The Wellcome Foundation lecture, 1986. The molecular control of normal and leukaemic granulocytes and macrophages

The development of semisolid culture methods supporting the clonal proliferation and maturation of granulocytes and macrophages led to the discovery of a group of specific glycoproteins, the colony-stimulating factors (CSFs), whose function it is to control the proliferation and functional activity of granulocytes, macrophages and associated blood cells. The four known CSFs in the mouse and man have been purified and complementary DNAs (cDNAs) for each have been cloned. The injection of bacterially synthesized recombinant CSF into mice has demonstrated that these CSFs can function in vivo to regulate granulocyte and macrophage formation. A major physiological role played by these CSFs is to control resistance to invading microorganisms through mechanisms capable of extremely rapid activation. Because the CSFs are the only known proliferative factors for these cells, the CSFs are involved in the initiation and the emergence of myeloid leukaemia but, conversely, at least one of the CSFs, G-CSF, is able to suppress myeloid leukaemic populations because of the ability of the CSFs to initiate differentiation commitment in responding granulocytic and macrophage populations. The CSFs are promising agents for clinical use in the treatment of infections in patients with depressed granulocyte-macrophage formation and possibly in the management of some types of myeloid leukaemia.     

Polypeptides controlling hematopoietic blood cell development and activation. II. Clinical results

Colony-stimulating factors (CSFs) have entered the clinical arena. Several investigators have explored, in first clinical phase I studies, different routes of administration to define the optimum biological dose, maximum tolerated dose, toxicity, and pharmacokinetics of these reagents. It has been demonstrated that recombinant human (rh) granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF) can be safely administered over a broad dose range to increase number of circulating granulocytes in man. More recently, GM-CSF and G-CSF have been involved in phase Ib/II studies to assess the granulopoietic responses of patients with granulocytopenia due to various underlying disease states including myelodysplastic syndrome, aplastic anemia, cyclic neutropenia, Kostmann's syndrome, and the acquired immuno-deficiency syndrome. Both factors were also investigated with respect to their potential to prevent chemotherapy induced granulocytopenia or to accelerate recovery from that condition. The short-term effects of rh GM-CSF after autologous bone marrow transplantation for various solid tumors and lymphoid malignancies were assessed as well. In this article we will focus on recent results that have emerged from in vivo studies utilizing CSFs.     

Clinical applications of recombinant human colony-stimulating factors.

The differentiation and maturation of hematopoietic progenitor cells are regulated by certain growth factors. Several of these glycoproteins have been characterized, and their amino acid sequences have been delineated. Modern DNA technology provides sufficient quantities of these hormones for testing in clinical trials. Erythropoietin (EPO) has been shown to increase the hemoglobin level and hematocrit in patients with end-stage renal disease. Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) can increase the numbers of neutrophils and monocytes, in a dose-dependent fashion. The function of granulocytes and monocytes is also enhanced. Clinical studies of the toxicity and activity of G-CSF and GM-CSF have been conducted in patients with acquired immune deficiency syndrome, aplastic anemia, myelodysplastic syndromes, and neutropenia due to cancer and chemotherapy. In almost all patients the neutrophil count increased within 24 hours after the start of treatment. Side effects of G-CSF and GM-CSF are infrequent and usually mild. Combinations of CSFs may be even more effective.     

Kinetics of the clonal proliferation of granulocytes and macrophages in cultures of mouse bone marrow cells as supported by two distinct types of colony-stimulating factors

Two different types of colony-stimulating factors (CSF) were used to support the clonal growth of myeloid progenitor cells (CFUc) in semi-solid agar or viscous methylcellulose cultures of mouse bone marrow cells. The cultures stimulated for 5 days with RSP-2-P3 cell CSF (CSFRSP) contained mainly granulocyte colonies, whereas the cultures stimulated for 10 days with human urine CSF (CSFhu) contained mainly monocyte/macrophage colonies. Four lines of study were carried out: 1) a kinetic study using combinations of the two types of CSFs in the same culture; 2) a study of transferring CFUc from the initial 3-day cultures to recipient cultures containing the same or different types of CSF; 3) an examination of the morphology over time of colonies that were confined by glass capillaries plunged in agar; and 4) electron microscopic observations on disintegrating granulocytes. The results of all these lines of study suggest that about one third of the CFUc can be stimulated both by CSFRSP and CSFhu while the other two thirds react specifically either with CSFRSP or with CSFhu. The present study also suggests that granulocytes in the culture stop proliferation and disintegrate while macrophages are still growing there. Thus, mixed-type colonies containing both macrophages and granulocytes later become macrophage colonies.     

Induction of colony-stimulating factors by Plasmodium cynomolgi components

Plasmodium cynomolgi total parasite antigens soluble in culture medium (P.c.SA), when injected in monkeys (Macaca mulatta) intravenously, induced the synthesis and secretion of serum colony-stimulating factors (CSFs). In vitro cultured monkey splenic macrophages and blood monocytes, following incubation with P.c.SA, also elaborated CSFs: the splenic macrophages responded more. Peak CSFs levels, both in vivo and in vitro, were attained after 8 hours of P.c.SA stimulation, and thereafter declined to baseline values within 48 hours. CSFs, both in serum and in conditioned medium, induced the formation of macrophage, granulocyte and granulocyte-macrophage colonies in vitro, in the same proportion, indicating that committed progenitor cells responded to CSF from both sources in a similar way. Polymyxin B treatment had no effect on P.c.SA stimulated CSF elaboration by macrophages, suggesting an LPS-independent mechanism of CSF induction. CSF synthesis appeared to be de novo, as cycloheximide treatment of macrophages completely inhibited CSF production. These observations indicate that P. cynomolgi components can induce CSF synthesis.     

Morphine modulation of plasmodial-antigens-induced colony-stimulating factors production by macrophages

Morphine abuse is known to cause immunosuppression and enhanced host susceptibility to malaria. We studied the effect of morphine on the Plasmodium berghei total-parasite-antigens soluble in culture medium (P.b.SA)-induced production of colony-stimulating factors (CSFs) by mouse peritoneal macrophages, in vitro. Morphine exerted a concentration-dependent biphasic modulatory effect; at 1 x 10(-4)-1 x 10 x 10(-6) M it slightly inhibited, whereas at 1 X 10(-8)-1 x 10(-10) M it augmented the production of CSFs. However, at 1 x 10(-12) M concentration the augmenting effect of morphine was significantly (p<0.05) diminished. Selective agonists of delta- (DPDPE) and mu- (DAGO) opioid receptors also respectively, inhibited and augmented the production of CSFs. The CSFs appear to be synthesized de novo as cycloheximide (50.0 microg/ml) completely inhibited their production. Naloxone ( 1 x 10(-5) M) lacked any effect on the inhibitory effect of morphine; however, at 1 x 10(-3) M it exerted partial blocking effect. Conversely, at 1 x 10(-5) M naloxone significantly (p<0.05) blocked the augmenting effect of morphine. These results suggest that morphine via opioid receptors, in a concentration-dependent biphasic manner, modulated the P.b.SA-induced de novo production of CSFs by macrophages, in vitro.     

Evaluation as a function of granulocytes in hyperimmunoglobulinemia E syndrome with recurrent infections

The hyper-IgE syndrome with recurrent infections (HIESRI) is characterized by skin and respiratory infections due to Staphylococcus aureus and several fungi infections which are frequently associated with tissue damage. A deficiency in the chemotaxis of phagocytic cells has been documented to explain these findings; however, the expression of adhesion molecules, the secretion of cytokines that activate granulocytes and the production of oxygen reactive molecules have not been evaluated in HIESRI. Six HIESRI patients were evaluated for the following parameters: (1) secretion of GM-CSF and IL-5 by mitogen and antigen-activated mononuclear cells, (2) the chemotactic response of FMLP-activated granulocytes, (3) the respiratory burst of PMA-activated granulocytes, and (4) the expression of L-selectin and CD11b in PMA-activated granulocytes. Human recombinant GM-CSF and culture supernatants were evaluated for capacity to modulate granulocytic function. Compared to controls, HIESRI patients showed a normal production of GM-CSF and an increase in the basal secretion of IL-5. No significant differences were observed for chemotaxis, respiratory burst or L-selectin and CD11b expression. The GM-CSF did not modulate these functions in granulocytes from HIESRI patients, but culture supernatants applied to granulocytes inhibited chemotaxis, increased respiratory burst and caused the shedding of L-selectin from the granulocyte surface. The 6 HIESRI patients were nonsymptomatic during the time of this research due to a program of continued treatment; findings suggest that granulocytes are activated more easily in response to proinflammatory factors and that production of these factors is higher in HIESRI.     

The role of phagocytes in the protective mechanisms of fish

Phagocytes are cells principally dedicated to the recognition and elimination of invading organisms and damaged tissue. Those described in fish are the granulocytes (particularly neutrophils) and mononuclear phagocytes (tissue macrophages and circulating monocytes). Their movement to sites of microbial invasion is an early event in the inflammatory response and the role of host-derived factors as attractants, such as eicosanoids, is discussed. Opsonins mediate the recognition between phagocyte and particle, and receptors for serum complement component C3 and the Fc fragment of opsonic antibody have been described. Fundamental to the protection offered by the phagocytes is their bactericidal larvacidal activity, which is closely associated with the production of oxygen free radicals. Phagocytes as accessory cells are discussed, including their role in antigen presentation. A knowledge of the modulation of phagocyte function, with activation by various substances and suppression by others, is important if protective responses are to be achieved by up-regulating phagocyte activity.     

Enkephalins-modulation of Plasmodium cynomolgi antigens-induced colony-stimulating factors elaboration by macrophages.

Methionine-enkephalin (M-Enk) and its analogue compound 82/205 (10(-5) and 10(-6) M) inhibited elaboration of Plasmodium cynomolgi total antigens soluble in culture medium (P.c.SA)-induced colony-stimulating factors (CSFs) by monkey blood monocyte-derived macrophages, in vitro. Paradoxically, lower concentrations (10(-7)-10(-9) M) of both the peptides greatly augmented CSFs elaboration; 82/205 appeared to be nearly 2.3-fold more potent. Naloxone (10(-5) M) pretreatment of macrophages inhibited only the M-Enk- and 82/205-induced enhanced CSFs elaboration, suggesting an opiate receptors-mediated mechanism of action. None of the peptides or naloxone (10(-5)-10(-9) M) had any direct effect on the CSF elaboration by unstimulated macrophages.     

IL-23 in inflammatory bowel diseases and colon cancer

Studies in recent years have identified a pivotal role of the cytokine IL-23 in the pathogenesis of inflammatory bowel diseases (IBD: Crohn´s disease, ulcerative colitis) and colitis-associated colon cancer. Genetic studies revealed that subgroups of IBD patients have single nucleotide polymorphisms in the IL-23R gene suggesting that IL-23R signaling affects disease susceptibility. Furthermore, increased production of IL-23 by macrophages, dendritic cells or granulocytes has been observed in various mouse models of colitis, colitis-associated cancer and IBD patients. Moreover, in several murine models of colitis, suppression of IL-12/IL-23 p40, IL-23 p19 or IL-23R function led to marked suppression of gut inflammation. This finding was associated with reduced activation of IL-23 target cells such as T helper 17 cells, innate lymphoid cells type 3, granulocytes and natural killer cells as well as with impaired production of proinflammatory cytokines. Based on these findings, targeting of IL-23 emerges as important concept for suppression of gut inflammation and inflammation-associated cancer growth. Consistently, neutralizing antibodies against IL-12/IL-23 p40 and IL-23 p19 have been successfully used in clinical trials for therapy of Crohn´s disease and pilot studies in ulcerative colitis are ongoing. These findings underline the crucial regulatory role of IL-23 in chronic intestinal inflammation and colitis-associated cancer and indicate that therapeutic strategies aiming at IL-23 blockade may be of key relevance for future therapy of IBD patients.     

Gadd45a and Gadd45b modulate innate immune functions of granulocytes and macrophages by differential regulation of p38 and JNK signaling

Gadd45 proteins function as stress sensors in response to various physiological and environmental stressors, interacting with other cellular proteins implicated in cellular stress responses, including p38 and JNK. This study shows that mice lacking either Gadd45a or Gadd45b are defective in the recruitment of granulocytes and macrophages to the intra-peritoneal cavity following intra-peritoneal administration of the bacterial cell wall pathogen-associated molecular pattern lipopolysaccharide (LPS). Bone marrow derived granulocytes and macrophages lacking either Gadd45a or Gadd45b are shown to be impaired in their chemotactic response to LPS, as well as other inflammatory stimuli such as N-formyl-methionine-leucine-phenylalanine and IL-8. Evidence was obtained also implicating Gadd45a and Gadd45b in other myeloid innate immune functions, including reactive oxygen species production, phagocytosis, and adhesion. Gadd45a and Gadd45b activation of p38 kinase was implicated in the response of granulocytes to LPS mediated chemotaxis, whereas Gadd45a and Gadd45b curtailment of JNK activation was linked to chemotaxis of macrophages in response to LPS. Collectively, these data highlight a novel role for both Gadd45a and Gadd45b in myeloid innate immune functions by differential modulation of p38 and JNK signaling in granulocytes compared to macrophages.     

Influence of HIV-infection on the phagocytic activity of monocytes/macrophages and granulocytes

Because of the great importance of phagocytosis as a key process in host defence, the influence of HIV-infection on the phagocytic activity of monocytes/macrophages (M0/MAC) and granulocytes was investigated. Therefore, blood samples from the peripheral blood of 70 HIV-infected individuals were incubated with fluorescein isothiocyanate (FITC) labeled Escherichia coli. The uptake of the bacteria was monitored by flow cytometer analysis. A strong and significant increase in the relative number of phagocytic granulocytes was observed ranging from 12.8% in an uninfected control collective to over 30% in AIDS patients. This effect was obtained for all patients and independent of the stage of disease. For monocytes, only marginal changes were found in their phagocytic function. These data suggest that the high susceptibility of HIV patients for secondary infections is not linked to a loss of phagocytic ability of monocytes/macrophages and/or granulocytes.     

IL-12 induction by a TH1-inducing adjuvant in vivo: dendritic cell subsets and regulation by IL-10

IL-12 induction is critical for immune responses against many viruses and intracellular bacterial pathogens. Recent studies suggest that IL-12-secreting dendritic cells (DC) are potent Th1-inducing APC. However, controversy exists concerning the function of DC subsets. Murine studies have suggested that CD8(+) DC preferentially induce Th1 responses, whereas CD8(-) DC induce Th2 development; in this model, different DC subsets prime different responses. Alternatively, the propensity of DC subsets to prime a Th1 response could depend upon the type of initial stimulus. We used a prototypic Th1-inducing adjuvant, heat-killed Brucella abortus (HKBA) to assess stimulation of DC subsets, relationship between Ag burden and IL-12 production, and down-regulation of DC subset IL-12 production by IL-10. In this study, we show that DC were sole producers of IL-12, although most HKBA uptake was by splenic macrophages and granulocytes. More CD8(-) than CD8(+) DC produced IL-12 after HKBA challenge, whereas only CD8(+) DC produced IL-12 after injection of another Th1-promoting microbial substance, soluble Toxoplasma gondii Ags. Studies in IL-10-deficient mice revealed that IL-10 down-regulates frequency and duration of IL-12 production by both DC subsets. In the absence of IL-10, IL-12 expression is enabled in CD11c(low) cells, but not in macrophages or granulocytes. These findings support the concept of DC as the major IL-12 producers in spleens, but challenge the notion that CD8(+) and CD8(-) DC are destined to selectively induce Th1 or Th2 responses, respectively. Thus, the nature of the stimulating substance is important in determining which DC subsets are activated to produce IL-12.     

Enhanced chemiluminescence ELISA for Listeria specific antigen in cerebrospinal fluid using an FITC-anti-FITC system

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of a soluble Listeria monocytogenes serogroup 4 antigen in cerebrospinal fluid samples (CSFs). In the ELISA an anti-Listeria monoclonal antibody, immobilized onto assay wells, was used to capture antigen from CSFs. the captured antigen was then reacted with a fluorescein isothiocyanate (FITC) conjugate of the same anti-Listeria antibody, which was detected with a horseradish peroxidase conjugate of a monoclonal antibody to FITC. The presence of antigen was detected by an enhanced chemiluminescence assay using a camera luminometer. Antigen was detected in the CSFs taken from five out of seven patients with culture proven L. monocytogenes serogroup 4 central nervous system infections, and in none of the CSFs taken from 25 other patients.     

Myelopoiesis during Zebrafish Early Development

Myelopoiesis is the process of producing all types of myeloid cells including monocytes/macrophages and granulocytes.Myeloid cells are known to manifest a wide spectrum of activities such as immune surveillance and tissue remodeling.Irregularities in myeloid cell development and their function are known to associate with the onset and the progression of a variety of human disorders such as leukemia.In the past decades,extensive studies have been carried out in various model organisms to elucidate the molecular mechanisms underlying myelopoiesis with the hope that these efforts will yield knowledge translatable into therapies for related diseases.Zebrafish has recently emerged as a prominent animal model for studying myelopoiesis,especially during early embryogenesis,largely owing to its unique properties such as transparent embryonic body and external development.This review introduces the methodologies used in zebrafish research and focuses on the recent research progresses of zebrafish myelopoiesis.     

Chlamydia pneumoniae Hides inside Apoptotic Neutrophils to Silently Infect and Propagate in Macrophages

Background

Intracellular pathogens have developed elaborate strategies for silent infection of preferred host cells. Chlamydia pneumoniae is a common pathogen in acute infections of the respiratory tract (e.g. pneumonia) and associated with chronic lung sequelae in adults and children. Within the lung, alveolar macrophages and polymorph nuclear neutrophils (PMN) are the first line of defense against bacteria, but also preferred host phagocytes of chlamydiae.

Methodology/Principal Findings

We could show that C. pneumoniae easily infect and hide inside neutrophil granulocytes until these cells become apoptotic and are subsequently taken up by macrophages. C. pneumoniae infection of macrophages via apoptotic PMN results in enhanced replicative activity of chlamydiae when compared to direct infection of macrophages, which results in persistence of the pathogen. Inhibition of the apoptotic recognition of C. pneumoniae infected PMN using PS- masking Annexin A5 significantly lowered the transmission of chlamydial infection to macrophages. Transfer of apoptotic C. pneumoniae infected PMN to macrophages resulted in an increased TGF-ß production, whereas direct infection of macrophages with chlamydiae was characterized by an enhanced TNF-α response.

Conclusions/Significance

Taken together, our data suggest that C. pneumoniae uses neutrophil granulocytes to be silently taken up by long-lived macrophages, which allows for efficient propagation and immune protection within the human host.     

The effect of the cell separation procedure on neutrophilic granulocyte function. II. Research on the bactericidal capacity and the NBT reduction ability of isolated granulocytes

The investigations were aimed at examining the impact of methodically simple separating and enriching techniques for gaining granulocytes on the bactericidal capacity and NBT reduction in isolated granulocytes. For this purpose, granulocytes were gained by the technique of sedimentation and flotation. Granulocytes isolated by the cell separator Haemonetics model 30 were used as reference material. The findings of the investigations together with those of previous studies revealed no indication of the granulocyte function and morphology being damaged by the separating techniques used.     

Phagocytosis and killing of Staphylococcus aureus by mouse granulocytes after interleukin-3 (IL-3) injection

Interleukin-3 is a multipotential hematopoietic growth factor, which like other colony stimulating factors (CSFs) is effective "in vitro" stimulation of the mature cells function. It was found that IL-3 synergistically with GM-CSF and G-CSF stimulated the proliferation of the granulocytes. Therefore the purpose of this investigation was the evaluation "in vivo" of the influence of IL-3 on the phagocytosis, bactericidal activity, and enzyme activities of granulocytes. IL-3 was injected into mice subcutaneously during 5 days in dose 1 microgram/kg/d. The examination of the percent of cells phagocytizing bacteria (Staphylococcus aureus), NBT test and bactericidal activity, were performed every day and evident increase of the tested parameters was found. Additionally the enzyme activities in primary granules were measured and showed on increase of acid phosphatase and peroxidase activity.