Cholesterol loading suppresses the atheroinflammatory gene polarization of human macrophages induced by colony stimulating factors.

In atherosclerotic lesions, blood-derived monocytes differentiate into distinct macrophage subpopulations, and further into cholesterol-filled foam cells under a complex milieu of cytokines, which also contains macrophage-colony stimulating factor (M-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF). Here we generated human macrophages in the presence of either M-CSF or GM-CSF to obtain M-MØ and GM-MØ, respectively. The macrophages were converted into cholesterol-loaded foam cells by incubating them with acetyl-LDL, and their atheroinflammatory gene expression profiles were then assessed. Compared with GM-MØ, the M-MØ expressed higher levels of CD36, SRA1, and ACAT1, and also exhibited a greater ability to take up acetyl-LDL, esterify cholesterol, and become converted to foam cells. M-MØ foam cells expressed higher levels of ABCA1 and ABCG1, and, correspondingly, exhibited higher rates of cholesterol efflux to apoA-I and HDL2. Cholesterol loading of M-MØ strongly suppressed the high baseline expression of CCL2, whereas in GM-MØ the low baseline expression CCL2 remained unchanged during cholesterol loading. The expression of TNFA, IL1B, and CXCL8 were reduced in LPS-activated macrophage foam cells of either subtype. In summary, cholesterol loading converged the CSF-dependent expression of key genes related to intracellular cholesterol balance and inflammation. These findings suggest that transformation of CSF-polarized macrophages into foam cells may reduce their atheroinflammatory potential in atherogenesis.

Pattern of human monocyte subpopulations in health and disease.

Monocytes are important cells of the innate system. They are a heterogeneous type of cells consisting of phenotypically and functionally distinct subpopulations, which play a specific role in the control, development and escalation of the immunological processes. Based on the expression of superficial CD14 and CD16 in flow cytometry, they can be divided into three subsets: classical, intermediate and non-classical. Variation in the levels of human monocyte subsets in the blood can be observed in patients in numerous pathological states, such as infections, cardiovascular and inflammatory diseases, cancer and autoimmune diseases. The aim of this review is to summarize current knowledge of human monocyte subsets and their significance in homeostasis and in pathological conditions.

[Undifferentiated carcinoma of the jejunum producing granulocyte colony-stimulating factor].

An 80-year-old man presented with abdominal fullness and vomiting. Laboratory data revealed severe anemia, an inflammatory response, and elevated white blood cell counts. Abdominal computed tomography indicated ileus caused by a jejunal tumor measuring 8cm in diameter. Although small-bowel endoscopy enabled visualization of the tumor, adequate biopsy specimens could not be obtained for accurate diagnosis. The patient's condition rapidly deteriorated, because of which surgical treatment could not be initiated. The patient died approximately 3 weeks after admission. High serum granulocyte colony-stimulating factor (G-CSF) levels were detected at autopsy. Immunohistochemical staining of the autopsy specimen indicated positive G-CSF levels in the jejunal tumor. On the basis of these findings, a final diagnosis of undifferentiated carcinoma of the jejunum producing G-CSF was made.

Sulodexide down-regulates the release of cytokines, chemokines, and leukocyte colony stimulating factors from human macrophages: role of glycosaminoglycans in inflammatory pathways of chronic venous disease.

Chronic venous disease (CVeD) is a debilitating condition that affects millions of individuals worldwide. The condition can result in varicose veins, or advance to severe skin changes and venous ulceration. The fundamental basis for CVeD is inflammation within the venous circulation and that it is subjected to increased hydrostatic pressure resulting in increased ambulatory venous pressure. The inflammation involves leukocytes, in particular macrophages and monocytes, inflammatory modulators and chemokines, cytokine expression, growth factors, metalloproteinase (MMP) activity, and many regulatory pathways that perpetuate inflammation. Sulodexide (SDX) is a glycosaminoglycan with pro-fibrinolytic and anti-thrombotic properties. We have previously demonstrated that SDX inhibits the secretion of pro-zymogen MMP-9 from human leukocytes without displacing high molecular complexes of MMP-9. The anti-inflammatory properties of SDX on activated leukocytes have not been well established. We hypothesized that SDX will reduce the secretion of inflammatory mediators from lipopolysaccharide (LPS)-stimulated macrophages. Therefore, we evaluated the effects of SDX on LPS-stimulated macrophage secretion of various inflammatory and anti-inflammatory cytokines, chemokines, and colony stimulating factors. We used microplatebased multiplex immunoassays. LPS-stimulated macrophages in vitro caused a substantial increase of interleukins, tumor necrosis factor, interferon, chemokines and colony stimulating factors. The addition of SDX caused both a dose-dependent and dose-independent decrease in nearly all of the inflammatory cytokines, chemokines and colony stimulating factors. These findings suggest that SDX has a significant effect on the release of inflammatory mediators from macrophages, and may be useful in the treatment of early and advanced CVeD.

Antigen presenting cells in a non-mammalian model system, the chicken.

The chicken has a different repertoire of tissues, cells and genes of the immune response compared to mammals, yet generally survives infection with viral, bacterial, protozoal and fungal pathogens, and also worms and ectoparasites, just like mammals. Poultry are also probably the most heavily vaccinated group of farmed animals. Antigen presentation to the adaptive immune response therefore obviously normally occurs efficiently in birds. Although comparatively much is known about macrophages and B cells in the chicken, there is as yet little work on the other, professional, antigen-presenting cells, the dendritic cells (DC). Birds also have at least two other sets of phagocytic cells, heterophils and thrombocytes, which may also have the ability to present antigen. Here we review the current state of knowledge about antigen presenting cells in the chicken, concentrating mainly on recent advances in our knowledge of DC.

Anticytokine autoantibodies in infectious diseases: pathogenesis and mechanisms.

Autoantibodies to cytokines occur in many different conditions and situations and can cause a wide range of disease, including pulmonary alveolar proteinosis, disseminated non-tuberculous mycobacterial disease, pure red-cell aplasia, and chronic mucocutaneous candidiasis. Anticytokine autoantibodies may also develop against exogenously administered cytokines, sometimes diminishing their effects or inhibiting the activity of the endogenous cytokine. Unlike primary congenital immunodeficiencies, autoantibodies may develop over time, wax and wane, and may change in titre or avidity. Naturally occurring autoantibodies to interferons α, ß, and γ, interleukins 1α, 2, 6, and 10, tumour necrosis factor, and granulocyte-macrophage colony-stimulating factor have been reported in healthy individuals and have been identified in rheumatological diseases, graft-versus-host disease, and cancer. Therapeutic antibodies, growth factors, other biological agents, and cytokines used to treat acute, chronic, malignant, and immune diseases may elicit or overcome autoantibodies, hence influencing the primary intended therapy. The increasing number of biologically active anticytokine autoantibodies being reported suggests that currently "idiopathic" diseases may someday be explained by neutralising or agonising autoantibodies. Their protean roles in causing, treating, preventing, and responding to disease, as well as simply maintaining normal homoeostasis, offer fascinating insights into the biology of immunity, inflammation, and infection.

Mycophenolic acid suppresses granulopoiesis by inhibition of interleukin-17 production.

Mycophenolic acid is a commonly used immunosuppressant after organ transplantation and in autoimmune diseases; however, myelosuppression is a major complication despite its largely favorable side-effect profile. Mycophenolic acid targets inosine monophosphate dehydrogenase, which is essential for T-cell proliferation. The T-cell cytokine interleukin-17 (IL-17 or IL-17A) and its receptor maintain normal neutrophilic granulocyte numbers in mice by induction of granulocyte-colony-stimulating factor. To test whether mycophenolic acid induces neutropenia by inhibiting IL-17-producing T cells, we treated C57Bl/6 mice with mycophenolate-mofetil (the orally available pro-drug) and found a dose-dependent decrease in blood neutrophils. This myelosuppressive effect was completely abolished in mice that lack the IL-17 receptor. Mycophenolic acid delayed myeloid recovery after bone marrow transplantation and decreased the percentage of IL-17-producing T cells in the spleen and thymus, and inhibited IL-17 production in human and mouse T cells in vitro. Injection of IL-17 during mycophenolic acid treatment overcame the suppression of the circulating neutrophil levels. Our study shows that mycophenolic acid suppresses neutrophil production by inhibiting IL-17 expression, suggesting that measurement of this interleukin might be useful in estimating the risk of neutropenia in clinical settings.

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.

The human cancer antigen mesothelin is more efficiently presented to the mouse immune system when targeted to the DEC-205/CD205 receptor on dendritic cells.

To develop a tumor vaccine directly targeting tumor antigen to dendritic cells in situ, we engineered human mesothelin (MSLN) into an antibody specific for mouse DEC-205, a receptor for antigen presentation. We then characterized both T cell and humoral responses to human MSLN and compared immunizing efficacy of DEC-205-targeted MSLN to nontargeted protein after a single-dose immunization. Targeting human MSLN to DEC-205 receptor induced stronger CD4(+) T-cell responses compared to high doses of mesothelin protein. Approximately 0.5% CD4(+) T cells were primed to produce IFN-gamma, tumor necrosis factor-alpha, and IL-2 via intracellular cytokine staining, and the T cells also could proliferate rapidly. The immune response exhibited breadth because the primed CD4(+) T cells responded to at least three epitopes in the H-2(b) background. Targeting MSLN protein to DEC-205 receptor also resulted in cross-presentation to CD8(+) T cells. Antibody responses against human MSLN were also detected in serum from primed mice by ELISA assays. In summary, targeting of MSLN to DEC-205 improves the induction of CD4(+) and CD8(+) T-cell immunity accompanied by an antibody response. DEC-205-targeting could be valuable for enhancing immunity to MSLN in cancers where this nonmutated protein is expressed.

Colony-stimulating factors in inflammation and autoimmunity.

Although they were originally defined as haematopoietic-cell growth factors, colony-stimulating factors (CSFs) have been shown to have additional functions by acting directly on mature myeloid cells. Recent data from animal models indicate that the depletion of CSFs has therapeutic benefit in many inflammatory and/or autoimmune conditions and as a result, early-phase clinical trials targeting granulocyte/macrophage colony-stimulating factor and macrophage colony-stimulating factor have now commenced. The distinct biological features of CSFs offer opportunities for specific targeting, but with some associated risks. Here, I describe these biological features, discuss the probable specific outcomes of targeting CSFs in vivo and highlight outstanding questions that need to be addressed.

Sarcoidosis--immunopathogenetic concepts.

Sarcoidosis is a chronic granulomatous disorder characterized by an accumulation of activated lymphocytes, predominantly T helper cells, expressing the Th1 phenotype and macrophages at sites of disease activity. Although the cause of sarcoidosis has not been elucidated, several lines of evidence suggest that granuloma formation results from exposure to one or more antigens, eliciting a T lymphocyte response. The induction and evolution of granuloma formation results from a complex interplay between diverse cell types, cytokines, and chemokines. Genetic polymorphisms may influence the clinical expression of the granuloma formation and disease outcome. This article discusses in depth the key cellular elements and signals that generate and orchestrate the sarcoid granulomatous response. The precise factors inciting the sarcoid granulomatous response have not yet been identified, but chronic exposure to microbial agents, their products, or inorganic substances may be important in the pathogenesis.

Vaccination of women with metastatic breast cancer, using a costimulatory gene (CD80)-modified, HLA-A2-matched, allogeneic, breast cancer cell line: clinical and immunological results.

MDA-MB-231, an HLA-A2(+), HER2/neu(+) allogeneic breast cancer cell line genetically modified to express the costimulatory molecule CD80 (B7-1), was used to vaccinate 30 women with previously treated stage IV breast cancer. Expression of CD80 conferred the ability to deliver a costimulatory signal and thereby improved the antigen presentation capability of the tumor cells to patient T cells in vitro. Patients were vaccinated with 10(7) or 10(8) irradiated gene-modified tumor cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) or BCG, three times at 2-week intervals and then monthly until progressive disease developed. GM-CSF-related flulike symptoms and minor injection site reactions were observed frequently. Prolonged disease stabilization was observed in four patients but no objective tumor regressions were seen. Immune responses were measured in matched peripheral blood samples collected before and after treatment from 9 of 15 patients treated at the 10(8) tumor cell dose. Four patients exhibited MHC class I-restricted cytokine production in response to the parental breast cancer cell line. One patient maintained an increased number of circulating tumor-specific, interferon gamma-secreting CD8(+) T cells for 24 months after the last vaccination. One patient exhibited a tumor-specific interleukin 5 response to an autologous tumor cell line. This immunization strategy proved to be safe and feasible, and induced tumor-specific immune responses in a minority of patients; however, no objective tumor regressions were observed.

Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodeficiency virus type 1 vaccines.

Heterologous "prime-boost" regimens that involve priming with plasmid DNA vaccines and boosting with recombinant viral vectors have been shown to elicit potent virus-specific cytotoxic T-lymphocyte responses. Increasing evidence, however, suggests that the utility of recombinant viral vectors in human populations will be significantly limited by preexisting antivector immunity. Here we demonstrate that the coadministration of plasmid chemokines and colony-stimulating factors with plasmid DNA vaccines markedly increases the immunogenicity of DNA prime-recombinant adenovirus serotype 5 (rAd5) boost and DNA prime-recombinant vaccinia virus (rVac) boost vaccine regimens in BALB/c mice. In mice with preexisting anti-Ad5 immunity, priming with the DNA vaccine alone followed by rAd5 boosting elicited only marginal immune responses. In contrast, cytokine-augmented DNA vaccine priming followed by rAd5 vector boosting was able to generate potent immune responses in mice with preexisting anti-Ad5 immunity. These data demonstrate that plasmid cytokines can markedly improve the immunogenicity of DNA prime-viral vector boost vaccine strategies and can partially compensate for antivector immunity.

Cytokines and the immune-testicular axis.

Cytokines are regulatory proteins involved in haematopoiesis, immune cell development, inflammation and immune responses. Several cytokines have direct effects on testicular cell functions, and a number of these are produced within the testis even in the absence of inflammation or immune activation events. There is compelling evidence that cytokines, in fact, play an important regulatory role in the development and normal function of the testis. Pro-inflammatory cytokines including interleukin-1 and interleukin-6 have direct effects on spermatogenic cell differentiation and testicular steroidogenesis. Stem cell factor and leukaemia inhibitory factor, cytokines normally involved in haematopoiesis, also play a role in spermatogenesis. Anti-inflammatory cytokines of the transforming growth factor-beta family are implicated in testicular development. Consequently, local or systemic up-regulation of cytokine expression during injury, illness or infection may contribute to the disruption of testicular function and fertility that frequently accompanies these conditions. The aim of this review is to provide a very brief summary of the extensive literature dealing with cytokines in testicular biology, and to follow this with some speculation concerning the significance of these molecules in interactions between the immune system and the testis.

Cytokines and HIV-1: interactions and clinical implications.

Cytokines play an important role in controlling the homoeostasis of the immune system. Infection with HIV results in dysregulation of the cytokine profile in vivo and in vitro. During the course of HIV-1 infection secretion of T-helper type 1 (Th1) cytokines, such as interleukin (IL)-2, and antiviral interferon (IFN)-gamma, is generally decreased, whereas production of T helper type 2 (Th2) cytokines, IL-4, IL-10, proinflammatory cytokines (IL-1, IL-6, IL-8) and tumour necrosis factor (TNF)-alpha, is increased. Such abnormal cytokine production contributes to the pathogenesis of the disease by impairing cell-mediated immunity. A number of cytokines have been shown to modulate in vitro HIV-1 infection and replication in both CD4 T lymphocytes and cells of macrophage lineage. HIV-inductive cytokines include: TNF-alpha, TNF-beta, IL-1 and IL-6, which stimulate HIV-1 replication in T cells and monocyte-derived macrophages (MDM), IL-2, IL-7 and IL-15, which upregulate HIV-1 in T cells, and macrophage-colony stimulating factor, which stimulates HIV-1 in MDM. HIV-suppressive cytokines include: IFN-alpha, IFN-beta and IL-16, which inhibit HIV-1 replication in T cells and MDM, and IL-10 and IL-13, which inhibit HIV-1 in MDM. Bifunctional cytokines such as IFN-gamma, IL-4 and granulocyte-macrophage colony-stimulating factor have been shown to have both inhibitory and stimulatory effects on HIV-1. The beta-chemokines, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta and RANTES are important inhibitors of macrophage-tropic strains of HIV-1, whereas the alpha-chemokine stromal-derived factor-1 suppresses infection of T-tropic strains of HIV-1. This review outlines the interactions between cytokines and HIV-1, and presents clinical applications of cytokine therapy combined with highly active antiretroviral therapy or vaccines.

Generation and characterization of a neutralizing monoclonal antibody against erythroid cell stimulating factor.

Erythroid cell stimulating factor (ESF) is present in mouse serum and has been reported to function in concert with erythropoietin (EPO) in the formation of erythroid cells in in vitro culture systems. We report here the generation and characterization of a monoclonal antibody (MAb) directed against ESF, with potent anti-ESF-neutralizing activity. A hybridoma-producing MAb to ESF was selected following enzyme-linked immunosorbent assay (ELISA)-based screening of 270 colonies obtained from a fusion of immunized mouse splenocytes with NS1 myeloma cells. Western blot analyses of mouse serum using this antibody specifically detected a single protein (approximate molecular weight of 60 kDa and 120 kDa, under reducing and nonreducing conditions, respectively) corresponding to ESF, with no reactivity to EPO. Furthermore, this MAb demonstrated reactivity to a protein similar in molecular mass, across species, showing reactivity in sera obtained from human, horse, goat, guinea pig, rabbit, and rat. Immuno-chemical characterization demonstrated this antibody to be of IgG3 isotype, bearing kappa light chains. Injection of this monoclonal anti-ESF antibody to exhypoxic polycythemic mice at 6 and 24 h after EPO injection significantly reduced 59Fe incorporation into red blood cells, demonstrating its ability to neutralize in vivo erythropoiesis in our mouse model system. Thus, this novel erythroid cell-specific MAb will be an invaluable tool for further delineating the physiological role of ESF in in vivo erythropoiesis.

Toxicology and pharmacokinetics of DTGM, a fusion toxin consisting of a truncated diphtheria toxin (DT388) linked to human granulocyte-macrophage colony-stimulating factor, in cynomolgus monkeys.

We developed a fusion toxin consisting of the catalytic and translocation domains of diphtheria toxin linked to human granulocyte-macrophage colony-stimulating factor (GM-CSF) (DTGM) for the treatment of patients with acute myeloid leukemia (AML). Our goal in this study was to determine the toxicity and pharmacokinetics of DTGM in cynomolgus monkeys (Macacca fascicularis), which possess cross-reactive GM-CSF receptors. Four groups of young adult monkeys (6 males and 12 females) were treated with five daily bolus iv infusions of 1, 5, 7.5, and 10 microgram/kg DTGM. Monkeys (2 males and 2 females) treated at 1 microgram/kg/day showed no significant side effects. Monkeys (2 males and 2 females) treated at 5 microgram/kg/day showed Grade 1-2 thrombopenia (NCI common toxicity criteria) on day 9. In contrast, monkeys (6 females) treated at 7.5 microgram/kg/day developed Grade 3 neutropenia, Grade 1-2 thrombopenia, Grade 1-3 anemia, and Grade 1-3 hypoalbuminemia. The neutropenia developed by day 4 in the 7.5 microgram/kg/day monkeys and by day 3 or 5 in the 10 microgram/kg/day monkeys and resolved in both groups by day 9, but the thrombopenia, anemia, and hypoalbuminemia persisted until day 16. Monkeys (2 male and 2 female) treated with 10 microgram/kg/day showed Grade 4 neutropenia that resolved by day 8 and Grade 2-3 anemia, hypoalbuminemia, and thrombopenia. Three of the animals developed sepsis. DTGM plasma half-life was 30 min with a peak concentration of 0.1 microgram/mL or 2 nM (1000-fold higher than the IC50 in vitro for AML blasts). Immune responses were minimal in all animals tested at 14 and 28 days with anti-DTGM levels <1 microgram/mL. All four animals at 10 microgram/kg died or were euthanized, and necropsies were performed. Animals necropsied on days 4 and 6 showed marked apoptosis and hypoplasia in the marrow, which was completely resolved for animals necropsied on day 9. No injury to other organs, including kidney, heart, liver, central nervous system, or lung, was seen. The drug was selectively toxic to malignant or differentiated myeloid cells with little toxicity to myeloid progenitors or other organs. Minimal effects in nontarget tissues make DTGM a promising candidate chemotherapeutic agent.

Cytokines, chemokines, and colony-stimulating factors in human milk: the 1997 update.

Epidemiologic studies conducted over the past 30 years to investigate the protective functions of human milk strongly support the notion that breast-feeding 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 due to 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. Nevertheless, 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 the immune system. Several different cytokines and chemokines have been discovered in human milk over 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.

A primer on cytokines: sources, receptors, effects, and inducers.

Protection against pathogens is a prerequisite for survival of most organisms. To cope with this continuous challenge, complex defense mechanisms have evolved. The construction, adaptation, and maintenance of these mechanisms are under control of an extensive network of regulatory proteins called cytokines. A great number of cytokines have been described over the last 2 decades. This review consists of an overview of cytokines that are involved in immune responses and describes some historical and general aspects as well as prospective clinical applications. Major biological effects together with information on cytokine receptors, producers, inducers, and biochemical and molecular characteristics are listed in tables. In addition, some basic information is given on cytokine receptor signal transduction. Finally, the recent discoveries of cytokine receptors functioning as coreceptors in the pathogenesis of human immunodeficiency virus are summarized.