Article

Suppression of activin A in autoimmune lung disease associated with anti-GM-CSF

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Abstract

Pulmonary alveolar proteinosis (PAP) is an autoimmune disorder characterized by neutralizing autoantibodies to granulocyte-macrophage colony stimulating factor (GM-CSF). Surfactant metabolism is severely dysregulated in PAP, resulting in a foam cell appearance of alveolar macrophages. Microarray analysis of RNA from PAP bronchoalveolar lavage (BAL) cells to explore autoimmune-related genes yielded evidence of a deficiency of activin A, a cytokine implicated in regulation of B-cell proliferation and reduction of foam cell formation. Subsequent studies confirmed a severe deficiency of activin A gene expression and protein secretion in PAP BAL cells and marked reduction of activin A protein in PAP BAL fluids compared to healthy controls. PAP cells, however, like those of healthy controls, were capable of elevated activin A production in response to GM-CSF. Treatment with activin A in vitro suppressed proliferation of PAP peripheral blood B-cells in a receptor-dependent manner and also reduced secretion of anti-GM-CSF autoantibody. These studies are the first to suggest that activin A may play a role in autoimmune disease.

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... In support, activin-A enhanced IgA secretion by mesenteric lymph node cells ex vivo, pointing to a role in the maintenance of gut homeostasis [38]. Of clinical relevance, activin-A inhibited phytohemagglutinin (PHA)-induced proliferation of B cells isolated from healthy individuals and patients with pulmonary alveolar proteinosis (PAP) and diminished the production of autoantibodies against GM-CSF [39]. ...
... In PAP, wherein autoantibodies neutralize GM-CSF activity and lead to dysregulated surfactant metabolism in the airways, loss of activin-A signaling was identified as a key event involved in disease pathogenesis. Studies in BAL samples demonstrated reduced activin-A levels in PAP patients, compared to HCs [39]. Moreover, PAP macrophages expressed reduced activin-A [39]. ...
... Studies in BAL samples demonstrated reduced activin-A levels in PAP patients, compared to HCs [39]. Moreover, PAP macrophages expressed reduced activin-A [39]. Importantly, activin-A stimulation inhibited the proliferation and autoantibody production from B cells of PAP patients [39] (Fig. 5). ...
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The TGF-β superfamily of cytokines plays pivotal roles in the regulation of immune responses protecting against or contributing to diseases, such as, allergy, autoimmunity and cancer. Activin-A, a member of the TGF-β superfamily, was initially identified as an inducer of follicle-stimulating hormone secretion. Extensive research over the past decades illuminated fundamental roles for activin-A in essential biologic processes, including embryonic development, stem cell maintenance and differentiation, haematopoiesis, cell proliferation and tissue fibrosis. Activin-A signals through two type I and two type II receptors which, upon ligand binding, activate their kinase activity, phosphorylate the SMAD2 and 3 intracellular signaling mediators that form a complex with SMAD4, translocate to the nucleus and activate or silence gene expression. Most immune cell types, including macrophages, dendritic cells (DCs), T and B lymphocytes and natural killer cells have the capacity to produce and respond to activin-A, although not in a similar manner. In innate immune cells, including macrophages, DCs and neutrophils, activin-A exerts a broad range of pro- or anti-inflammatory functions depending on the cell maturation and activation status and the spatiotemporal context. Activin-A also controls the differentiation and effector functions of Th cell subsets, including Th9 cells, TFH cells, Tr1 Treg cells and Foxp3+ Treg cells. Moreover, activin-A affects B cell responses, enhancing mucosal IgA secretion and inhibiting pathogenic autoantibody production. Interestingly, an array of preclinical and clinical studies has highlighted crucial functions of activin-A in the initiation, propagation and resolution of human diseases, including autoimmune diseases, such as, systemic lupus erythematosus, rheumatoid arthritis and pulmonary alveolar proteinosis, in allergic disorders, including allergic asthma and atopic dermatitis, in cancer and in microbial infections. Here, we provide an overview of the biology of activin-A and its signaling pathways, summarize recent studies pertinent to the role of activin-A in the modulation of inflammation and immunity, and discuss the potential of targeting activin-A as a novel therapeutic approach for the control of inflammatory diseases.
... Previously we reported that healthy human AMs synthesize activin A in response to GM-CSF but AMs of patients with PAP are deficient in activin A [16]. In addition, PAP AMs are deficient in the nuclear transcription factor, Peroxisome Proliferator-activated Receptor, (PPARγ), a regulator of lipid and glucose metabolism that is restored by GM-CSF treatment [17]. ...
... Because of the phenotypic similarities between human PAP and the GM-CSF knockout mouse, this study was undertaken to investigate activin A regulation in the lung. Initially, it was hypothesized that activin A might be impaired in GM-CSF knockout mice based upon previous data from PAP studies [16]. ...
... Activin A and IFNγ are intrinsically elevated in GM-CSF knockout mice as compared to wild-type mice Unlike previous findings of activin A deficiency in human PAP [16], activin A mRNA expression of BAL cells was significantly (p <0.005) elevated in GM-CSF knockout mice compared to wild-type controls ( Figure 1A). Quantification of activin A protein in BAL fluids confirmed mRNA findings with significantly (p < 0.05) elevated protein levels in GM-CSF knockout compared to wild-type ( Figure 1B of follistatin, an inhibitor of activin A [21], was similar to wild-type mice (data not shown) and thus could not account for the striking elevation of activin A. Intrinsic factors that could potentially affect activin A levels were subsequently investigated in GM-CSF knockout mice. ...
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Activin A is a pleiotrophic regulatory cytokine, the ablation of which is neonatal lethal. Healthy human alveolar macrophages (AMs) constitutively express activin A, but AMs of patients with pulmonary alveolar proteinosis (PAP) are deficient in activin A. PAP is an autoimmune lung disease characterized by neutralizing autoantibodies to Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF). Activin A can be stimulated, however, by GM-CSF treatment of AMs in vitro. To further explore pulmonary activin A regulation, we examined AMs in bronchoalveolar lavage (BAL) from wild-type C57BL/6 compared to GM-CSF knockout mice which exhibit a PAP-like histopathology. Both human PAP and mouse GM-CSF knockout AMs are deficient in the transcription factor, peroxisome proliferator activated receptor gamma (PPARgamma). In sharp contrast to human PAP, activin A mRNA was elevated in mouse GM-CSF knockout AMs, and activin A protein was increased in BAL fluid. Investigation of potential causative factors for activin A upregulation revealed intrinsic overexpression of IFNgamma, a potent inducer of the M1 macrophage phenotype, in GM-CSF knockout BAL cells. IFNgamma mRNA was not elevated in PAP BAL cells. In vitro studies confirmed that IFNgamma stimulated activin A in wild-type AMs while antibody to IFNgamma reduced activin A in GM-CSF knockout AMs. Both IFNgamma and Activin A were also reduced in GM-CSF knockout mice in vivo after intratracheal instillation of lentivirus-PPARgamma compared to control lentivirus vector. Examination of other M1 markers in GM-CSF knockout mice indicated intrinsic elevation of the IFNgamma-regulated gene, inducible Nitrogen Oxide Synthetase (iNOS), CCL5, and interleukin (IL)-6 compared to wild-type. The M2 markers, IL-10 and CCL2 were also intrinsically elevated. Data point to IFNgamma as the primary upregulator of activin A in GM-CSF knockout mice which in addition, exhibit a unique mix of M1-M2 macrophage phenotypes.
... Mouse A-MØ exhibit much higher expression of PPARγ than other macrophages in the steady-state (34), and its GM-CSF-dependent expression is essential for their differentiation and maturation from fetal monocytes (24). In fact, PPARγ expression in A-MØ is lost in GM-CSF-deficient mice and in patients with pulmonary alveolar proteinosis (PAP), a pathology derived from a defective expression or activity of GM-CSF (23,41) and associated with suppressed activin A expression (42). However, it is currently unknown whether PPARγ is required for maintenance of A-MØ throughout adult life (43). ...
... Assessment of the function of PPARγ in human macrophages has now revealed that (1) the range of PPARγ target genes differs between proinflammatory (GM-MØ) and anti-inflammatory (M-MØ) monocyte-derived macrophages; (2) PPARG1/3 and PPARG2 are preferentially expressed by human GM-MØ; (3) activin A mediates the GM-CSF-induced expression of PPARγ in in vitro generated GM-MØ and ex vivo isolated A-MØ; and (4) PPARγ shapes the gene signature of GM-CSF-conditioned human macrophages in the absence of exogenous agonists. The involvement of activin A in PPARγ expression in GM-CSF-dependent human macrophages points toward a connection between Smad2/3 activation and PPARG gene expression, whose existence has been already suggested in PAP patients, which exhibit a deficiency in PPARγ and a severe reduction in Activin A expression and secretion (42). Thus, our results provide a molecular explanation for such a correlation, and support the existence of a functional GM-CSF/activin A/PPARγ axis in human macrophages. ...
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GM-CSF promotes the functional maturation of lung alveolar macrophages (A-MØ), whose differentiation is dependent on the peroxisome proliferator-activated receptor gamma (PPARγ) transcription factor. In fact, blockade of GM-CSF-initiated signaling or deletion of the PPARγ-encoding gene PPARG leads to functionally defective A-MØ and the onset of pulmonary alveolar proteinosis. In vitro, macrophages generated in the presence of GM-CSF display potent proinflammatory, immunogenic and tumor growth-limiting activities. Since GM-CSF upregulates PPARγ expression, we hypothesized that PPARγ might contribute to the gene signature and functional profile of human GM-CSF-conditioned macrophages. To verify this hypothesis, PPARγ expression and activity was assessed in human monocyte-derived macrophages generated in the presence of GM-CSF [proinflammatory GM-CSF-conditioned human monocyte-derived macrophages (GM-MØ)] or M-CSF (anti-inflammatory M-MØ), as well as in ex vivo isolated human A-MØ. GM-MØ showed higher PPARγ expression than M-MØ, and the expression of PPARγ in GM-MØ was found to largely depend on activin A. Ligand-induced activation of PPARγ also resulted in distinct transcriptional and functional outcomes in GM-MØ and M-MØ. Moreover, and in the absence of exogenous activating ligands, PPARγ knockdown significantly altered the GM-MØ transcriptome, causing a global upregulation of proinflammatory genes and significantly modulating the expression of genes involved in cell proliferation and migration. Similar effects were observed in ex vivo isolated human A-MØ, where PPARγ silencing led to enhanced expression of genes coding for growth factors and chemokines and downregulation of cell surface pathogen receptors. Therefore, PPARγ shapes the transcriptome of GM-CSF-dependent human macrophages (in vitro derived GM-MØ and ex vivo isolated A-MØ) in the absence of exogenous activating ligands, and its expression is primarily regulated by activin A. These results suggest that activin A, through enhancement of PPARγ expression, help macrophages to switch from a proinflammatory to an anti-inflammatory polarization state, thus contributing to limit tissue damage and restore homeostasis.
... Robson et al. [127] demonstrated that NK cells express types I and II activin receptors and that activin-A directly regulates NK cell functions by downregulating expression of the T-box transcription factor T-bet, secretion of IFN-γ, and expression of CD25 [127]. Finally, B lymphocytes have not been exempt from the regulatory influences of activin-A as it has been shown that through direct action on resting B cells, activin-A could enhance IgG and IgE production [108], induce IgA production from mouse mesenteric lymph node cells [128] and decrease proliferation and autoantibody production from B cells of individuals with pulmonary alveolar proteinosis [129]. ...
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During the 26 years that have elapsed since its discovery, activin-A, a member of the transforming growth factor β super-family originally discovered from its capacity to stimulate follicle-stimulating hormone production by cultured pituitary gonadotropes, has been established as a key regulator of various fundamental biological processes, such as development, homeostasis, inflammation, and tissue remodeling. Deregulated expression of activin-A has been observed in several human diseases characterized by an immuno-inflammatory and/or tissue remodeling component in their pathophysiology. Various cell types have been recognized as sources of activin-A, and plentiful, occasionally contradicting, functions have been described mainly by in vitro studies. Not surprisingly, both harmful and protective roles have been postulated for activin-A in the context of several disorders. Recent findings have further expanded the functional repertoire of this molecule demonstrating that its ectopic overexpression in mouse airways can cause pathology that simulates faithfully human acute respiratory distress syndrome, a disorder characterized by strong involvement of neutrophils. This finding when considered together with the recent discovery that neutrophils constitute an important source of activin-A in vivo and earlier observations of upregulated activin-A expression in diseases characterized by strong activation of neutrophils may collectively imply a more intimate link between activin-A expression and neutrophil reactivity. In this review, we provide an outline of the functional repertoire of activin-A and suggest that this growth factor functions as a guardian of homeostasis, a modulator of immunity and an orchestrator of tissue repair activities. In this context, a relationship between activin-A and neutrophils may be anything but coincidental.
... Supernatant and cells were harvested after culture. ATP was measured in mol/mL with BacTiterGlo chemiluminescence assay (Promega, Madison, WI) as a surrogate for cellular activity [35] and therefore growth kinetics [36]. To account for intrasubject variability the data is presented as a ratio with negative control as the denominator. ...
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... PBMCs: Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy control individuals as previously described using Institutional IRB approval [17]. We experimented with the protocol to optimize the yield of monocytes in anticipation of obtaining limited blood volumes from CF patients. ...
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... Both the neutralising activity and total anti-GM-CSF levels in BAL correlated with improvement in HRCT scans, suggesting that autoantibody levels in the target organ correlate with pathophysiology. Although the source of the antibody may be the circulation, B-cells are found in the lung [31] and our previous studies have noted a decrease in activin A, a negative regulator of B-cell proliferation and activity, in PAP BAL fluid [32]. Our results support the concept that the pathology of PAP is mediated by GM-CSF autoantibody, as suggested by results from passive transfer of a highly purified, PAP patient-derived, anti-GM-CSF preparation to primates [33,34]. ...
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Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by the accumulation of lipoproteinaceous material within the alveoli. Recent data suggest that granulocyte-macrophage colony- stimulating factor (GM-CSF) may be involved in the pathogenesis of PAP. To extend this understanding and clarify whether GM-CSF replacement confers benefit, we report the preliminary results for the first four patients in an open-label study of GM-CSF treatment for moderate exacerbation of PAP. All four patients had idiopathic PAP confirmed by open lung biopsy. Subcutaneous GM-CSF was self-administered once daily for 12 wk (dose escalation from 3 to 9 microg/kg/d). Response was assessed from symptom scores, arterial blood gas measurements, pulmonary function testing, and chest radiographs. Three of the four patients experienced symptomatic, physiologic, and radiographic improvement with GM-CSF. Responders experienced sufficient improvement in oxygenation as to eliminate the need for supplemental oxygen, and one patient was removed from the waiting list for lung transplantation. Improved oxygenation was not apparent until 8 to 12 wk after the start of therapy. Notably, expected increases in the peripheral white blood cell count did not occur, suggesting lack of a hematopoietic response to exogenous GM-CSF in PAP. We conclude that GM-CSF appears to benefit a subset of patients with adult PAP, and may represent an alternative to whole-lung lavage in treating the disease.
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Pulmonary alveolar proteinosis (PAP; the accumulation of surfactant lipids and proteins in the alveoli) has a number of infectious and environmental causes but is usually idiopathic. The clinical presentation of PAP is nonspecific; thus, the diagnosis is frequently missed, leading to inappropriate therapy and unnecessary morbidity. Recent advances suggest that a deficiency in granulocyte-macrophage colony-stimulating factor (GM-CSF) activity may lead to this surfactant accumulation. Anti-GM-CSF antibodies have been found in PAP patients, fueling speculation that PAP may be an autoimmune disease. These findings are being translated into novel forms of therapy.
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Activin A, a homodimeric protein (betaAbetaA) and a member of the TGF-beta superfamily, is involved in the inflammatory repair process. Using cDNA microarray analysis, we discovered strong induction of the activin betaA gene in human mast cells (MC) on stimulation with PMA and calcium ionophore (A23187). Activin betaA mRNA was also highly induced in primary cultured murine bone marrow MC (BMMC) after stimulation by IgE receptor cross-linking. Secretion of activin A was evident in human mast cell-1 line cells 3 h after stimulation and progressively increased over time. Activin A was present in the cytoplasm of activated but not unstimulated murine bone marrow MC as demonstrated by immunofluorescence studies, suggesting that secretion of activin A by MC was due to de novo synthesis rather than secretion of preformed protein. Activin A also colocalized with human lung MC from patients with asthma by double-immunofluorescence staining. Furthermore, secretion of activin A was significantly increased in the airway of wild-type mice after OVA sensitization followed by intranasal challenge. Secretion of activin A, however, was greatly reduced in MC-deficient WBB6F(1)-W/W(v) mice as compared with wild-type mice, indicating that MC are an important contributor of activin A in the airways of a murine asthma model. Additionally, activin A promoted the proliferation of human airway smooth muscle cells. Taken together, these data suggest that MC-derived activin A may play an important role in the process of airway remodeling by promoting the proliferation of airway smooth muscle.
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Granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically implicated in lung homeostasis in the GM-CSF knockout mouse model. These animals develop an isolated lung lesion reminiscent of pulmonary alveolar proteinosis (PAP) seen in humans. The development of the adult form of human alveolar proteinosis is not due to the absence of a GM-CSF gene or receptor defect but to the development of an anti-GM-CSF autoimmunity. The role of GM-CSF in the development of PAP is unknown. Studies in the GM-CSF knockout mouse have shown that lack of PU.1 protein expression in alveolar macrophages is correlated with decreased maturation, differentiation, and surfactant catabolism. This study investigates PU.1 expression in vitro and in vivo in human PAP alveolar macrophages as well as the regulation of PU.1 by GM-CSF. We show for the first time that PU.1 mRNA expression in PAP bronchoalveolar lavage cells is deficient compared with healthy controls. PU.1-dependent terminal differentiation markers CD32 (FCgammaII), mannose receptor, and macrophage colony-stimulating factor receptor (M-CSFR) are decreased in PAP alveolar macrophages. In vitro studies demonstrate that exogenous GMCSF treatment upregulated PU.1 and M-CSFR gene expression in PAP alveolar macrophages. Finally, in vivo studies showed that PAP patients treated with GM-CSF therapy have higher levels of PU.1 and M-CSFR expression in alveolar macrophages compared with healthy control and PAP patients before GM-CSF therapy. These observations suggest that PU.1 is critical in the terminal differentiation of human alveolar macrophages.
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Pulmonary alveolar proteinosis (PAP) is characterized by the accumulation of lipoproteinaceous material within the lung alveoli. Recent studies indicate that PAP is an autoimmune disease characterized by a neutralizing anti-granulocyte macrophage colony stimulating factor (GM-CSF) antibody. At present the only definitive diagnostic test for PAP is open lung biopsy. We have previously published that anti-GM-CSF is diagnostic for PAP and correlates with disease pathogenesis using a traditional serial anti-GM-CSF antibody titer format (T. L. Bonfield, M. S. Kavuru, and M. J. Thomassen, Clin. Immunol. 105:342-350, 2002). Titer analysis is a semiquantitative method, and often subtle changes in antibody titer are not detectable. In this report we present data to support anti-GM-CSF detection by a quantitative highly sensitive multiplexed particle-based assay which has the potential to be a clinical diagnostic test.
Article
Inhibin is a gonadal protein that specifically inhibits the secretion of pituitary follicle-stimulating hormone (FSH). Two forms of inhibin (A and B) have been purified from porcine follicular fluid1 and characterized as heterodimers of relative molecular mass (Mr) 32,000 (ref. 2). Each inhibin is comprised of an identical α-subunit of Mr 18,000 and a distinct but related β-subunit of Mr 13,800-14,700 linked by interchain disulphide bond(s). Throughout the purification of inhibins, we consistently observed two fractions which stimulated the secretion of pituitary FSH. We report here the isolation of one of the FSH-releasing proteins; it has a Mr of 24,000 and its N-terminal sequences up to residue 32 are identical to those of each β-subunit of inhibins A and B. In the presence of reducing agents, SDS-polyacrylamide gel electrophoresis resolves the FSH-releasing substance into two subunits which are identical in their migration behaviour to the reduced β-subunits of inhibins A and B. Based on the N-terminal sequence data and Mr of the intact and reduced molecules, we propose that the FSH-releasing substance, which is active in picomolar concentrations, is a heterodimeric protein composed of the two β-subunits of inhibins A and B linked by interchain disulphide bond(s). The structural organization of the FSH-releasing substance is homologous to that of transforming growth factor-β (TGF-β)3, which also possesses FSH-releasing activity in the same bioassay4. We suggest that the substance be called activin to signify the fact that it has opposite biological effects to inhibin.
Article
Inhibin is a 32-kD dimeric glycoprotein consisting of an α subunit and one of two β subunits (βA or βB), which was isolated and cloned on the basis of its ability to inhibit FSH release from the pituitary. Activin results from the combination of two inhibins. Activins can cause stimulation of FSH release from pituitary cells both in vitro and in vivo, and in addition are involved in embryogenesis, erythropoiesis, and reproductive function. The inhibin-related peptides, and their receptors, are present in the testis and ovary from early gestation through adulthood. An additional level of control of the activity of growth factors is afforded by specific binding which may regulate protein turnover, localization and bioactivity. To date, two distinct binding proteins for inhibin and activin have been identified, both of which are expressed in the testes and other tissues and are present in the circulation. In serum, inhibin is primarily found associated with α2-macroglobulin (α2M), a high-capacity, low-affinity binding protein which binds many cytokines and growth factors. Binding to α2M does not appear to alter immuno- or bioactivity of inhibin or activin. The second binding protein, follistatin, is produced in many of the same tissues which produce the activin and inhibin. This molecule may function primarily as a regulator of activin bioavailability and bioactivity. The affinity of follistatin for activin ( < 1.0 nM) is similar to that of the high-affinity activin receptors. Thus, dynamic changes in the relative levels of the amount of any of these components could act to modulate activin and inhibin bioavailability in both a developmentally and tissue-restricted pattern in the testes or ovary.Copyright © 1996 S. Karger AG, Basel
Article
In studies of the regulation of hematopoiesis, increasing attention has focused on the role of bone marrow stromal cells as rich sources of various cytokines. Present studies indicate that marrow stromal cells and monocytes produce activin A, implicating this new cytokine in the paracrine control of hematopoiesis. Activin A, which was initially recognized as a beta A beta A dimeric gonadal protein, was found to potentiate the proliferation and differentiation of erythroid progenitors; both purified erythroid colony-forming units (CFU-E) and K562 cells possess high affinity receptors specific for activin A. Present studies using Western and Northern blots demonstrate the presence of beta A subunits of activin A in the conditioned medium of monocytes and stromal cells and its RNA transcripts in these cells. The presence of functional and homodimeric beta A beta A activin molecule was confirmed through bioassay with or without a blocking antiserum against activin A or an activin binding protein, follistatin; its presence is further supported by a specific enzyme-linked immunosorbent assay (ELISA) in which a monoclonal antibody reacted only with the beta A beta A dimeric form of this molecule. In other experiments, the production of activin A was found to be regulated by various cytokines and regulators. The production of activin A in monocytes was stimulated more than ninefold by treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). Activin A expression was also stimulated, albeit less potently, by bacterial lipopolysaccharide (LPS) and gamma-interferon. On the other hand, the expression of activin A in marrow stromal cells was upregulated by incubation with tumor necrosis factor-alpha (TNF-alpha), LPS, and interleukin 1 alpha (IL-1 alpha). Therefore, we propose that the local production of activin A in the microenvironment within bone marrow may fine tune the regulation of steady-state hematopoiesis. In addition, this factor may normally be produced at minimal levels, but under certain situations may be further induced to provide important biological functions.
Article
A variety of hypophysiotropic peptides or proteins have been reported to be present in mammalian gonads. Inhibin, a hormone that under most circumstances selectively suppresses the secretion of follicle-stimulating hormone (FSH) but not luteinizing hormone (LH), has been isolated from the gonadal fluids of several species and characterized as a heterodimeric protein consisting of alpha- and beta-polypeptides associated by disulphide bonds. The complete amino-acid sequences of the precursors of porcine and human inhibin alpha-subunits and two distinct porcine inhibin beta-subunits (beta A and beta B) have been deduced from complementary DNA sequences. Gonadotropin releasing peptides have also been found in the gonad and have generally been shown to be active in radioreceptor assays for gonadotropin releasing hormone (GnRH) but to exhibit different chromatographic and immunological characteristics from those of GnRH. During our purification of inhibin from porcine follicular fluid, we noted fractions that could stimulate the secretion of FSH by cultured anterior pituitary cells. We report here the purification of an FSH releasing protein (FRP) and its characterization by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions and by partial sequence analysis. Our results indicate that porcine gonadal FRP is a homodimer consisting of two inhibin beta A-chains linked by disulphide bonds. FRP is highly potent (50% effective concentration (EC50) approximately 25 pM) in stimulating the secretion and biosynthesis of FSH but not of LH or any other pituitary hormone. In contrast to the effects of GnRH and other reported gonadal gonadotropin releasing fractions, the action of FRP is not mediated by GnRH receptors.
Article
Inhibin is a gonadal protein that specifically inhibits the secretion of pituitary follicle-stimulating hormone (FSH). Two forms of inhibin (A and B) have been purified from porcine follicular fluid and characterized as heterodimers of relative molecular mass (Mr) 32,000 (ref. 2). Each inhibin is comprised of an identical alpha-subunit of Mr 18,000 and a distinct but related beta-subunit of Mr 13,800-14,700 linked by interchain disulphide bond(s). Throughout the purification of inhibins, we consistently observed two fractions which stimulated the secretion of pituitary FSH. We report here the isolation of one of the FSH-releasing proteins; it has a Mr of 24,000 and its N-terminal sequences up to residue 32 are identical to those of each beta-subunit of inhibins A and B. In the presence of reducing agents, SDS-polyacrylamide gel electrophoresis resolves the FSH-releasing substance into two subunits which are identical in their migration behaviour to the reduced beta-subunits of inhibins A and B. Based on the N-terminal sequence data and Mr of the intact and reduced molecules, we propose that the FSH-releasing substance, which is active in picomolar concentrations, is a heterodimeric protein composed of the two beta-subunits of inhibins A and B linked by interchain disulphide bond(s). The structural organization of the FSH-releasing substance is homologous to that of transforming growth factor-beta (TGF-beta), which also possesses FSH-releasing activity in the same bioassay. We suggest that the substance be called activin to signify the fact that it has opposite biological effects to inhibin.
Article
Activin A is a homodimeric protein structurally and functionally related to transforming growth factor beta (TGF-beta), and the expression of activin A is modulated by TGF-beta. Here, we demonstrate the expression of activin A in normal and bleomycin (BLM)-treated murine lungs. ICR mice were treated with BLM intraperitoneally for 10 days, whereas saline vehicle was injected into control mice. Intra-alveolar fibrotic changes were observed in the lung tissue obtained from the mice at day 14 after the final BLM administration. Immunohistochemical studies using a polyclonal antibody to activin A revealed the presence of activin A in the bronchiolar epithelium and smooth muscle cells of veins in both control and BLM-treated mice. In the BLM-treated mice at days 7 and 14, the marked infiltration of immunoreactive alveolar macrophages was observed in the area of fibrotic changes. Bioactivity of activin A measured by erythroid differentiation factor assay in the conditioned medium of alveolar macrophages obtained from BLM-treated mice at day 14 was significantly increased. These findings indicate that alveolar macrophages are a potent source of activin A after BLM treatment. The present study demonstrates for the first time the abundant expression of activin A in murine lung tissues after BLM administration, suggesting that activin A may play a role in the pathogenesis of BLM-induced pulmonary fibrosis.
Article
The capacity of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), glucocorticoids or all-trans-retinoic acid to modulate production of activin A by human monocytes was studied. It was shown that GM-CSF stimulated monocytes to accumulate activin A RNA after as few as 4 hr of incubation, reaching a peak of stimulation at approximately 16 hr of incubation. The activin A transcripts accumulated in the monocytes after stimulation with only 5 U/ml of GM-CSF and reached a maximum plateau level of expression between 25 and 50 U/ml of GM-CSF. Biologically active activin A molecules were detected in the conditioned media by a bioassay, performed both in the absence and presence of a neutralizing antiserum for activin A. Accumulation of bioactive activin A in conditioned medium of monocyte cultures was detected after 24 hr of incubation with GM-CSF and high levels of activin A were maintained for 72 hr. The production of the dimeric beta A beta A in these monocytes was further confirmed by sandwich enzyme-linked immunosorbent assay (ELISA) specific for activin A. In contrast to the stimulatory effect of GM-CSF, hydrocortisone, dexamethasone or all-trans-retinoic acid at 1 x 10(-7) to 1 x 10(-5) M inhibited the constitutive expression of activin A and greatly suppressed the GM-CSF-stimulated production. Thus, the expression of activin A is modulated in monocytes by different agents. These observations may imply new roles for activin A at sites of inflammation where monocytes accumulate.
Article
Inhibin is a 32-kD dimeric glycoprotein consisting of an alpha subunit and one of two beta subunits (beta A or beta B), which was isolated and cloned on the basis of its ability to inhibit FSH release from the pituitary. Activin results from the combination of two inhibins. Activins can cause stimulation of FSH release from pituitary cells both in vitro and in vivo, and in addition are involved in embryogenesis, erythropoiesis, and reproductive function. The inhibin-related peptides, and their receptors, are present in the testis and ovary from early gestation through adulthood. An additional level of control of the activity of growth factors is afforded by specific binding which may regulate protein turnover, localization and bioactivity. To date, two distinct binding proteins for inhibin and activin have been identified, both of which are expressed in the testes and other tissues and are present in the circulation. In serum, inhibin is primarily found associated with alpha 2-macroglobulin (alpha 2M), a high-capacity, low-affinity binding protein which binds many cytokines and growth factors. Binding to alpha 2M does not appear to alter immuno- or bioactivity of inhibin or activin. The second binding protein, follistatin, is produced in many of the same tissues which produce the activin and inhibin. This molecule may function primarily as a regulator of activin bioavailability and bioactivity. The affinity of follistatin for activin (< 0.1 nM) is similar to that of the high-affinity activin receptors. Thus, dynamic changes in the relative levels of the amount of any of these components could act to modulate activin and inhibin bioavailability in both a developmentally and tissue-restricted pattern in the testes and ovary.
Article
Activins, members of the transforming growth factor-beta family, have been implicated in the regulation of growth and differentiation of various types of cells. We have recently found that activin A induces apoptotic cell death of plasmacytic cells including B cell hybridoma cells and myeloma cells. In the present study, we demonstrated that activin A caused cell-cycle arrest in the G1 phase before appearance of apoptotic cells in mouse B cell hybridoma cells. Phosphorylation of retinoblastoma protein (Rb) and in vitro Rb kinase activity of cyclin-dependent kinase (CDK)4 was inhibited in activin A-treated cells. Analysis of expression of genes regulating Rb phosphorylation revealed that activin A suppressed cyclin D2, the sole D-type cyclin gene expressed in the hybridoma cells, and activated p21CIP1/WAF1 but had no effect on expression of cyclin-dependent kinases (CDK2, CDK4, CDK6) and other CDK inhibitors (p27KIP1, p16INK4a, p15INK4b). Modulation of cyclin D2 and p21CIP1/WAF1 expression resulted in a decrease in level of cyclin D2-CDK4 complex and an increase in level of CDK4 complexed with p21CIP1/WAF1. Moreover, overexpression of cyclin D2 partially abrogated inhibition of Rb phosphorylation and G1 arrest in the hybridoma cells.
Article
Macrophage (M phi) foam cell formation is a characteristic event that occurs in the early stage of atherosclerosis. To examine the roles of activin-A, a member of the transforming growth factor-beta superfamily, and follistatin, the binding protein for activin-A, in M phi function, we investigated their effects on foam cell formation of THP-1 M phi s. When THP-1 M phi s were treated with activin-A (5 nmol/L), foam cell formation and cellular cholesteryl ester accumulation were decreased. This downregulation was paralleled by a reduction in cell association and degradation of acetylated LDL. The inhibitory effect of activin-A on cell association and degradation was dose dependent, and the effect was blocked by concomitant addition of follistatin. Activin-A (5 nmol/L) also decreased the Bmax for acetylated LDL and scavenger receptor mRNA expression. Follistatin showed an effect opposite to that of activin-A and promoted M phi foam cell formation and cellular cholesteryl ester accumulation. It increased binding, cell association, and degradation of acetylated LDL and upregulated scavenger receptor mRNA expression. Because follistatin is the binding protein for activin-A, follistatin's effect is considered to be mediated by blocking the inhibitory effect of intrinsic activin-A. These results indicate that activin-A inhibits and follistatin promotes M phi foam cell formation by regulating scavenger receptor mRNA expression. We conclude that activin-A and follistatin play important roles in the process of atherosclerosis by regulating M phi foam cell formation.
Article
Human marrow stromal cells were analysed with immunocytochemical staining, Northern blot, and functional bioassay for production of activin A. Although Northern blot and immunocytochemical staining did not detect the alpha subunit of inhibin in human marrow stromal cells, RT-PCR analyses confirmed its presence, along with the expected activin beta A PCR products. Present studies showed that human marrow fibroblastoid cells were reactive with anti-activin A antibodies and that the production of beta A RNA was upregulated by pro-inflammatory cytokines/regulators like interleukin 1 alpha (IL-1 alpha), tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) or 12-O-tetradecanoylphorbol 13-acetate (TPA). IL-1 alpha or TNF-alpha stimulated-marrow stromal cells accumulated beta A RNA after 2 h of incubation, reaching a peak stimulation at approximately 8 h. Biologically active activin A molecules were detected in the conditioned media by a bioassay, and their activity was specifically inhibited by a blocking antibody or an activin-binding protein, follistatin. Accumulation of bioactive activin A in conditioned medium of human marrow stromal cells increased after incubation with IL-1 alpha or TNF-alpha. Nuclear run-off assays with TNF-alpha stimulated marrow stromal cells showed that the enhanced expression of activin A was related to an increase in its rate of transcription. In contrast to the stimulatory effect of pro-inflammatory cytokines, hydrocortisone and dexamethasone at 1 x 10(-7) to 1 x 10(-6) M inhibited both the constitutive and the cytokine-stimulated expression of activin beta A RNA, and also the production of bioactive activin A protein. The upregulation of activin A production by cytokines and its suppression by glucocorticoids imply that activin A may also act as a moderator in diverse functions including host defences.
Article
Granulocyte-macrophage colony stimulating factor (GM-CSF) deficient mice develop a pulmonary alveolar proteinosis (PAP) syndrome which is corrected by the administration/expression of GM-CSF. These observations implicate GM-CSF in the pathogenesis of human PAP. We hypothesized that human PAP may involve an intrinsic cellular defect in monocytes/macrophages with an inability to produce GM-CSF and/or respond to GM-CSF. Thus, we investigated the cytokine responses to GM-CSF and LPS from peripheral blood monocytes and alveolar macrophages from patients with idiopathic PAP and healthy controls. Macrophage inflammatory protein-1-alpha (MIP) was measured from GM-CSF-stimulated cells and GM-CSF was measured from LPS-stimulated cells by ELISA. The MIP and GM-CSF production by monocytes and alveolar macrophages did not differ between PAP patients and healthy controls. Growth of the GM-CSF-dependent human myeloid cell line TF-1 was inhibited by serum from all patients studied (n = 10) and all patients had anti-GM-CSF antibody in their serum. The BAL from PAP patients had less detectable GM-CSF by ELISA than healthy controls (P = 0.05); in contrast, the inhibitory cytokine, interleukin-10 (IL-10), was increased in PAP compared to controls (P = 0.04). IL-10 is a potent inhibitor of LPS-stimulated GM-CSF production from healthy control alveolar macrophages. These studies are the first to demonstrate that circulating monocytes and alveolar macrophages from PAP patients are able to synthesize GM-CSF and respond to GM-CSF, suggesting no intrinsic abnormalities in GM-CSF signaling. In addition, these observations suggest that PAP in a subset of patients is the result of decreased availability of GM-CSF due to GM-CSF blocking activity and reduced GM-CSF production by IL-10.
Article
Pulmonary alveolar proteinosis is a rare clinical syndrome that was first described in 1958. Subsequently, over 240 case reports and small series have described at least 410 cases in the literature. Characterized by the alveolar accumulation of surfactant components with minimal interstitial inflammation or fibrosis, pulmonary alveolar proteinosis has a variable clinical course ranging from spontaneous resolution to death with pneumonia or respiratory failure. The most effective proven treatment--whole lung lavage--was described soon after the first recognition of this disease. In the last 8 years, there has been rapid progress toward elucidation of the molecular mechanisms underlying both the congenital and acquired forms of pulmonary alveolar proteinosis, following serendipitous discoveries in gene-targeted mice lacking granulocyte-macrophage colony-stimulating factor (GM-CSF). Impairment of surfactant clearance by alveolar macrophages as a result of inhibition of the action of GM-CSF by blocking autoantibodies may underlie many acquired cases, whereas congenital disease is most commonly attributable to mutations in surfactant protein genes but may also be caused by GM-CSF receptor defects. Therapy with GM-CSF has shown promise in approximately half of those acquired cases treated, but it is unsuccessful in congenital forms of the disease, consistent with the known differences in disease pathogenesis.
Article
Pulmonary alveolar proteinosis (PAP) is characterized clinically by nonresolving pulmonary infiltrates and hypoxemia in patients in their third and fourth decades, and it occurs with a male predominance. This disease is quite remarkable for flooding of the alveoli with lipoproteinaceous material, yet with minimal local lung inflammation or distortion of architecture on biopsy, for reasons that are unknown. The last 8 years have seen some remarkable advances into the pathophysiology of this disease, through transgenic murine models that have clearly established that hematopoietic growth factor (granulocyte-macrophage colony stimulating factor [GM-CSF]) is critical for local regulation of surfactant homeostasis. Additional human studies have clearly linked the presence of a circulating, neutralizing anti–GM-CSF antibody in adults with idiopathic PAP.1–² Therefore, in the current paradigm, there are three major clinical subtypes of PAP: the most common adult idiopathic variety, which is presumably autoimmune (with a circulating anti–GM-CSF antibody); a neonatal variety that is likely due to a defect in surfactant proteins B or C or the common β-chain of the GM-CSF receptor; and secondary PAP associated with occupational exposures or immunologic disorders. To date, there have been no reports of the presence of an antibody in the neonatal variety. Preliminary reports from several groups indicate that a subset of adult patients with idiopathic PAP respond favorably to GM-CSF therapy.3–⁴
Article
Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-activated, nuclear transcription factor that regulates genes involved in lipid and glucose metabolism, inflammation, and other pathways. The hematopoietic growth factor, granulocyte macrophage colony-stimulating factor (GM-CSF), is essential for lung homeostasis and is thought to regulate surfactant clearance, but mechanisms involved are unknown. GM-CSF is reported to stimulate PPAR-gamma, but the activation status of PPAR-gamma in human alveolar macrophages has not been defined. In pulmonary alveolar proteinosis (PAP), a rare interstitial lung disease, surfactant accumulates in alveolar airspaces, resident macrophages become engorged with lipoproteinaceous material, and GM-CSF deficiency is strongly implicated in pathogenesis. Here we show that PPAR-gamma mRNA and protein are highly expressed in alveolar macrophages of healthy control subjects but severely deficient in PAP in a cell-specific manner. Further, we show that the PPAR-gamma-regulated lipid scavenger receptor, CD36, is also deficient in PAP. PPAR-gamma and CD36 deficiency are not intrinsic to PAP alveolar macrophages, but can be upregulated by GM-CSF therapy. Moreover, GM-CSF treatment of patients with PAP fully restores PPAR-gamma to healthy control levels. Based upon these novel findings, we hypothesize that GM-CSF regulates lung homeostasis via PPAR-gamma-dependent pathways.
Article
Pulmonary Alveolar Proteinosis (PAP) is characterized by the accumulation of surfactant phospholipids and proteins within the alveoli of lungs. Currently, no specific therapy exists for PAP, and sequential whole lung lavage is standard treatment. Over the past 5 years, important advances have been made in our understanding of alveolar proteinosis, offering new directions for research as well as patient management. First, genetically altered mice that are homozygous for a disrupted granulocyte-macrophage colony-stimulating factor (GM-CSF) gene developed a lung lesion with histologic resemblance to PAP, along with normal hematopoiesis. The biochemical properties of the material filling the airspaces in these mutant mice are similar to those of patients with PAP. Surfactant is thought to be cleared or catabolized mostly by alveolar macrophages, a process dependent on GM-CSF. Second, a neutralizing autoantibody against GM-CSF was found in bronchoalveolar lavage fluid and serum of patients with idiopathic PAP, but not healthy controls. These observations raise the previously unsuspected notion that human alveolar proteinosis may be an autoimmune disease, and suggest that GM-CSF antibody has a potential role as a diagnostic test. The relationship between the antibody and disease pathogenesis remains unknown. Additional data suggest that exogenous therapy with GM-CSF may improve the lung disease in some patients with PAP. Intervention directed at treating a relative GM-CSF deficiency or lowering the antibody (i.e., by plasmapheresis or immunosuppression) may have promise in the therapy of this disease. Alveolar proteinosis may be the first human disease wherein a circulating antibody against a growth factor is linked to disease pathogenesis. Over a relatively short time, studies from ;;knock-out'' mice have been translated to human studies for a new approach to diagnosis and therapy for this disease.
PPARg is deficient in alveolar macrophages from patients with alveolar proteinosis
  • Tl Bonfield
  • Cf Farver
  • Bp Barna
  • A Malur
  • S Abraham
  • B Raychaudhuri
Bonfield TL, Farver CF, Barna BP, Malur A, Abraham S, Raychaudhuri B, et al. PPARg is deficient in alveolar macrophages from patients with alveolar proteinosis. Am J Respir Cell Mol Biol 2003;29:677e82.
PPARγ is deficient in alveolar macrophages from patients with alveolar proteinosis
  • Bonfield
Functional analysis of activins during mammalian development
  • Matzuk