Effects of Decreased Calmodulin Protein on the Survival Mechanisms of Alveolar Macrophages during Pneumocystis Pneumonia

ABSTRACT Pneumocystis infection causes increased intracellular levels of reactive oxygen species (ROS) and the subsequent apoptosis of alveolar macrophages (Amø). Assessments of key prosurvival molecules in Amø and bronchoalveolar lavage fluids from infected rats and mice showed low levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and reduced activation of phosphoinositide-3 kinase (PI-3K). Ubiquitous calcium-sensing protein calmodulin protein and mRNA levels were also reduced in Amø during Pneumocystis pneumonia (Pcp). Calmodulin has been implicated in control of GM-CSF production and PI-3K activation in other immune cell types. Experiments to determine the control of GM-CSF and PI-3K by calmodulin in Amø showed that GM-CSF expression and PI-3K activation could not be induced when calmodulin was inhibited. Calmodulin inhibition also led to increased levels of ROS and apoptosis in cells exposed to bronchoalveolar lavage fluids from infected animals. Supplementation of Amø with exogenous calmodulin increased survival signaling via GM-CSF and PI-3K and reduced ROS and apoptosis. These data support the hypotheses that calmodulin levels at least partially control survival signaling in Amø and that restoration of GM-CSF or PI-3K signaling will improve host response to the organism.

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Selective Expansion of Alveolar Macrophages In Vivo by Adenovirus-Mediated Transfer of the Murine Granulocyte-Macrophage Colony-Stimulating Factor cDNA

Blood ◽

10.1182/blood.v93.2.655.402k02_655_666 ◽

1999 ◽

Vol 93 (2) ◽

pp. 655-666 ◽

Cited By ~ 2

Author(s):

Stefan Worgall ◽

Ravi Singh ◽

Philip L. Leopold ◽

Robert J. Kaner ◽

Neil R. Hackett ◽

...

Keyword(s):

Bronchoalveolar Lavage ◽

Alveolar Macrophages ◽

Genetically Modify ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Based on the hypothesis that genetic modification of freshly isolated alveolar macrophages (AM) with the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA would induce AM to proliferate, this study focuses on the ability of adenoviral (Ad) vectors to transfer and efficiently express the murine (m) GM-CSF cDNA in murine AM with consequent expansion in the number of AM in vitro and in vivo. To demonstrate that an Ad vector can effectively transfer and express genes in AM, murine AM recovered by bronchoalveolar lavage from the lung of Balb/c mice were infected with an Ad vector coding for green fluorescent protein (GFP) in vitro and expressed GFP in a dose-dependent fashion. Infection of AM with an Ad vector containing an expression cassette coding for mGM-CSF led to GM-CSF expression and to AM proliferation in vitro. When AM infected with AdGFP were returned to the respiratory tract of syngeneic recipient mice, GFP-expressing cells could still be recovered by bronchoalveolar lavage 2 weeks later. In vitro infection of AM with AdmGM-CSF and subsequent transplantation of the genetically modified AM to the lungs of syngeneic recipients led to GM-CSF expression in vivo. Strikingly, the AM recovered by lavage 5 weeks after transplantation demonstrated an increased rate of proliferation, and the total number of alveolar macrophages was 1.9-fold greater than controls. Importantly, the increase in the numbers of AM was selective (ie, other inflammatory cell numbers were unchanged), and there was no modification to the lung architecture. Thus, it is feasible to genetically modify AM with Ad vectors and to use this strategy to modify the behavior of AM in vivo. Based on the importance of AM in the primary defense of the respiratory epithelial surface, this strategy may be useful in enhancing pulmonary defenses in immunodeficiency states.

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Selective Expansion of Alveolar Macrophages In Vivo by Adenovirus-Mediated Transfer of the Murine Granulocyte-Macrophage Colony-Stimulating Factor cDNA

Blood ◽

10.1182/blood.v93.2.655 ◽

1999 ◽

Vol 93 (2) ◽

pp. 655-666 ◽

Cited By ~ 32

Author(s):

Stefan Worgall ◽

Ravi Singh ◽

Philip L. Leopold ◽

Robert J. Kaner ◽

Neil R. Hackett ◽

...

Keyword(s):

Bronchoalveolar Lavage ◽

Alveolar Macrophages ◽

Genetically Modify ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Abstract Based on the hypothesis that genetic modification of freshly isolated alveolar macrophages (AM) with the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA would induce AM to proliferate, this study focuses on the ability of adenoviral (Ad) vectors to transfer and efficiently express the murine (m) GM-CSF cDNA in murine AM with consequent expansion in the number of AM in vitro and in vivo. To demonstrate that an Ad vector can effectively transfer and express genes in AM, murine AM recovered by bronchoalveolar lavage from the lung of Balb/c mice were infected with an Ad vector coding for green fluorescent protein (GFP) in vitro and expressed GFP in a dose-dependent fashion. Infection of AM with an Ad vector containing an expression cassette coding for mGM-CSF led to GM-CSF expression and to AM proliferation in vitro. When AM infected with AdGFP were returned to the respiratory tract of syngeneic recipient mice, GFP-expressing cells could still be recovered by bronchoalveolar lavage 2 weeks later. In vitro infection of AM with AdmGM-CSF and subsequent transplantation of the genetically modified AM to the lungs of syngeneic recipients led to GM-CSF expression in vivo. Strikingly, the AM recovered by lavage 5 weeks after transplantation demonstrated an increased rate of proliferation, and the total number of alveolar macrophages was 1.9-fold greater than controls. Importantly, the increase in the numbers of AM was selective (ie, other inflammatory cell numbers were unchanged), and there was no modification to the lung architecture. Thus, it is feasible to genetically modify AM with Ad vectors and to use this strategy to modify the behavior of AM in vivo. Based on the importance of AM in the primary defense of the respiratory epithelial surface, this strategy may be useful in enhancing pulmonary defenses in immunodeficiency states.

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IL-1 receptor antagonist release is regulated differently in human alveolar macrophages than in monocytes

Journal of Applied Physiology ◽

10.1152/jappl.1992.73.4.1686 ◽

1992 ◽

Vol 73 (4) ◽

pp. 1686-1692 ◽

Cited By ~ 12

Author(s):

J. N. Kline ◽

M. M. Monick ◽

G. W. Hunninghake

Keyword(s):

Receptor Antagonist ◽

Alveolar Macrophages ◽

Interleukin 1 ◽

Calf Serum ◽

Cell Types ◽

Cell Group ◽

Blood Monocytes ◽

Gm Csf ◽

Monocytes And Macrophages ◽

Macrophage Colony

These studies compared the release of interleukin-1 receptor antagonist (IL-1 RA) from alveolar macrophages and peripheral blood monocytes. The cells were cultured in medium containing various amounts of heat-inactivated fetal calf serum (FCS), granulocyte-macrophage colony-stimulating factor (GM-CSF), lipopolysaccharide (LPS), and immunoglobulin G (IgG). In serum-free medium alone, IL-1 RA release was similar from macrophages and monocytes. Increasing FCS concentration caused a significant upregulation of IL-1 RA release in macrophages but not in monocytes. GM-CSF caused a small increase in both cell types. LPS caused downregulation of IL-1 RA release from monocytes but not from macrophages. IgG did not affect IL-1 RA release in either cell group. These studies demonstrate that regulation of IL-1 RA release is different in monocytes and macrophages.

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Toll-like receptors stimulate human neutrophil function

Blood ◽

10.1182/blood-2003-04-1078 ◽

2003 ◽

Vol 102 (7) ◽

pp. 2660-2669 ◽

Cited By ~ 591

Author(s):

Fumitaka Hayashi ◽

Terry K. Means ◽

Andrew D. Luster

Keyword(s):

Immune Cell ◽

Germ Line ◽

Quantitative Polymerase Chain Reaction ◽

Neutrophil Function ◽

Human Neutrophils ◽

Toll Like Receptors ◽

Gm Csf ◽

Latex Beads ◽

Macrophage Colony ◽

And Function

Abstract The first immune cell to arrive at the site of infection is the neutrophil. Upon arrival, neutrophils quickly initiate microbicidal functions, including the production of antimicrobial products and proinflammatory cytokines that serve to contain infection. This allows the acquired immune system enough time to generate sterilizing immunity and memory. Neutrophils detect the presence of a pathogen through germ line-encoded receptors that recognize microbe-associated molecular patterns. In vertebrates, the best characterized of these receptors are Toll-like receptors (TLRs). We have determined the expression and function of TLRs in freshly isolated human neutrophils. Neutrophils expressed TLR1, 2, 4, 5, 6, 7, 8, 9, and 10—all the TLRs except TLR3. Granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment increased TLR2 and TLR9 expression levels. The agonists of all TLRs expressed in neutrophils triggered or primed cytokine release, superoxide generation, and L-selectin shedding, while inhibiting chemotaxis to interleukin-8 (IL-8) and increasing phagocytosis of opsonized latex beads. The response to the TLR9 agonist nonmethylated CpG-motif-containing DNA (CpG DNA) required GM-CSF pretreatment, which also enhanced the response to the other TLR agonists. Finally, using quantitative polymerase chain reaction (QPCR), we demonstrate a chemokine expression profile that suggests that TLR-stimulated neutrophils recruit innate, but not acquired, immune cells to sites of infection. (Blood. 2003;102:2660-2669)

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In vitro p53 and/or Rb antisense oligonucleotide treatment in association with growth factors induces the proliferation of peripheral hematopoietic progenitors

Journal of Cell Science ◽

10.1242/jcs.108.3.1287 ◽

1995 ◽

Vol 108 (3) ◽

pp. 1287-1293

Author(s):

T. Mahdi ◽

A. Brizard ◽

C. Millet ◽

P. Dore ◽

J. Tanzer ◽

...

Keyword(s):

Tumor Suppressor ◽

Cell Types ◽

Suppressor Gene ◽

Antisense Oligodeoxynucleotide ◽

Signal Pathways ◽

Semisolid Medium ◽

Gm Csf ◽

Colony Forming Units ◽

Macrophage Colony

In this work we intended to determine whether p53 and/or retinoblastoma (Rb) tumor suppressor genes are involved at specific stages in the process of in vitro human peripheral stem cell hematopoiesis. Mononuclear peripheral blood cells were depleted of adherent cells and T lymphocytes (A-T-PMCs). Cells were then cultured in semisolid medium, under conditions that favor the growth of specific progenitor cell types. A-T-PMCs were exposed to p53 and/or Rb sense, scrambled DNA and antisense oligodeoxynucleotides. p53 and/or Rb antisenses (but not their senses or scrambled DNA) treatment of A-T-PMCs resulted in a significantly increase in the number of granulocyte/macrophage colony-forming units (CFU-GM) in the presence of interleukin-3 (IL-3) and/or granulocyte/macrophage colony-stimulating factor (GM-CSF). After antisense treatment, blast forming units/erythroblasts (BFU-E) derived from A-T-PMCs cultured in the presence of IL-3 + erythropoietin (Epo) were also increased whereas colony forming units/erythroblasts (CFU-E) were not markedly affected in the presence of Epo only. Megakaryocytic colony (CFU-Meg) formation from A-T-PMCs in the presence of interleukin-6 (IL-6) + IL-3 + Epo was also increased after antisense oligodeoxynucleotide treatment. These results are consistent with the hypothesis that p53 and Rb tumor suppressor gene products are involved in the control of distinct signal pathways in different peripheral progenitor cells.

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Depletion of Alveolar Macrophages Does Not Prevent Hantavirus Disease Pathogenesis in Golden Syrian Hamsters

Journal of Virology ◽

10.1128/jvi.00304-16 ◽

2016 ◽

Vol 90 (14) ◽

pp. 6200-6215 ◽

Cited By ~ 5

Author(s):

Christopher D. Hammerbeck ◽

Rebecca L. Brocato ◽

Todd M. Bell ◽

Christopher W. Schellhase ◽

Steven R. Mraz ◽

...

Keyword(s):

Alveolar Macrophages ◽

Immune Cell ◽

Cell Types ◽

Protective Role ◽

Hantavirus Infection ◽

Specific Cell ◽

Disease Pathogenesis ◽

Hamster Model ◽

Syrian Hamsters ◽

Antiviral Responses

ABSTRACTAndes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, dysregulation of components of the immune response is often suggested as a possible cause. Alveolar macrophages are found in the alveoli of the lung and represent the first line of defense to many airborne pathogens. To determine whether alveolar macrophages play a role in HPS pathogenesis, alveolar macrophages were depleted in an adult rodent model of HPS that closely resembles human HPS. Syrian hamsters were treated, intratracheally, with clodronate-encapsulated liposomes or control liposomes and were then challenged with ANDV. Treatment with clodronate-encapsulated liposomes resulted in significant reduction in alveolar macrophages, but depletion did not prevent pathogenesis or prolong disease. Depletion also did not significantly reduce the amount of virus in the lung of ANDV-infected hamsters but altered neutrophil recruitment, MIP-1α and MIP-2 chemokine expression, and vascular endothelial growth factor (VEGF) levels in hamster bronchoalveolar lavage (BAL) fluid early after intranasal challenge. These data demonstrate that alveolar macrophages may play a limited protective role early after exposure to aerosolized ANDV but do not directly contribute to hantavirus disease pathogenesis in the hamster model of HPS.IMPORTANCEHantaviruses continue to cause disease worldwide for which there are no FDA-licensed vaccines, effective postexposure prophylactics, or therapeutics. Much of this can be attributed to a poor understanding of the mechanism of hantavirus disease pathogenesis. Hantavirus disease has long been considered an immune-mediated disease; however, by directly manipulating the Syrian hamster model, we continue to eliminate individual immune cell types. As the most numerous immune cells present in the respiratory tract, alveolar macrophages are poised to defend against hantavirus infection, but those antiviral responses may also contribute to hantavirus disease. Here, we demonstrate that, like in our prior T and B cell studies, alveolar macrophages neither prevent hantavirus infection nor cause hantavirus disease. While these studies reflect pathogenesis in the hamster model, they should help us rule out specific cell types and prompt us to consider other potential mechanisms of disease in an effort to improve the outcome of human HPS.

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Mechanisms regulating the mRNA levels of interleukin-5 and two other coordinately expressed lymphokines in the murine T lymphoma EL4.23

Blood ◽

10.1182/blood.v83.12.3620.3620 ◽

1994 ◽

Vol 83 (12) ◽

pp. 3620-3628 ◽

Cited By ~ 29

Author(s):

H Naora ◽

IG Young

Keyword(s):

Gene Transcription ◽

De Novo ◽

Mrna Levels ◽

Interleukin 5 ◽

Mrna Stabilization ◽

Gm Csf ◽

Mrna Induction ◽

Run Off ◽

T Lymphoma ◽

Macrophage Colony

Abstract The mechanisms that regulate the mRNA levels of interleukin-5 (IL-5) were compared with those regulating the mRNA levels of two other coordinately expressed lymphokines in the murine T lymphoma EL4.23. Our results indicate that IL-5 mRNA levels are independently regulated from those of IL-2 and granulocyte-macrophage colony-stimulating factor (GM- CSF) mRNAs. The induction of IL-5 mRNA by phorbol 12-myristate 13- acetate (PMA) stimulation was found to be cyclosporin A-resistant, in contrast to the induction of IL-2 and GM-CSF mRNAs. Although the three lymphokine mRNAs were not detected in unstimulated cells by Northern blot analysis, the GM-CSF gene was found by nuclear run-off analysis to be constitutively transcribed. However, the IL-2 and IL-5 genes were transcriptionally inactive in the absence of PMA stimulation. The induction of IL-5 mRNA by PMA stimulation primarily involved increased transcriptional activity. In contrast, GM-CSF mRNA induction predominantly involved enhanced mRNA stability. Both transcriptional and mRNA stabilization mechanisms appeared to regulate IL-2 mRNA induction. The activation of IL-2 and IL-5 gene transcription was dependent on de novo protein synthesis. Cellular treatment with cycloheximide enhanced IL-2 gene transcription once activation was initiated, implicating the involvement of a labile repressor(s). Furthermore, IL-5 mRNA was more stable than IL-2 and GM-CSF mRNAs. These latter two species were stabilized by cycloheximide, suggesting that a labile mechanism may regulate their degradation.

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GM-CSF primes cardiac inflammation in a mouse model of Kawasaki disease

Journal of Experimental Medicine ◽

10.1084/jem.20151853 ◽

2016 ◽

Vol 213 (10) ◽

pp. 1983-1998 ◽

Cited By ~ 36

Author(s):

Angus T. Stock ◽

Jacinta A. Hansen ◽

Matthew A. Sleeman ◽

Brent S. McKenzie ◽

Ian P. Wicks

Keyword(s):

Kawasaki Disease ◽

Mouse Model ◽

Immune Cell ◽

Cardiac Fibroblasts ◽

Developed Countries ◽

Water Soluble ◽

Cardiac Inflammation ◽

Gm Csf ◽

Cytokines And Chemokines ◽

Macrophage Colony

Kawasaki disease (KD) is the leading cause of pediatric heart disease in developed countries. KD patients develop cardiac inflammation, characterized by an early infiltrate of neutrophils and monocytes that precipitates coronary arteritis. Although the early inflammatory processes are linked to cardiac pathology, the factors that regulate cardiac inflammation and immune cell recruitment to the heart remain obscure. In this study, using a mouse model of KD (induced by a cell wall Candida albicans water-soluble fraction [CAWS]), we identify an essential role for granulocyte/macrophage colony-stimulating factor (GM-CSF) in orchestrating these events. GM-CSF is rapidly produced by cardiac fibroblasts after CAWS challenge, precipitating cardiac inflammation. Mechanistically, GM-CSF acts upon the local macrophage compartment, driving the expression of inflammatory cytokines and chemokines, whereas therapeutically, GM-CSF blockade markedly reduces cardiac disease. Our findings describe a novel role for GM-CSF as an essential initiating cytokine in cardiac inflammation and implicate GM-CSF as a potential target for therapeutic intervention in KD.

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Phase II trial of pembrolizumab (PEM) plus granulocyte macrophage colony stimulating factor (GM-CSF) in advanced biliary cancers (ABC).

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.4_suppl.386 ◽

2018 ◽

Vol 36 (4_suppl) ◽

pp. 386-386 ◽

Cited By ~ 3

Author(s):

Robin Kate Kelley ◽

Emily Mitchell ◽

Spencer Behr ◽

Jimmy Hwang ◽

Bridget Keenan ◽

...

Keyword(s):

Immune Cell ◽

Progression Free Survival ◽

Immune Effector ◽

Effector Cells ◽

Gm Csf ◽

Phase 2 Trial ◽

Best Response ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stage 1

386 Background: The efficacy of immune checkpoint inhibition (CPI) has not been established in ABC. GM-CSF modulates immune effector cells and has demonstrated safety and improved survival (OS) in combination with ipilimumab in melanoma. This phase 2 trial aims to evaluate the efficacy and safety of PEM in combination with GM-CSF in ABC. Methods: Design: Simon’s 2-stage. Key eligibility: ABC with progression/intolerance on ≥ 1 standard therapy, no prior CPI, bilirubin ≤1.5xULN. Treatment: PEM 200 mg IV Q21 days plus 2 cycles of GM-CSF 250 µg SC D1-14 Q21 days in cycles 2 and 3 (Stage 1 Safety Cohort) or in cycles 1 and 2 (Stage 2). Endpoints: 1◦: Progression-free survival at 6 months (PFS6) with H0 25% vs. H1 50%. Key 2◦: Safety, overall response rate (ORR) and duration (DOR), OS, PD-L1 expression. Exploratory: PBMC and tumor immune cell profiles, tumor genotype, microsatellite (in)stability (MSI or MSS). Results: Accrual has completed with 27 patients (pts) enrolled 5/2016-6/2017: F/M 13/14; median age 61 (range 37-77); intrahepatic 19 (70%), extrahepatic 7 (26%), mixed 1 (4%) cholangiocarcinoma; stage IVA/B 85%, II/III 15%; median prior therapies 2 (range 1-6). Adverse events (AE): Related grade(Gr) ≥3 AE occurred in 4/27 (15%) pts including immune-related (ir)AE of Gr4 diabetes mellitus and Gr3 thrombocytopenia in 1 pt each. Gr≤2 irAE in ≥5% were: arthralgia (33%), dry eye/mouth (15%), hyperthyroid/thyroiditis (15%), hypothyroid (15%), neuropathy (11%), rash (11%), and adrenal insufficiency (7%). Steroids were required in 3/27 (11%) pts. Disposition: 19 pts removed for PD, 1 for Gr2 irAE; 7 pts remain active on treatment. Median time on treatment: 6 cycles (range 2-22+). Best response by RECIST 1.1: Partial response (PR) in 5/24 (21%) evaluable pts (1 MSI, 4 MSS); minor regression and ≥50% CA 19-9 decline in 2 additional MSS pts for 11+ and 16+ months. PBMC analyses show changes in expression of activating and inhibitory markers including PD-1 on various immune cell populations. Conclusions: PEM plus induction GM-CSF is safe and tolerable in ABC. Durable radiographic and tumor marker responses including MSS pts warrant further study. PFS6, OS, and correlative analyses are ongoing. Clinical trial information: NCT02703714.

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Growth factor-dependent inhibition of normal hematopoiesis by N-ras antisense oligodeoxynucleotides.

Journal of Experimental Medicine ◽

10.1084/jem.175.3.743 ◽

1992 ◽

Vol 175 (3) ◽

pp. 743-750 ◽

Cited By ~ 26

Author(s):

T Skorski ◽

C Szczylik ◽

M Z Ratajczak ◽

L Malaguarnera ◽

A M Gewirtz ◽

...

Keyword(s):

Cell Types ◽

Antisense Oligodeoxynucleotides ◽

Antisense Oligodeoxynucleotide ◽

Semisolid Medium ◽

Gm Csf ◽

Cd34 Cells ◽

Dependent Inhibition ◽

Normal Human ◽

Macrophage Colony

To determine whether N-ras expression is required at specific stages of the process of in vitro normal human hematopoiesis, adherent- and T lymphocyte-depleted mononuclear marrow cells (A-T-MNC) or highly purified progenitors (CD34+ cells) were cultured in semisolid medium, under conditions that favor the growth of specific progenitor cell types, after exposure to N-ras sense and antisense oligodeoxynucleotides. N-ras antisense, but not sense, oligodeoxynucleotide treatment of A-T-MNC and CD34+ cells resulted in a significantly decreased number of granulocyte/macrophage colony-forming units (CFU-GM) induced by interleukin 3 (IL-3) or granulocyte/macrophage colony-stimulating factor (GM-CSF) and of macrophage colonies (CFU-M) induced by M-CSF, but not of granulocytic colonies induced with G-CSF or IL-5. However, the same treatment significantly inhibited colony formation induced by each of the above factors in combination with IL-3. Megakaryocytic colony (CFU-Meg) formation from A-T-MNC or CD34+ cells in the presence of IL-6 + IL-3 + erythropoietin (Epo) was also markedly decreased after antisense oligodeoxynucleotide treatment. Erythroid colonies derived from A-T-MNC in the presence of Epo (CFU-E) were not inhibited upon antisense treatment, whereas those arising from A-T-MNC or CD34+ cells in the presence of IL-3 + Epo (BFU-E) were markedly affected. These results are consistent with the hypothesis that distinct signal transduction pathways, involving N-ras or not, are activated by different growth factors in different hematopoietic progenitor cells.

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