independent but protein synthesis-dependent cell death signal. Furthermore, they suggest that TNF-alpha activates a caspase-dependent pathway that negatively regulates reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity.

Case-Control Studies↗
PubMed2002

Lineage-specific growth factors can compensate for stem and progenitor cell deficiencies at the postprogenitor cell level: an analysis of doubly TPO- and G-CSF receptor-deficient mice.

Multiple lines of evidence indicate that thrombopoietin (TPO) substantially impacts the number of hematopoietic stem cells and progenitors of all myeloid lineages. Nevertheless, tpo knock-out mice (T(-)) display thrombocytopenia only; blood erythroid and neutrophil levels are normal despite 60% to 85% reductions in stem and progenitor cells. The compensatory mechanism(s) for these deficiencies remains uncertain; lineage-specific cytokines such as erythropoietin or granulocyte colony-stimulating factor (G-CSF) have been postulated but never proven to be responsible. To directly test whether G-CSF can compensate for the myeloid progenitor cell reduction in the T(-) model of hematopoietic deficiency, T(-) and G-CSF-receptor knock-out (GR(-)) mice were crossed, and F1 animals bred to obtain doubly nullizygous mice (T(-)GR(-)). This experiment also allowed us to test the hypothesis that G-CSF contributes to the residual platelet production in T(-) mice. We found that T(-)GR(-) F2 mice displayed similar blood platelet levels as that seen in T(-) mice, indicating that G-CSF does not account for the residual megakaryopoiesis in T(-) mice. However, we also noted excessive perinatal mortality of T(-)GR(-) animals, caused by infection due to a profound and significant decrease in marrow and peripheral blood neutrophils, far greater than that seen in either T(-) or GR(-) mice. These data indicate that in the additional absence of GR, T(-) mice cannot compensate for their 62% reduction in myeloid progenitors and become profoundly neutropenic, supporting the hypothesis that G-CSF can compensate for the myeloid effects of TPO deficiency by expanding the pool of cells between the granulocyte-macrophage colony-forming unit and mature neutrophil stages of granulopoiesis.

Animals↗
PubMed2002

Therapeutic use of cytokines to modulate phagocyte function for the treatment of infectious diseases: current status of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and interferon-gamma.

The innate immune system represents the initial arm of host defense against pathogenic bacteria, fungi, and parasites. Neutrophils, monocytes, and tissue-based macrophages are major cellular components of this system. The potential ability to augment activity of the innate immune system has increased dramatically during the past 2 decades, with the discovery and development of cytokines. Four cytokines, namely granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interferon (IFN)-gamma, have received increasing attention as potential adjunctive agents for the treatment of infectious diseases. In various animal models of infection, therapeutic administration of each of the 4 cytokines has been shown to enhance pathogen eradication and to decrease morbidity and/or mortality. However, variable therapeutic efficacy has been reported in clinical trials conducted to date. This review summarizes the current status of the use of G-CSF, GM-CSF, M-CSF, and IFN-gamma in the treatment of infectious diseases.

Animals↗
PubMed2002

Apoptosis of vascular smooth muscle cells is induced by Fas ligand derived from monocytes/macrophage.

Fas and its ligand (FasL), are a receptor-ligand pair identified as promoting cell death in several tissues. Apoptosis of vascular smooth muscle cells (VSMCs) in human atherosclerotic plaque may contribute to weakening of the fibrous cap, ultimately resulting in plaque rupture. We investigated the ability of monocytes to induce apoptosis of cultured VSMCs through Fas/FasL pathway. In addition, we examined the association of FasL with apoptosis in human coronary plaques. Both activated monocytes and the supernatant obtained from activated monocytes were able to kill cultured VSMCs. The apoptotic response of VSMCs was almost completely blocked by the caspase inhibitor z-VAD-fmk and was partially blocked by incubation with antagonistic anti-Fas IgG1 which suggests that Fas/FasL system was involved in the induction of cell death. An approximate 30 kDa protein, which represents a cleaved, soluble form of FasL, was identified in culture medium from activated monocytes, but not in culture medium from control, unactivated monocytes. Immunohistochemical analysis of human atherosclerotic coronary lesions showed that FasL is expressed by macrophages, and microvessels in the adventitia as well as in the plaque. Finally, double-staining with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and FasL antibody showed that FasL enriched lesions always included a number of TUNEL-positive cells. These data suggest that Fas/FasL pathway can be employed by monocytes/macrophages to induce VSMC apoptosis in the atherosclerotic lesions.

Antigen-Antibody Complex↗
PubMed2002

Granulocyte transfusion therapy for infections in candidates and recipients of HPC transplantation: a comparative analysis of feasibility and outcome for community donors versus related donors.

BACKGROUND: Feasibility, response to granulocyte transfusion therapy, and clinical outcome were compared among HPC transplant recipients enrolled in a prospective study of a community blood bank-based unrelated donors program, a prospective granulocyte study using family donors, and matched control patients without granulocyte transfusion therapy. STUDY DESIGN AND METHODS: Overall, 40 patients (327 collections) received granulocyte concentrates from unrelated donors, 34 patients (219 collections) received granulocyte concentrates from related donors, and 74 patients served as controls. Study entry criteria for patients included an absolute neutrophil count (ANC) of less than 200 per microL and documented invasive fungal or bacterial infections. RESULTS: There was a median delay of 3 days (range, 0-14) in patients receiving transfusions from unrelated donors between day of diagnosis of infection and start of granulocyte transfusion therapy as compared with a median delay of 5 days (range, 0-25) in patients receiving transfusions from related donors (p = 0.01). The ANC increment after the first, second, and seventh transfusions in patients who had community donors was significantly higher or comparable to patients who had family donors. Overall, clinical outcome was comparable between the three patient groups. Kaplan-Meier analysis revealed no difference between all cohorts in overall 6-month survival (p = 0.28, log-rank) or event-free survival (p = 0.17, log-rank). CONCLUSION: These results suggest that future efficacy trials should consider inclusion of unrelated community donors for timely institution of granulocyte transfusion therapy.

Adolescent↗
PubMed2002

Mutant elastase in pathogenesis of cyclic and severe congenital neutropenia.

Severe neutropenia is characterized by maturation arrest of myeloid cells at the promyelocyte stage of hematopoiesis. We reported that accelerated apoptosis of bone marrow myeloid progenitor cells was observed in both cyclic (CN) and severe congenital neutropenia (SCN). Short and long-term cultures of bone marrow CD34+ cells revealed reduced production of multipotent progenitors in SCN. In contrast, production of these cells was slightly elevated in CN compared with CD34+ cells from healthy volunteers. Production of myeloid-committed progenitor cells was significantly reduced in both CN and SCN. FACS analysis of CD34+ cells revealed G /G cell cycle arrest in SCN but not in CN.(0) (1) All CN patients and more than 90% of SCN patients have mutation in the neutrophil elastase (NE) gene. Molecular modeling of NE tertiary structure indicates that mutations observed in SCN are primarily located around the glycosylation sites, whereas CN mutations affect predominantly the active site. Transient expression of CN- or SCN-specific mutant NE cDNA results in impaired survival of human myeloid progenitor cells compared with control cells transfected with intact NE cDNA. We hypothesize that abnormal processing and subcellular localization of mutant NE might predetermine the etiology of cyclic or severe congenital neutropenia.

Hematopoiesis↗