Granulocyte-macrophage Colony-stimulating Factor Deficiency Research Articles

Recombinant GM-CSF for diseases of GM-CSF insufficiency: Correcting dysfunctional mononuclear phagocyte disorders.

Endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF), identified by its ability to support differentiation of hematopoietic cells into several types of myeloid cells, is now known to support maturation and maintain the metabolic capacity of mononuclear phagocytes including monocytes, macrophages, and dendritic cells. These cells sense and attack potential pathogens, present antigens to adaptive immune cells, and recruit other immune cells. Recombinant human (rhu) GM-CSF (e.g., sargramostim [glycosylated, yeast-derived rhu GM-CSF]) has immune modulating properties and can restore the normal function of mononuclear phagocytes rendered dysfunctional by deficient or insufficient endogenous GM-CSF. We reviewed the emerging biologic and cellular effects of GM-CSF. Experts in clinical disease areas caused by deficient or insufficient endogenous GM-CSF examined the role of GM-CSF in mononuclear phagocyte disorders including autoimmune pulmonary alveolar proteinosis (aPAP), diverse infections (including COVID-19), wound healing, and anti-cancer immune checkpoint inhibitor therapy. We discuss emerging data for GM-CSF biology including the positive effects on mitochondrial function and cell metabolism, augmentation of phagocytosis and efferocytosis, and immune cell modulation. We further address how giving exogenous rhu GM-CSF may control or treat mononuclear phagocyte dysfunction disorders caused or exacerbated by GM-CSF deficiency or insufficiency. We discuss how rhu GM-CSF may augment the anti-cancer effects of immune checkpoint inhibitor immunotherapy as well as ameliorate immune-related adverse events. We identify research gaps, opportunities, and the concept that rhu GM-CSF, by supporting and restoring the metabolic capacity and function of mononuclear phagocytes, can have significant therapeutic effects. rhu GM-CSF (e.g., sargramostim) might ameliorate multiple diseases of GM-CSF deficiency or insufficiency and address a high unmet medical need.

Pathogenic Tconvs promote inflammatory macrophage polarization through GM-CSF and exacerbate abdominal aortic aneurysm formation.

Abdominal aortic aneurysms (AAAs) elicit massive inflammatory leukocyte recruitment to the aorta. CD4+ T cells, which include regulatory T cells (Tregs) and conventional T cells (Tconvs), are involved in the progression of AAA. Tregs have been reported to limit AAA formation. However, the function and phenotype of the Tconvs found in AAAs remain poorly understood. We characterized aortic Tconvs by bulk RNA sequencing and discovered that Tconvs in aortic aneurysm highly expressed Cxcr6 and Csf2. Herein, we determined that the CXCR6/CXCL16 signaling axis controlled the recruitment of Tconvs to aortic aneurysms. Deficiency of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), encoded by Csf2, markedly inhibited AAA formation and led to a decrease of inflammatory monocytes, due to a reduction of CCL2 expression. Conversely, the exogenous administration of GM‐CSF exacerbated inflammatory monocyte infiltration by upregulating CCL2 expression, resulting in worsened AAA formation. Mechanistically, GM‐CSF upregulated the expression of interferon regulatory factor 5 to promote M1‐like macrophage differentiation in aortic aneurysms. Importantly, we also demonstrated that the GM‐CSF produced by Tconvs enhanced the polarization of M1‐like macrophages and exacerbated AAA formation. Our findings revealed that GM‐CSF, which was predominantly derived from Tconvs in aortic aneurysms, played a pathogenic role in the progression of AAAs and may represent a potential target for AAA treatment.

Macrophage Deficiency Makes Intestinal Epithelial Cells Susceptible to NSAID-Induced Damage

Objectives In Crohn's disease (CD), the mechanisms underlying the regulation by granulocyte-macrophage colony-stimulating factor (GM-CSF) of mucosal barrier function in the ileum are unclear. We analyzed the molecular mechanisms underlying the regulation by GM-CSF of the mucosal barrier function. Methods We examined the role of GM-CSF in the intestinal barrier function in CD at the molecular-, cellular-, and animal-model levels. Results Macrophages directly secreted GM-CSF, promoting intestinal epithelial proliferation and inhibiting apoptosis, which maintained intestinal barrier function. Macrophages were absent in NSAID-induced ileitis, causing GM-CSF deficiency, increasing the apoptosis rate, decreasing the proliferation rate, increasing inter- and paracellular permeabilities, decreasing the TJP levels, and reducing the numbers of mesenteric lymph nodes, memory T cells, and regulatory T cells in Csf1op/op transgenic mice. Conclusions GM-CSF is required for the maintenance of intestinal barrier function. Macrophages directly secrete GM-CSF, promoting intestinal epithelial proliferation and inhibiting apoptosis.

PULMONARY ALVEOLAR PROTEINOSIS PRESENTING AS INTRACEREBRAL NOCARDIOSIS

SESSION TITLE: Diffuse Lung Disease SESSION TYPE: Med Student/Res Case Report PRESENTED ON: 10/09/2018 07:30 am - 08:30 am INTRODUCTION: Pulmonary alveolar proteinosis (PAP) is a rare disorder with an incidence of 0.5 / million people [1]. PAP is caused by the accumulation of surfactant in the alveoli, leading to hypoxemic respiratory failure and increased risk of opportunistic infections [2]. First-line therapy after infection eradication is whole-lung lavage. Treatment with exogenous granulocyte macrophage colony stimulating factor (GM-CSF) is under investigation. PAP can be a diagnostic challenge due to its low prevalence and nonspecific presentation. Here we describe a unique presentation that was diagnosed as PAP. CASE PRESENTATION: A 56 year old male farmer, never smoker, with no past medical history presented with right-sided weakness, two months of dyspnea, and dry cough. Brain MRI demonstrated multiple cerebral rim-enhancing abscesses (Fig 1A). He underwent a craniotomy and evacuation of cerebral abscesses with acid fast cultures positive for Nocardia farcinica. Chest CT showed a "crazy paving" pattern consistent with PAP (Fig 1B, C). Serum anti GM-CSF antibody was positive. Bronchoscopy showed opaque fluid on bronchoalveolar lavage, with negative cultures, and clumps of amorphous proteinaceous material consistent with PAP. Disseminated Nocardia was treated with ciprofloxacin for 1 year and sulfamethoxazole/ trimethoprim for 6 months. He underwent a whole lung lavage with resolution of symptoms. DISCUSSION: PAP is caused by surfactant accumulation due to incomplete removal by alveolar macrophages. Autoimmune PAP (primary PAP) accounts for the majority of cases [1]. Autoimmune PAP is caused by anti GM-CSF antibodies that disrupt macrophage activation, antigen presentation, and phagocytosis [2]. Secondary PAP is thought due to a relative deficiency of GM-CSF usually in the setting of hematologic diseases or dust exposures. The ensuing macrophage dysfunction leads to increased infection risk. Opportunistic infections including Nocardia, Mycobacterium, and fungi in otherwise immunocompetent patients with PAP have been previously reported [2]. In a report of 7 patients with disseminated Nocardiosis without PAP, 5 patients were found to have anti GM-CSF antibodies; two of these patients were also found to have radiographic characteristics of PAP [3]. CONCLUSIONS: Primary PAP is a rare autoimmune disorder that confers an immunocompromised state due to impaired alveolar macrophage activation. Opportunistic infections such as Nocardiosis are frequently seen with PAP. Eradication of the opportunistic infections, and targeted whole lung lavage are effective at relieving hypoxia in PAP. A high diagnostic suspicion is essential in the work-up of PAP. Reference #1: Clinics in Chest Medicine. 2016;37(3):431-440. https://doi.org/10.1016/j.ccm.2016.04.006 Reference #2: Journal of Infectio. 2012;65(2):173-179. https://doi.org/10.1016/j.jinf.2012.03.020 Reference #3: Clinical Infectious Disease. 2014;60(7):1017-1025. https://doi.org/10.1093/cid/ciu968 DISCLOSURES: No relevant relationships by David Dennis, source=Web Response No relevant relationships by Ulysses Magalang, source=Web Response No relevant relationships by Virgil Secasanu, source=Web Response

Better approach for autoimmune pulmonary alveolar proteinosis treatment: inhaled or subcutaneous granulocyte-macrophage colony-stimulating factor: a meta-analyses

BackgroundAutoimmune pulmonary alveolar proteinosis (aPAP) is a rare pulmonary disease caused by functional deficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF therapy in aPAP has been reported effective in some studies. This meta-analyses aimed to evaluate whether GM-CSF therapy, including inhaled and subcutaneous GM-CSF have therapeutic effect in aPAP patients.MethodsWe analyzed 10 studies searched from PubMed, EmBase, Web of Science, Wiley Online Library and Cochrane Collaboration databases to evaluate the pooled effects of GM-CSF treatment in aPAP patients.ResultsTen observational studies involving 115 aPAP patients were included. The pooled analyses of response rate (81%, p < 0.001), relapse rate (22%, p = 0.009), PaO2 (13.76 mmHg, p < 0.001) and P(A-a)O2 (19.44 mmHg, p < 0.001) showed that GM-CSF treatment was effective on aPAP patients. Further analyses showed that inhaled GM-CSF treatment was more effective than subcutaneous GM-CSF therapy, including a higher response rate (89% vs. 71%, p = 0.023), more improvements in PaO2 (21.02 mmHg vs. 8.28 mmHg, p < 0.001) and P(A-a)O2 (19.63 mmHg vs. 9.15 mmHg, p < 0.001).ConclusionsAs two routes of exogenous GM-CSF treatment, inhaled and subcutaneous were both proven to have effect on aPAP patients. Furthermore, inhaled GM-CSF therapy showed a higher response rate, more improvements on PaO2 and P(A-a)O2 than subcutaneous GM-CSF treatment in aPAP patients, suggesting inhaled GM-CSF therapy could have more benefits on aPAP patients. Therefore, GM-CSF therapy, especially inhaled GM-CSF, might be a promising therapeutic option in treating aPAP.

Granulocyte–macrophage colony-stimulating factor: not just another haematopoietic growth factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is often used to treat leucopenia. Other haematopoietins may increase the number of circulating leucocytes with higher efficiency, but GM-CSF has additional effects that may be far more relevant than its haematopoietic activity. GM-CSF induces differentiation, proliferation and activation of macrophages and dendritic cells which are necessary for the subsequent T helper cell type 1 and cytotoxic T lymphocyte activation. GM-CSF haematopoietic and non-haematopoietic functions have pro-inflammatory and immune regulatory potential to treat a variety of autoimmune diseases and tumours. On the other hand, GM-CSF deficiency leads to various immune dysfunctions and the current utilization of GM-CSF as haematopoietic factor might be an accurate but very incomplete indication for a cytokine with vast clinical potential.

Effectiveness of Granulocyte-Macrophage Colony-Stimulating Factor Therapy in Autoimmune Pulmonary Alveolar Proteinosis: A Meta-analysis of Observational Studies

Autoimmune pulmonary alveolar proteinosis (aPAP) is a rare pulmonary disease caused by functional deficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF). Administration of GM-CSF represents a potential therapeutic strategy in management of aPAP. Herein, we systematically review the efficacy of GM-CSF therapy in aPAP.We searched the PubMed and EmBase databases for studies reporting the use of GM-CSF in aPAP. We calculated the proportion with 95% CI to assess the response and relapse rates of GM-CSF therapy in individual studies and pooled them using a random-effects model. Statistical heterogeneity was assessed using the I(2) and Cochran Q tests. Publication bias was analyzed using funnel plot and Egger and Begg-Mazumdar tests.Our initial searches yielded 1,585 studies. Of these, five observational studies (involving 94 patients) were included for analysis. Three studies used the subcutaneous route, and two studies used the inhalational route for GM-CSF administration. The response rate of GM-CSF varied from 43% to 92%, with the pooled response rate being 58.6% (95% CI, 42.7-72.9). The relapse rate in GM-CSF responders was 29.7% (95% CI, 10.5-60.4). There was no evidence of statistical heterogeneity or publication bias for the outcome of response. GM-CSF therapy was associated with minor complications, such as fever and local complications at the site of administration.GM-CSF represents a useful approach in the treatment of aPAP. The optimal indication, dose and duration of therapy, and the factors predicting response and relapse need to be defined by future studies.

Lentivirus-ABCG1 instillation reduces lipid accumulation and improves lung compliance in GM-CSF knock-out mice

We have shown decreased expression of the nuclear transcription factor, peroxisome proliferator-activated receptor-gamma (PPARγ) and the PPARγ-regulated ATP-binding cassette transporter G1 (ABCG1) in alveolar macrophages from patients with pulmonary alveolar proteinosis (PAP). PAP patients also exhibit neutralizing antibodies to granulocyte–macrophage colony stimulating factor (GM-CSF), an upregulator of PPARγ. In association with functional GM-CSF deficiency, PAP lung is characterized by surfactant-filled alveolar spaces and lipid-filled alveolar macrophages. Similar pathology characterizes GM-CSF knock-out (KO) mice. We reported previously that intratracheal instillation of a lentivirus (lenti)-PPARγ plasmid into GM-CSF KO animals elevated ABCG1 and reduced alveolar macrophage lipid accumulation. Here, we hypothesized that instillation of lenti-ABCG1 might be sufficient to decrease lipid accumulation and improve pulmonary function in GM-CSF KO mice. Animals received intratracheal instillation of lenti-ABCG1 or control lenti-enhanced Green Fluorescent Protein (eGFP) plasmids and alveolar macrophages were harvested 10days later. Alveolar macrophage transduction efficiency was 79% as shown by lenti-eGFP fluorescence. Quantitative PCR analyses indicated a threefold (p=0.0005) increase in ABCG1 expression with no change of PPARγ or ABCA1 in alveolar macrophages of lenti-ABCG1 treated mice. ABCG1 was unchanged in control lenti-eGFP and PBS-instilled groups. Oil Red O staining detected reduced intracellular neutral lipid in alveolar macrophages from lenti-ABCG1 treated mice. Extracellular cholesterol and phospholipids were also decreased as shown by analysis of bronchoalveolar lavage fluid. Lung compliance was diminished in untreated GMCSF KO mice but improved significantly after lenti-ABCG1 treatment. Data demonstrate that in vivo instillation of lenti-ABCG1 in GM-CSF KO mice is sufficient to restore pulmonary homeostasis by: (1) upregulating ABCG1; (2) reducing intra and extracellular lipids; and (3) improving lung function. Results suggest that the ABCG1 lipid transporter is the key downstream target of GM-CSF-induced PPARγ necessary for surfactant catabolism.

Pulmonary Alveolar Proteinosis

Pulmonary alveolar proteinosis is a rare but potentially treatable disease, characterized by impaired surfactant metabolism that leads to accumulation in the alveoli of proteinaceous material rich in surfactant protein and its component. Novel insights from an animal model aided the discovery of granulocyte macrophage colony stimulating factor (GM-CSF) antibodies as a pathogenetic mechanism in human pulmonary alveolar proteinosis. The vast majority of pulmonary alveolar proteinosis occurs as an autoimmune disease; less commonly, it is congenital or secondary to an underlying disorder such as infection, hematological malignancy, or immunodeficiency. The subacute indolent course of this disease often delays the diagnosis by months to years. Crazy-paving appearance in a geographic distribution is a characteristic feature of this disease visible on high-resolution computed tomography (CT). A definitive diagnosis, however, requires lung biopsy, which typically shows partial or complete filling of alveoli with periodic-acid-Schiff-positive granular and eosinophilic material in preserved alveolar architecture. Patients with minimal symptoms are managed conservatively, whereas patients with hypoxemia require a more aggressive approach. Whole-lung lavage is the most widely accepted therapy for symptomatic pulmonary alveolar proteinosis. Correction of GM-CSF deficiency with exogenous GM-CSF is an alternative therapy. The combination of a systemic treatment (GM-CSF) and a local treatment (whole-lung lavage) augmenting the action of one another is a promising new approach. As the knowledge about this rare disease increases, the role of novel therapies is likely to be better defined and optimized.

Alveolar proteinosis syndrome: pathogenesis, diagnosis, and management

This review discusses the most recent clinical and basic research literature on pulmonary alveolar proteinosis (PAP) as it relates to pathogenesis, diagnosis, and management. The discovery of Granulocyte macrophage-colony stimulating factor (GM-CSF) and the alveolar macrophage as critical regulators of surfactant protein and lipid homeostasis has led to significant advances in PAP. Adults affected by PAP have circulating neutralizing anti-GM-CSF antibodies. Reduced localized GM-CSF activity in the lung (from neutralizing anti-GM-CSF antibodies), decreases alveolar macrophage surfactant degradation with surfactant excess and accumulation. Cause, source of antibodies or downstream effects of GM-CSF deficiency is speculative. GM-CSF antibodies above a threshold level have proved to be a useful diagnostic test. Research towards therapy has focused on improving the technique for therapeutic whole lung lavage as well as overcoming effects of neutralizing anti-GM-CSF, which include GM-CSF therapy (systemic and inhaled) and anecdotal reports of anti-B cell therapy. Whereas this approach has been somewhat successful for primary PAP, other causes of PAP (i.e. alveolar macrophage dysfunction, surfactant protein alterations) are still without therapy. Understanding of the pathogenesis of PAP has greatly increased in the last decade; study has brought better comprehension of lung biology and recognition of the critical role for GM-CSF and alveolar macrophage in surfactant clearance. Balance between resident immune cell population and normal lung function still needs further study. Resident alveolar macrophages have an essential role in surfactant homeostasis. With this knowledge more effective diagnostic tests (e.g. anti-GM-CSF antibody) and therapies for PAP are under investigation.

Pulmonary Alveolar Proteinosis: A Bench-to-Bedside Story of Granulocyte-Macrophage Colony-Stimulating Factor Dysfunction

Pulmonary alveolar proteinosis (PAP) is a rare disorder characterized by ineffective clearance of surfactant by alveolar macrophages. Through recent studies with genetically altered mice, the etiology of this idiopathic disease is becoming clearer. Functional deficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF) appears to contribute to disease pathogenesis because mutant mice deficient in GM-CSF or its receptor spontaneously develop PAP. Recent human studies further suggest a connection between PAP and defective GM-CSF activity because inactivating anti-GM-CSF autoantibodies are observed in all patients with idiopathic PAP, and additional rare cases of PAP in children have been accompanied by genetic defects in the alpha chain of the GM-CSF receptor. In patients and mouse models of PAP, deficient GM-CSF activity appears to result in defective alveolar macrophages that are unable to maintain pulmonary surfactant homeostasis and display defective phagocytic and antigen-presenting capabilities. The most recent studies also suggest that neutrophil dysfunction additionally contributes to the increased susceptibility to lung infections seen in PAP. Because the phenotypic and immunologic abnormalities of PAP in mouse models can be corrected by GM-CSF reconstituting therapies, early clinical trials are underway utilizing administration of GM-CSF to potentially treat human PAP. The development of novel treatment approaches for PAP represents a dramatic illustration in pulmonary medicine of the "bench-to-bedside" process, in which basic scientists, translational researchers, and clinicians have joined together to rapidly take advantage of the unexpected observations frequently made in the modern molecular biology research laboratory.

125. REGULATION OF STRESS PROTEIN GENES DURING PRE-IMPLANTATION EMBRYO DEVELOPMENT IN MICE BY GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF)

In vitro culture has been shown to be detrimental for pre-implantation embryo development and this has been associated with culture stress and elevated expression of apoptotic genes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to promote development and survival of both human and mouse pre-implantation embryos. To investigate the mechanism of action of GM-CSF in mouse embryos, gene expression was examined in in vitro cultured blastocysts with and without recombinant mouse GM-CSF (rmGM-CSF) and in vivo blastocysts flushed from Csf2 null mutant and wild-type mice. Microarray analysis of the effect of GM-CSF on transcription profile implicated apoptosis and stress response gene pathways in blastocyst responses to rmGM-CSF in vitro. Groups of 30 blastocysts were collected from in vitro cultured and in vivo developed blastocyst were analysed using quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR analysis of in vitro blastocysts revealed that addition of rmGM-CSF causes differential expression of several genes associated with apoptosis and cellular stress pathway, including Cbl, Hspa5, Hsp90aa1, Hsp90ab1 and Gas5. Immunocytochemical analysis of common proteins of the apoptosis and cellular stress response pathways BAX, BCL2, TRP53 (p53) and HSPA1A/1B (Hsp70) in in vitro blastocysts revealed that HSPA1A/1B and BCL2 proteins were less abundant in embryos cultured in rmGM-CSF, but BAX and TRP53 were unchanged. In in vivo developed blastocysts, Csf2 null mutation resulted in elevated levels of only the heat shock protein Hsph1, suggesting that in vivo, other cytokines can compensate for GM-CSF deficiency as the absence of GM-CSF has a lesser effect on the stress response pathway. We conclude that GM-CSF is a regulator of the apoptosis and cellular stress response pathways influencing mouse pre-implantation embryo development to facilitate embryo growth and survival, and the effects of GM-CSF are particularly evident in in vitro culture media in the absence of other cytokines.

GM‐CSF deficiency delays neointima formation in a normolipidemic mouse model of endoluminal endothelial damage

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been implicated in atherogenesis and has been shown to have both pro- and antiatherogenic properties. Neointimal thickening is a prominent feature of early atherogenesis. This study aimed to examine the role of GM-CSF in neointimal formation induced by endothelial injury using a GM-CSF(-/-) mouse model. Neointimal thickening was induced by endothelial damage in the common iliac arteries of normolipidemic C57Bl/6 (wild-type) and GM-CSF(-/-) mice. Arteries were collected weekly for 3-7 weeks following surgery. A significant delay in neointimal formation in the GM-CSF(-/-) compared with wild-type mice was detected by morphometric analysis of the intimal area. Neointimal size was approximately 10% smaller in GM-CSF(-/-) mice at 4-6 weeks post-surgery, compared with wild-type mice. The neointima was composed predominantly of smooth muscle cells and there was no difference in the extent of endothelial cell coverage between the wild-type and GM-CSF(-/-) mice. Using immunohistochemistry, reduced macrophages (F4/80(+) cells), proliferating cells (proliferating cell nuclear antigen (PCNA)(+) cells) and platelet-derived growth factor-B were detected within the arteries of GM-CSF(-/-) mice compared with wild types at 4 weeks post-surgery. GM-CSF(-/-) mice had reduced connective tissue within the neointima compared with wild types at 5 weeks post-surgery, determined by trichrome staining. We conclude that GM-CSF deficiency reduces neointimal formation in a normolipidemic model, primarily due to reduced macrophage recruitment.

Lysyl Oxidase Inhibition Is Responsible for the Vascular Elastic Fiber Phenotype

To the Editor: In the July issue of Hypertension , Mercier et al1 demonstrated a rat model in which the structural vascular phenotype is modified in response to amine oxidase inhibition by semicarbazide. Under these conditions, the elastic lamellae presented globular masses along their periphery, and focal disorganization was observed in the ascending aorta. Immunoconfocal microscopy demonstrated branching of elastic lamellae and a sparse hairy aspect of some elastic fibers after amine oxidase inhibition. Because the above-described abnormalities are not found in semicarbazide-sensitive amine oxidase–deficient mice, the authors conclude …

ABCG1 is deficient in alveolar macrophages of GM-CSF knockout mice and patients with pulmonary alveolar proteinosis

Patients with pulmonary alveolar proteinosis (PAP) display impaired surfactant clearance, foamy, lipid-filled alveolar macrophages, and increased cholesterol metabolites within the lung. Neutralizing autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF) are also present, resulting in virtual GM-CSF deficiency. We investigated ABCG1 and ABCA1 expression in alveolar macrophages of PAP patients and GM-CSF knockout (KO) mice, which exhibit PAP-like pulmonary pathology and increased pulmonary cholesterol. Alveolar macrophages from both sources displayed a striking similarity in transporter gene dysregulation, consisting of deficient ABCG1 accompanied by highly increased ABCA1. Peroxisome proliferator-activated receptor gamma (PPARgamma), a known regulator of both transporters, was deficient, as reported previously. In contrast, the liver X receptor alpha, which also upregulates both transporters, was highly increased. GM-CSF treatment increased ABCG1 expression in macrophages in vitro and in PAP patients in vivo. Overexpression of PPARgamma by lentivirus-PPARgamma transduction of primary alveolar macrophages, or activation by rosiglitazone, also increased ABCG1 expression. These results suggest that ABCG1 deficiency in PAP and GM-CSF KO alveolar macrophages is attributable to the absence of a GM-CSF-mediated PPARgamma pathway. These findings document the existence of ABCG1 deficiency in human lung disease and highlight a critical role for ABCG1 in surfactant homeostasis.

Granulocyte Macrophage Colony-Stimulating Factor Deficiency Affects Vascular Elastin Production and Integrity of Elastic Lamellae

Background: Granulocyte macrophage colony-stimulating factor (GM-CSF) deficiency affects the production and fiber assembly/organization of the vascular collagenous matrix; structural alterations to the elastic system were observed. The present study elaborates the effect of GM-CSF deficiency on the vascular elastin system. Methods and Results: Histological examination of the aorta of GM-CSF-deficient mice revealed structurally altered elastic fibers. The elastic fiber area was significantly enhanced, whereas the remaining medial area was not affected. Aortic size was significantly increased. Reverse transcription polymerase chain reaction demonstrated decreased expression levels of tropoelastin, lysyl oxidase and bone morphogenetic protein 1 (BMP-1). Cell culture studies on vascular smooth muscle cells showed that after clearance of GM-CSF with GM-CSF antibodies, the tropoelastin mRNA expression was markedly reduced. Concomitantly, lysyl oxidase and BMP-1 mRNA levels were decreased. Treatment with GM-CSF stimulated the expression of these mRNAs. Conclusions: Our studies demonstrate that disorganization of elastic lamellae as induced by GM-CSF deficiency is associated with adaptive vascular remodeling. The decreased tropoelastin expression observed is associated with elastic fiber hypertrophy. This paradox effect may be explained by decreased expression levels of lysyl oxidase and BMP-1, both mediating cross-linkage and thus assembly and organization of elastic fibers. From our data, we conclude that GM-CSF is a prerequisite for the maintenance of structural integrity of the vessel wall.

GM-CSF regulation of embryo development and pregnancy

The reproductive tissues undergo profound structural changes and major immune adaptation to accommodate pregnancy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of an array of cytokines with pivotal roles in embryo implantation and subsequent development. Several cell lineages in the reproductive tract and gestational tissues synthesise GM-CSF under direction by ovarian steroid hormones and signalling agents originating in male seminal fluid and the conceptus. The pre-implantation embryo, invading placental trophoblast cells and the abundant populations of leukocytes controlling maternal immune tolerance are all subject to GM-CSF regulation. GM-CSF deficiency in pregnancy adversely impacts fetal and placental development, as well as progeny viability and growth after birth, highlighting this cytokine as a central maternal determinant of pregnancy outcome with clinical relevance in human fertility.

Positional specificity of lysosomal phospholipase A2

Lysosomal phospholipase A(2) (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A(1) and phospholipase A(2) activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.

Elevated BALF concentrations of α- and β-defensins in patients with pulmonary alveolar proteinosis

Defensins are endogenous antibiotics and regulators of inflammation, immunity and wound repair. Their concentrations are substantially increased in bronchoalveolar lavage fluid (BALF) of patients with infectious lung diseases. alpha-defensin (HAD) levels are also elevated in patients with idiopathic pulmonary fibrosis (IPF) and correlated with the decline in pulmonary function tests, suggesting the association of defensins with the pathogenesis of interstitial lung diseases. The aim of this study was to determine the profile of defensins in interstitial lung diseases. Serum and BALF levels of HAD and beta-defensin 1 and 2 (HBD-1, and -2) were measured by radioimmunoassay in 63 patients with interstitial lung diseases, including idiopathic pulmonary alveolar proteinosis (PAP), IPF, nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP) and pulmonary sarcoidosis, and in 9 healthy volunteers as controls. Levels of HAD in BALF of patients with PAP were significantly higher than those in controls and patients with COP and sarcoidosis. Serum levels of HAD in all groups were significantly higher than those in controls. Levels of HBD-1 and -2 in BALF of patients with PAP were extremely high in all subjects. Serum levels of HBD-1 were higher in all patient groups, with the exception of those with PAP, and those of HBD-2 were also higher in patients with IPF and sarcoidosis, compared with controls. BALF of PAP patients, but not IPF patients and controls, expressed antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. Our findings suggest different kinetics of HAD and HBD-1 and -2 in serum and BALF of interstitial lung diseases and that these antimicrobial peptides in the airway lumen may contribute to prevention of bacterial airway infections in PAP.

Granulocyte–Macrophage Colony-Stimulating Factor Is Essential for Normal Wound Healing

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent growth factor, which plays an important role during the process of wound healing. In clinical settings it has occasionally been employed in the treatment of cutaneous wounds of diverse etiologies. In a previous study, we have shown the positive influence of GM-CSF on full thickness excisional wounds in transgenic mice overexpressing GM-CSF in the basal layer of the epidermis. Direct GM-CSF action as well as indirect processes through the induction of secondary cytokines were proposed to contribute towards the beneficial effects. In this study, we analyzed the process of wound healing in transgenic mice overexpressing a GM-CSF antagonist in the epidermis. These mice not only exhibited a delayed scab rejection and reepithelialization but also neovascularization was reduced. The newly formed tissue was of poor quality as exhibited by the presence of extensive fibrosis. We suggest that the presence of GM-CSF in the repair process is of basic importance and its absence leads not only to delayed wound healing but it is also detrimental for the quality of the newly formed tissue.