Colony stimulating factors in the nervous system.

Although traditionally seen as regulators of hematopoiesis, colony-stimulating factors (CSFs) have emerged as important players in the nervous system, both in health and disease. This review summarizes the cellular sources, patterns of expression and physiological roles of the macrophage (CSF-1, IL-34), granulocyte-macrophage (GM-CSF) and granulocyte (G-CSF) colony stimulating factors within the nervous system, with a particular focus on their actions on microglia. CSF-1 and IL-34, via the CSF-1R, are required for the development, proliferation and maintenance of essentially all CNS microglia in a temporal and regional specific manner. In contrast, in steady state, GM-CSF and G-CSF are mainly involved in regulation of microglial function. The alterations in expression of these growth factors and their receptors, that have been reported in several neurological diseases, are described and the outcomes of their therapeutic targeting in mouse models and humans are discussed.

The Protective and Reparative Role of Colony-Stimulating Factors in the Brain with Cerebral Ischemia/Reperfusion Injury.

Stroke is a debilitating disease and has the ability to culminate in devastating clinical outcomes. Ischemic stroke followed by reperfusion entrains cerebral ischemia/reperfusion (I/R) injury, which is a complex pathological process and is associated with serious clinical manifestations. Therefore, the development of a robust and effective poststroke therapy is crucial. Granulocyte colony-stimulating factor (GCSF) and erythropoietin (EPO), originally discovered as hematopoietic growth factors, are versatile and have transcended beyond their traditional role of orchestrating the proliferation, differentiation, and survival of hematopoietic progenitors to one that fosters brain protection/neuroregeneration. The clinical indication regarding GCSF and EPO as an auspicious therapeutic strategy is conferred in a plethora of illnesses, including anemia and neutropenia. EPO and GCSF alleviate cerebral I/R injury through a multitude of mechanisms, involving antiapoptotic, anti-inflammatory, antioxidant, neurogenic, and angiogenic effects. Despite bolstering evidence from preclinical studies, the multiple brain protective modalities of GCSF and EPO failed to translate in clinical trials and thereby raises several questions. The present review comprehensively compiles and discusses key findings from in vitro, in vivo, and clinical data pertaining to the administration of EPO, GCSF, and other drugs, which alter levels of colony-stimulating factor (CSF) in the brain following cerebral I/R injury, and elaborates on the contributing factors, which led to the lost in translation of CSFs from bench to bedside. Any controversial findings are discussed to enable a clear overview of the role of EPO and GCSF as robust and effective candidates for poststroke therapy.

Role of the colony-stimulating factor (CSF)/CSF-1 receptor axis in cancer.

Cancer cells employ a variety of mechanisms to evade apoptosis and senescence. Pre-eminent among these is the aberrant co-expression of growth factors and their ligands, forming an autocrine growth loop that promotes tumour formation and progression. One growth loop whose transforming potential has been repeatedly demonstrated is the CSF-1/CSF-1R axis. Expression of CSF-1 and/or CSF-1R has been documented in a number of human malignancies, including breast, prostate and ovarian cancer and classical Hodgkin's lymphoma (cHL). This review summarizes the large body of work undertaken to study the role of this cytokine receptor system in malignant transformation. These studies have attributed a key role to the CSF-1/CSF-1R axis in supporting tumour cell survival, proliferation and enhanced motility. Moreover, increasing evidence implicates paracrine interactions between CSF-1 and its receptor in defining a tumour-permissive and immunosuppressive tumour-associated stroma. Against this background, we briefly consider the prospects for therapeutic targeting of this system in malignant disease.

Maternal immune activation by poly I:C induces expression of cytokines IL-1ß and IL-13, chemokine MCP-1 and colony stimulating factor VEGF in fetal mouse brain.

BACKGROUND: Maternal viral infection during pregnancy is associated with an increase in the incidence of psychiatric disorders with presumed neurodevelopmental origin, including autism spectrum disorders and schizophrenia. The enhanced risk for developing mental illness appears to be caused by deleterious effects of innate immune response-associated factors on the development of the central nervous system, which predispose the offspring to pathological behaviors in adolescence and adulthood. To identify the immune response-associated soluble factors that may affect central nervous system development, we examined the effect of innate immune response activation by polyriboinosinic-polyribocytidylic acid (poly(I:C)), a synthetic analogue of viral double-stranded RNA, on the expression levels of pro- and anti-inflammatory cytokines, chemokines and colony stimulating factors in fetal and postnatal mouse brain 6 h and 24 h after treatment. METHODS: C57BL/6J pregnant mice (gestational day 16) or newborn mice (postnatal day 4) received a single intraperitoneal injection of the synthetic analogue of viral double-stranded RNA poly(I:C) (20 mg/kg). Thirty-two immune response-associated soluble factors, including pro- and anti-inflammatory cytokines, chemokines and colony stimulating factors, were assayed 6 h and 24 h after poly(I:C) injection using multiplexed bead-based immunoassay (Milliplex Map) and processed in a Luminex 100 IS instrument. RESULTS: Maternal exposure to poly(I:C) at gestational day 16 induced a significant increase in cytokines interleukin (IL)-1ß, IL-7 and IL-13; chemokines monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1α, interferon gamma-induced protein (IP)-10 and monokine induced by IFN-gamma (MIG); and in the colony stimulating factor vascular endothelial growth factor (VEGF) in the fetal brain. IL-1ß showed the highest concentration levels in fetal brains and was the only cytokine significantly up-regulated 24 h after maternal poly(I:C) injection, suggesting that IL-1ß may have a deleterious impact on central nervous system development. In contrast, poly(I:C) treatment of postnatal day 4 pups induced a pronounced rise in chemokines and colony stimulating factors in their brains instead of the pro-inflammatory cytokine IL-1ß. CONCLUSIONS: This study identified a significant increase in the concentration levels of the cytokines IL-1ß and IL-13, the chemokine MCP-1 and the colony stimulating factor VEGF in the developing central nervous system during activation of an innate immune response, suggesting that these factors are mediators of the noxious effects of maternal immune activation on central nervous system development, with potential long-lasting effects on animal behavior.

Growth of factor-dependent hemopoietic precursor cell lines.

Cell lines have been produced from long-term cultures of mouse bone marrow that require a factor, present in WEHI-3 conditioned medium (CM) or in spleen CM, for their sustained growth. The cell lines were obtained from nonvirus-treated cultures, are nonleukemic, maintain a normal karyotype, and form colonies showing granulocyte maturation when plated in soft agar. Granulocyte/macrophage (GM) colony-stimulating factor is not the inductive moiety involved in the maintenance of proliferation of these cells. It is suggested that the cell lines represent a self-renewing population of cells ancestral to GM colony-forming cells, which may be responding to a hitherto unrecognized regulator.

Identification of leukemia-associated inhibitory activity as acidic isoferritins. A regulatory role for acidic isoferritins in the production of granulocytes and macrophages.

Acidic isoferritins have been identified as leukemia-associated inhibitory activity (LIA), which suppresses colony and cluster formation of colony-forming unit-granulocyte macrophages from normal donors but not from patients with leukemia. LIA was detected in all ferritin preparations tested, including ferritin isolated from normal heart, spleen, liver, and placental tissues, and from the spleens of patients with chronic myelogenous leukemia and Hodgkin's disease. Purified preparations of LIA were composed almost entirely of acidic isoferritins, as determined by immunoassay, radioimmunoassay, and isoelectric focusing. The inhibitory activity in the LIA and ferritin samples was inactivated by a battery of antisera specific for ferritin, including those prepared against acidic isoferritins from normal heart and spleen tissues from patients with Hodgkin's disease, and those previously absorbed with basic isoferritins. Antisera absorbed with acidic isoferritins did not inactivate the inhibitory activity. Separation of LIA and chronic myelogenous leukemia and normal spleen ferritin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing confirmed that the regions of peak inhibitory activity corresponded in each to an apparent molecular weight of approximately 550,000 and to a pI value of 4.7. Similar physicochemical characteristics included inactivation by methods that dissociate ferritin molecules into subunits and by treatment with trypsin, chymotrypsin, pronase, and periodate. The purified preparations were extremely stable to heat treatment. The glycoprotein nature of the inhibitory activity was substantiated because it bound to concanavalin A-Sepharose and was eluted off by alpha-methyl mannose. Inhibitory activity of the activity of the acidic isoferritins was detected at concentrations as low as 10(-17)-10(-19) M and iron saturation did not appear to be necessary for its action. These results implicate acidic isoferritins in the regulation of normal myelopoiesis and suggest a role for them in the progression of leukemia.

Cytokines in chronic inflammatory arthritis. I. Failure to detect T cell lymphokines (interleukin 2 and interleukin 3) and presence of macrophage colony-stimulating factor (CSF-1) and a novel mast cell growth factor in rheumatoid synovitis.

Because previous studies showed low levels of IFN-gamma in rheumatoid arthritis (RA) synovial fluid (SF) and synovial tissue (ST) explant supernatants, we assayed RA SF and ST for IL-2 and IL-3-like activity. Using an IL-2 dependent murine CTLL line, 6 of 14 RA SF caused increased thymidine uptake (greater than three times control). The activity was distinct from IL-2 because it was not blocked by antibody to IL-2-R. In addition, IL-2 was not detected (less than 50 pg/ml) in 16 joint samples using an ELISA. Multi-colony-stimulating factor (CSF) activity was measured using two assays that can detect murine IL-3 (mast cell proliferation, and bone marrow CSF). In the mast cell assay, [3H]TdR uptake was 493 +/- 67 cpm for medium, 2,910 +/- 329 cpm in the presence of RA SF (p less than 0.001), 1,246 +/- 156 cpm in the presence of SF from patients with seronegative spondyloarthropathies (p less than 0.001), and 736 +/- 100 cpm in the presence of osteoarthritis SF (p greater than 0.1). In the CSF assay, four of five RA SF and five of five RA ST induced colony formation from bone marrow nonadherent cells. Macrophage colonies were most common, although mixed colonies and granulocytes were occasionally observed. The multi-CSF activity in RA is not due to IL-3 since human rIL-3 was not active in either murine assay, and IL-3 mRNA was not detected in RA synovium. Sephadex column chromatography of RA SF revealed that the mast cell growth factor (approximately 6 x 10(3) mol wt) and the CSF (approximately 40 and 100 x 10(3) mol wt) are distinct. The colony-stimulating aspect of the "IL-3-like" activity in RA SF is likely due to CSF-1 because it is the appropriate mol wt and because the activity was neutralized by specific anti-CSF-1 antibody. Finally, an RIA detected 1.6-25 ng/ml of CSF-1 in RA SF and ST and CSF-1 mRNA was detected in four of five RA synovial tissue samples tested.

Antibody-dependent antitumor cytotoxicity by human monocytes cultured with recombinant macrophage colony-stimulating factor. Induction of efficient antibody-mediated antitumor cytotoxicity not detected by isotope release assays.

Macrophage colony-stimulating factor (M-CSF) is known to stimulate proliferation of monocyte/macrophage progenitors and enhance in vitro antitumor cytotoxicity by murine macrophages. In this paper we have shown that recombinant human M-CSF causes human peripheral blood monocytes to differentiate in culture into metabolically active macrophage-like cells. These cells mediate very efficient antibody-dependent cellular cytotoxicity (ADCC) against human melanoma and neuroblastoma cell lines in the presence of two murine IgG3 mAbs (3F8 and R24). They also mediate antibody-independent cytotoxicity (or cytostasis) to a lesser extent. Human serum had an inconsistent effect on ADCC, but often induced similar high levels of ADCC. Cytotoxicity was measured using a novel ELISA to detect surviving tumor cells after ADCC. Two conventional isotope-release assays (51Cr and [3H]TdR) underestimated or entirely failed to detect ADCC by M-CSF-activated monocytes. Optimal activation occurred with 100-300 U/ml of M-CSF, and required 9-11 d for completion. Most of the M-CSF cultured monocytes expressed the low-affinity Fc receptor (CD16). ADCC by cells of the monocyte/macrophage lineage using murine IgG3 mAbs may have significance for the immunotherapy of human malignancies.

Granulocyte/macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells.

A panning method has been developed to enrich Langerhans cells (LC) from murine epidermis. In standard culture media, the enriched populations progressively lose viability over a 3-d interval. When the cultures are supplemented with keratinocyte-conditioned medium, LC viability is improved and the cells increase in size and number of dendritic processes. Accessory function, as monitored by stimulating activity in the mixed lymphocyte reaction (MLR), increases at least 10-20-fold. The conditioned media of stimulated macrophages and T cells also support the viability and maturation of cultured LC. A panel of purified cytokines has been tested, and only granulocyte/macrophage colony-stimulating factor (GM-CSF) substitutes for bulk-conditioned medium. The recombinant molecule exhibits half-maximal activity at 5 pM. Without activity are: IL-1-4; IFN-alpha/beta/gamma; cachectin/TNF; M- and G-CSF. A rabbit anti-GM-CSF specifically neutralizes the capacity of keratinocyte-conditioned medium to generate active LC. However, GM-CSF is not required for LC function during the MLR itself. We conclude that the development of immunologically active LC in culture is mediated by GM-CSF. The observation that these dendritic cells do not respond to lineage-specific G- and M-CSFs suggests that LC represent a distinct myeloid differentiation pathway. Because GM-CSF can be made by nonimmune cells and can mediate the production of active dendritic cells, this cytokine provides a T-independent mechanism for enhancing the sensitization phase of cell-mediated immunity.

Synthesis of granulocyte colony-stimulating factor and its requirement for terminal divisions in chronic myelogenous leukemia.

In this paper we demonstrate that maturing neoplastic cells from patients with chronic myelogenous leukemia (CML) constitutively produce G-CSF and are also receptive for this molecule. G-CSF functions as an autocrine growth factor in stable phase CML, and thus is responsible for divisions of maturing leukemic cells leading to an expansion of the compartment of mature cells. This observation is well in line with in vivo features of CML in stable phase, i.e., the hyperplasia of the mature granulocyte compartment. In acute blastic phase of CML expression of the G-CSF gene seems to be less common and not related to autonomous blast growth.

Gamma interferon and lymphotoxin, released by activated T cells, synergize to inhibit granulocyte/monocyte colony formation.

We have shown that lymphocytes stimulated by PHA produce colony-forming unit of granulocyte/monocyte (CFU-GM)-stimulating and -inhibiting activities, IFN-gamma, and lymphotoxin (LT). IFN-gamma is necessary for inhibition of CFU-GM by PHA-conditioned medium (CM), as shown by experiments in which removal of IFN-gamma from PHA-CM abrogated inhibition. However, experiments in which rIFN-gamma was added to IFN-gamma-depleted PHA-CM revealed the presence, in PHA-CM, of other factors that act in synergy with IFN-gamma to inhibit CFU-GM. Fractionation of PHA-CM on a Sephadex G-100 column was used to separate IFN-gamma and LT. Colony-inhibiting activity was eluted in fractions that contained both IFN-gamma and LT activities, identifying LT as a factor present in PHA-CM that synergizes with IFN-gamma to inhibit CFU-GM. Treatment of PHA-CM with mAb against either IFN-gamma or LT completely abrogated the colony-inhibiting activity, demonstrating a requirement for both lymphokines in PHA-CM-induced inhibition of CFU-GM. Experiments using rIFN-gamma and preparations of purified LT confirmed that neither lymphokine alone, when added to bone marrow cells at the concentrations present in PHA-CM, strongly inhibited day 7 or day 14 CFU-GM, but that the two lymphokines, added together, behaved synergistically to inhibit CFU-GM by up to 70%. The inhibition observed using purified preparations of lymphokines shows that synergy between IFN-gamma and LT is sufficient to explain PHA-CM-induced inhibition of CFU-GM. Our findings suggest that activated T cells regulate hematopoiesis through the release of inhibitory as well as stimulatory factors, and that the simultaneous production of IFN-gamma and LT may represent a mechanism of suppression of hematopoiesis in the cases of bone marrow failure associated with the presence of activated T cells.

Cytokines in human milk.

Epidemiologic studies conducted in the past 30 years to investigate the protective functions of human milk strongly support the notion that breastfeeding prevents infantile infections, particularly those affecting the gastrointestinal and respiratory tracts. However, more recent clinical and experimental observations also suggest that human milk not only provides passive protection, but also can directly modulate the immunological development of the recipient infant. The study of this remarkable defense system in human milk has been difficult because of its biochemical complexity, the small concentration of certain bioactive components, the compartmentalization of some of these agents, the dynamic quantitative and qualitative changes of milk during lactation, and the lack of specific reagents to quantify these agents. However, a host of bioactive substances, including hormones, growth factors, and immunological factors such as cytokines, have been identified in human milk. Cytokines are pluripotent polypeptides that act in autocrine/paracrine fashions by binding to specific cellular receptors. They operate in networks and orchestrate the development and functions of immune system. Several different cytokines and chemokines have been discovered in human milk in the past years, and the list is growing very rapidly. This article will review the current knowledge about the increasingly complex network of chemoattractants, activators, and anti-inflammatory cytokines present in human milk and their potential role in compensating for the developmental delay of the neonate immune system.

Immune factors in human embryo culture and their significance.

There is increasing evidence that human development before implantation is regulated by embryonically and maternally derived growth factors. The "regulators" of embryonic origin such as soluble human leukocyte antigen G, platelet-activating factor, Th1/Th2 cytokines, insulin-like growth factor, epidermal growth factor, transforming growth factor alpha, colony-stimulating factor, platelet-derived growth factor may be used as indicators of embryo viability and implantation potential. The data prove the influence of growth factors on the development and growth of preimplantation embryos. Though there is a lot of research in the field of biomarkers during folliculogenesis and maternal-fetal interface, only few of them deal with regulators derived from embryonic cells to the cultivation medium. The aim of our study was to summarize the research dealing with immune markers produced by embryos in vitro and to estimate their impact on the cell growth, viability and implantation potential.

[A two-phase culture system for megakaryocyte differentiation of human mobilized peripheral blood CD34+ cells].

In our study, a two-phase culture system was developed to acquire large amount of CD41+ and polyploidy cells. Human mobilized peripheral blood CD34+ (PB CD34+) cells were first cultured in expansion medium (Cocktail or CC100 medium) for 3,4,5 or 6 days, and then cultured in megakaryocytic differentiation medium containing TPO and SCF for additional 7, 8 or 9 days. Cell expansion, morphology, CD41+ cell percentage and DNA content were investigated to evaluate the protocol. The result showed that more CD41+ and polyploidy cells could be obtained following the two-phase culture with Cocktail medium than with CC100. Moreover, with 3 days expansion in Cocktail medium plus 7 days in differentiation medium, the initial CD 34+ cells obtained 16-fold expansion of CD41+ cells and 3-fold expansion of polyploidy cells, such obtained level being significantly higher than that of culturing cells with only one step in TPO or TPO+SCF. We conclude that with the two-phase culture system, PB CD34+ cells can expand and differentiate to more CD41+ and polyploidy cells than those cultured only in accordance to the one-stage culture protocol, so a new and highly efficient megakaryocyte differentiation model for megakaryocyte and platelet related researches is provided already.

Limitation of excessive myelopoiesis by the intrinsic modulation of macrophage-derived prostaglandin E.

The clonal proliferation of the committed granulocyte-macrophage stem cell is controlled by a balance between mutually opposing factors, colony stimulating factor and prostaglandin E, both of monocyte-macrophage derivation. Increases beyond a critical concentration of colony stimulating factor within the local milieu of the mononuclear phagocyte induces the coincident elaboration of prostaglandin E, a self-regulated response which serves to limit the unopposed humoral stimulation of myelopoiesis.

The granulocyte-macrophage colony-stimulating factors.

The granulocyte-macrophage colony-stimulating factors are well-characterized specific glycoproteins that interact to control the production, differentiation, and function of two related white cell populations of the blood, the granulocytes and monocyte-macrophages. Widely produced in the body, these regulators probably play an important role in resistance to infections. The proliferation of myeloid leukemia cells remains dependent on stimulation by colony-stimulating factors, although one of them also has the ability to suppress leukemic populations by inducing terminal differentiation.

Simultaneous effects of erythropoietin and colony-stimulating factor on bone marrow cells.

Erythropoietin or colony-stimulating factor, or both, were added to rat or mouse marrow cell cultures, and the responses to each inducer were measured. Colony-stimulating factor caused the suppression of erythropoietin-stimulated hemoglobin synthesis, and erythropoietin caused the suppression of the granulocyte-macrophage colony formation that is dependent on colony-stimulating factor. The extent of suppression by each inducer was dose-dependent. Marrow cells from plethoric rats were more sensitive to suppression of erythropoietin action by colony-stimulating factor than were normal marrow cells. These findings suggest that either (i) the receptors for erythropoietin and for colony-stimulating factor have overlapping specificities and that the "wrong" inducer may bind without having an inductive effect, or (ii) the target cells for erythropoietin and colony-stimulating factor are very closely related or are the same.

Control of granulocytes and macrophages: molecular, cellular, and clinical aspects.

The production and functional activity of two important white blood cells, the granulocytes and macrophages, are regulated mainly by a group of glycoprotein colony-stimulating factors. The colony-stimulating factors have been mass-produced with recombinant technology and are now proving of value in preventing or suppressing infections in a variety of individuals with subnormal or defective formation of blood cells.

The molecular control of blood cell development.

The establishment of a cell culture system for the clonal development of blood cells has made it possible to identify the proteins that regulate the growth and differentiation of different blood cell lineages and to discover the molecular basis of normal and abnormal cell development in blood forming tissues. A model system with myeloid blood cells has shown that (i) normal blood cells require different proteins to induce cell multiplication (growth inducers) and cell differentiation (differentiation inducers), (ii) there is a hierarchy of growth inducers as cells become more restricted in their developmental program, and (iii) a cascade of interactions between proteins determines the correct balance between immature and mature cells in normal blood cell development. Gene cloning has shown that there is a family of different genes for these proteins. Normal protein regulators of blood cell development can control the abnormal growth of certain types of leukemic cells and suppress malignancy by inducing differentiation to mature nondividing cells. Chromosome abnormalities that give rise to malignancy in these leukemic cells can be bypassed and their effects nullified by inducing differentiation, which stops cells from multiplying. These blood cell regulatory proteins are active in culture and in the body, and they can be used clinically to correct defects in blood cell development.