Restoration of the acute phase response after infection in cyclophosphamide-treated mice by granulocyte colony-stimulating factor

The therapeutic effect of granulocyte colony-stimulating factor (G-CSF) against intramuscular infection withPseudomonas aeruginosa in cyclophosphamide (CY)-treated mice was analyzed by measuring plasma levels of amyloid P-component (APC) and proinflammatory cytokine levels. CY (100mg/kg) treatment of mice significantly suppressed plasma concentrations of APC and tumor-necrosis factor- (TNF-) following infection withP. aeruginosa, in associated with enhanced susceptibility of the treated mice to this bacterium. A 4-day treatment of CY-treated mice with recombinant human G-CSF (rhG-CSF) increased resistance of CY-treated mice, together with the marked restoration of APC and TNF- productions. The capacity to produce interleukin 1- and TNF- of peritoneal macrophages and also that to produce IL-6 of spleen cells were significantly enhanced by thein vivo administration of rhG-CSF in CY-treated mice. These results indicate that G-CSF may increase the functions of monocytes/macrophages directly or indirectlyin vivo. Therefore, the therapeutic effect of rhG-CSF seems to consist of not only increases in the number and functions of neutrophills but also enhancement of monocyte/macrophage functions.Abbreviations rhG-CSF recombinant human granulocyte-colony stimulating factor - PMNs polymorphonuclear leukocytes - CY cyclophosphamide - HBSS Hanks' balanced salt solution - APC amyloid P-component - IEP immunoelectrophoresis - CFU colony-forming units - TNF- tumor-necrosis factor- - d IL interleukin     

Production of granulocyte colony-stimulating factor by head and neck carcinomas

Detectable levels of G-CSF by enzyme-linked immunosorbent assay (ELISA) were found in sera of 4 out of 15 patients with head and neck carcinomas. Also cells prepared from the tumors of these 4 patients secreted G-CSF. The supernatants of cells derived from all 15 patients did not contain granulocyte-monocyte CSF, monocyte CSF, tumor necrosis factor-, transforming growth factor- ₁, epidermal growth factor, interleukin (IL)-1 and IL-6. These findings suggest that leukocytosis in patients with carcinomas might be due to the production of G-CSF by tumor cells.Abbreviations CSF colony stimulating factor - EGF epidermal growth factor - ELISA Enzyme-linked immunosorbent assay - G granulocyte - GM granulocyte-monocyte - IL interluekin - M monocyte - TGF transforming growth factor - TNF tumor necrosis factor     

Production of granulocyte/macrophage colony-stimulating factor by cultured astrocytes

We investigated the production of interleukin-3 (IL-3)-like factor by murine astrocytes. Supernatants from lipopolysaccharide (LPS)-stimulated astrocytes induced proliferation of IC-2, an IL-3- and granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent cell line. This activity was completely neutralized by the antibody against GM-CSF but not by the anti-IL-3 monoclonal antibody. Northern blot analysis revealed the expression of GM-CSF mRNA, but not of IL-3 mRNA, in cultured astrocytes. These results indicate that with proper stimuli murine astrocytes produce GM-CSF.     

Production of soluble granulocyte colony-stimulating factor receptors from myelomonocytic cells

It has been speculated that a soluble form of G-CSFR might be physiologically present in humans, since G-CSFR mRNA that lacks a transmembrane domain has been identified from a human myelomonocytic cell line. Here, we demonstrate human soluble G-CSFR (sG-CSFR) of two different molecular sizes (80 and 85 kDa) on an immunoblot analysis using Abs generated against the amino-terminal, extracellular domain of the full-length G-CSFR. Both isoforms of sG-CSFR were able to bind recombinant human G-CSF (rhG-CSF). RT-PCR analysis with primers targeted outside of the transmenbrane region revealed that membrane-anchored G-CSFR is expressed at all maturation stages of purified myeloid cells, including CD34+CD13+ cells (blasts), CD11b-CD15+ cells (promyelocytes or myelocytes), CD11b+CD15+ cells (metamyelocytes and mature neutrophils), and CD14+ cells (monocytes). On the other hand, sG-CSFR mRNA was detectable in CD11b-CD15+, CD11b+CD15+, and CD14+ cells, but not in the CD34+CD13+ blast population. The serum concentration of both isoforms of sG-CSFR appeared to be correlated with the numbers of neutrophils/monocytes before and after rhG-CSF treatment in normal individuals. Thus, two isoforms of sG-CSFR are physiologically secreted from relatively mature myeloid cells and might play an important role in myelopoiesis through their binding to serum G-CSF.     

Nuclear lncRNA stabilization in the host response to bacterial infection

Long non‐coding RNAs (lncRNAs) play important roles in many cellular pathways, but their contribution to the defense of eukaryotic cells against pathogens remains poorly understood. A new study from Imamura et al in The EMBO Journal reports that Salmonella infection in human cells impacts nuclear RNA decay, which in turn drives the accumulation of otherwise unstable nuclear lncRNAs, some of which may have protective effects against this common bacterial pathogen. These unexpected findings demand more efforts to fully decrypt the molecular functions of lncRNAs in innate and adaptive immunity.     

Endogenous granulocyte colony-stimulating factor: a biomarker in acute ischemic stroke

Granulocyte colony-stimulating factor (G-CSF) may protect ischemic brain injury either in animal or human. No studies have reported that endogenous G-CSF (enG-CSF) level is related to the severity of ischemic stroke. This study was designed to assess the severity of ischemic patients correlated with the alteration of enG-CSF on the 1st day after an ischemic event. Patient's plasma enG-CSF and scoring of National Institute of Health Stroke Scale were measured on the 1st day after ischemic stroke. The acute ischemic stroke could significantly induce enG-GCF secretion as compared with healthy control group (16.77 vs. 22.86 μg/L, p = 0.001). Elevated enG-CSF concentration was positively correlated with the severity of stroke patients on day 1 after the event (p = 0.006; Spearman correlation coefficient = 0.268). The enG-CSF is a good biomarker for prediction of severity of acute ischemic stroke.     

Involvement of the acute phase protein alpha 1-acid glycoprotein in nonspecific resistance to a lethal gram-negative infection

Resistance to gram-negative infection can be induced by pretreating animals with several agents such as turpentine and interleukin (IL)-1. Because these agents are powerful inducers of acute phase proteins, we wondered whether these proteins, more particularly alpha(1)-acid glycoprotein (alpha(1)-AGP), are involved in nonspecific resistance to infection. Turpentine and IL-1 protect completely against a lethal challenge of Klebsiella pneumoniae when given 48 and 12-48 h before the challenge, respectively. alpha(1)-AGP induction in the serum reached peak values 48 h after turpentine and 12-48 h after IL-1 injection. Administration of alpha(1)-AGP, 2 h before a challenge of K. pneumoniae, significantly increased the survival. Numbers of bacteria cultured from blood and organs were significantly lower in mice pretreated with a protective dose of turpentine, IL-1, or alpha(1)-AGP. These data suggest that alpha(1)-AGP is a possible mediator in turpentine- or IL-1-induced protection because time points of maximal induction of alpha(1)-AGP by turpentine or IL-1 and of optimal protection by alpha(1)-AGP coincide. Transgenic overexpression of rat alpha(1)-AGP protected mice from a K. pneumoniae infection. Bacterial counts in blood and organs were significantly lower in transgenic mice, and only in control mice were large necrotic areas, apoptosis, and blood clots observed in the spleen. Our data suggest that alpha(1)-AGP prevents gram-negative infections and may be an essential component in nonspecific resistance to infection.     

Correlation between the proliferative response to granulocyte colony-stimulating factor and the positivity of transferrin receptor in acute myeloblastic leukemia cells.

The use of granulocyte colony-stimulating factor (G-CSF) after chemotherapy for acute myeloblastic leukemia (AML) has been reported. However, there is a drawback in that G-CSF may stimulate the proliferation of AML progenitors. To determine the parameter(s) indicative of responsiveness of AML blasts to G-CSF, various surface phenotypes of blasts were examined in relation to the blast colony formation stimulated by G-CSF in 39 AML patients. A correlation was found only with transferrin receptor positivity among the various phenotypes studied. The population mean of percentages of transferrin receptor-positive blasts in the group responding to G-CSF in vitro was significantly higher than that of blasts in the group not responding to G-CSF. A further correlation was found between transferrin receptor positivity and the number of G-CSF receptors on the blasts; that is, blasts expressing more G-CSF receptors have greater transferrin receptor positivity. In our previous study, we observed that blasts with a large number of G-CSF receptors produce more colonies in response to G-CSF. These results indicated that blasts expressing more transferrin receptors have a larger number of G-CSF receptors and may show more active proliferation in response to G-CSF. Therefore, the proliferative response of blasts to G-CSF can be predicted by examining transferrin receptor positivity. The clinical use of G-CSF in AML patients may be recommended when the patient's blasts have a low level of transferrin receptor expression. The measurement of transferrin receptors on blasts, instead of the rather complicated G-CSF receptor determination, would be a useful indicator for the safer application of G-CSF in AML patients.     

Helminth-primed dendritic cells alter the host response to enteric bacterial infection

To examine whether intestinal helminth infection may be a risk factor for enteric bacterial infection, a murine model was established using the intestinal helminth Heligomosomoides polygyrus and a murine pathogen Citrobacter rodentium, which causes infectious colitis. Using this model we recently have shown that coinfection with the Th2-inducing H. polygyrus and C. rodentium promotes bacterial-associated disease and colitis. In this study, we expand our previous observations and examine the hypothesis that dendritic cells (DC) stimulated by helminth infection may play an important role in the regulation of the intestinal immune response to concurrent C. rodentium infection as well as in the modulation of the bacterial pathogenesis. We show that H. polygyrus infection induces DC activation and IL-10 expression, and that adoptive transfer of parasite-primed DC significantly impairs host protection to C. rodentium infection, resulting in an enhanced bacterial infection and in the development of a more severe colonic injury. Furthermore, we demonstrate that adoptive transfer of parasite-primed IL-10-deficient DCs fails to result in the development of a significantly enhanced C. rodentium-mediated colitis. Similarly, when the DC IL-10 response was neutralized by anti-IL-10 mAb treatment in mice that received parasite-primed DC, no deleterious effect of the parasite-primed DC on the host intestinal response to C. rodentium was detected. Thus, our results provide evidence to indicate that the H. polygyrus-dependent modulation of the host response to concurrent C. rodentium infection involves IL-10-producing DCs.     

Recombinant human granulocyte colony-stimulating factor enhances superoxide release in human granulocytes stimulated by the chemotactic peptide

Recombinant human granulocyte colony-stimulating factor (G-CSF) by itself was not an effective stimulus for inducing the release of superoxide (O-2) in human granulocytes. However, G-CSF was able to prime human granulocytes, and enhanced O-2 release stimulated by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). The preincubation with G-CSF for 5-10 min at 37 degrees C was sufficient for priming the cells. The optimal enhancing effect was obtained at 25 ng/ml of G-CSF. The enhancement of O-2 release by G-CSF was observed over the complete range of effective concentrations of FMLP (10(-8)-10(-6) M). These findings indicate that G-CSF is a potent activator of mature granulocyte functions.     

Lipid A diversity and the innate host response to bacterial infection

Lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria, is a potent immunostimulatory molecule which activates the innate host defense system. Over the past few years progress has been made in identifying the molecular mechanisms of host recognition of lipid A (a component of lipopolysaccharide), the identification of the genes required for Escherichia coli lipid A biosynthesis, and the role of lipid A acylation when viable bacteria are presented to host cells. Recent data indicate that bacteria can regulate this molecule in response to different host microenvironments. Host factors that induce lipid A modifications and the resultant changes in host response remain to be determined.     

The effects of granulocyte colony-stimulating factor and neutrophil recruitment on the pulmonary chemokine response to intratracheal endotoxin

Although G-CSF has been shown to increase neutrophil (polymorphonuclear leukocyte, PMN) recruitment into the lung during pulmonary infection, relatively little is known about the local chemokine profiles associated with this enhanced PMN delivery. We investigated the effects of G-CSF and PMN recruitment on the pulmonary chemokine response to intratracheal LPS. Rats pretreated twice daily for 2 days with an s.c. injection of G-CSF (50 microg/kg) were sacrificed at either 90 min or 4 h after intratracheal LPS (100 microg) challenge. Pulmonary recruitment of PMNs was not observed at 90 min post LPS challenge. Macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) concentrations in bronchoalveolar lavage (BAL) fluid were similar in animals pretreated with or without G-CSF at this time. G-CSF pretreatment enhanced pulmonary recruitment of PMNs (5-fold) and greatly reduced MIP-2 and CINC levels in BAL fluid at 4 h after LPS challenge. In vitro, the presence of MIP-2 and CINC after LPS stimulation of alveolar macrophages was decreased by coculturing with circulating PMNs but not G-CSF. G-CSF had no direct effect on LPS-induced MIP-2 and CINC mRNA expression by alveolar macrophages. Pulmonary recruited PMNs showed a significant increase in cell-associated MIP-2 and CINC. Cell-associated MIP-2 and CINC of circulating PMNs were markedly increased after exposure of these cells to the BAL fluid of LPS-challenged lungs. These data suggest that recruited PMNs are important cells in modulating the local chemokine response. G-CSF augments PMN recruitment and, thereby, lowers local chemokine levels, which may be one mechanism resulting in the subsidence of the host proinflammatory response.     

Functional domains of the granulocyte colony-stimulating factor receptor.

The granulocyte colony-stimulating factor (G-CSF) receptor has a composite structure consisting of an immunoglobulin(Ig)-like domain, a cytokine receptor-homologous (CRH) domain and three fibronectin type III (FNIII) domains in the extracellular region. Introduction of G-CSF receptor cDNA into IL-3-dependent murine myeloid cell line FDC-P1 and pro-B cell line BAF-B03, which normally do not respond to G-CSF, enabled them to proliferate in response to G-CSF. On the other hand, expression of the G-CSF receptor cDNA in the IL-2-dependent T cell line CTLL-2 did not enable it to grow in response to G-CSF, although G-CSF could transiently stimulate DNA synthesis. Mutational analyses of the G-CSF receptor in FDC-P1 cells indicated that the N-terminal half of the CRH domain was essential for the recognition of G-CSF, but the Ig-like, FNIII and cytoplasmic domains were not. The CRH domain and a portion of the cytoplasmic domain of about 100 amino acids in length were indispensable for transduction of the G-CSF-triggered growth signal.     

Establishment of a human anaplastic thyroid cancer cell line secreting granulocyte colony-stimulating factor in response to cytokines

Summary A human anaplastic thyroid cancer cell line K-119, derived from a 77-yr-old woman who had developed marked neutrophilia and underwent surgery for anaplastic thyroid cancer, has been established. The spindlelike and polygonal cells in shape are stably proliferating since the beginning of its culture 2 yr ago. The cells grow rapidly and the population doubling time is 26 h. The chromosomes show many abnormalities and many marker chromosomes have been observed. Heterotransplantation of the cells into nude mice has resulted in the formation of tumors that are histologically interpreted as anaplastic cancer. The most noteworthy characteristics of the cell line are the many Ki-67-positive cells (86.3%) and that the cell line spontaneously secretes granulocyte colony-stimulating factor (G-CSF) and releases increased amounts of G-CSF in response to the stimulation of tumor necrosis factor, interleukin 1α, and interleukin 1β. The conditioned medium obtained from K-119 cells contains an autocrine factor stimulating the proliferation of themselves.     

Intraclonal diversity of fibrosarcoma cells for the production of macrophage colony-stimulating factor and granulocyte colony-stimulating factor

A new cell line was established from fibrosarcoma that had spontaneously developed in a mouse. The cells were maintained growing in culture for two years and constantly produced both macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF). Cloning of the cells by anchorage-independent colony formation gave subclones showing the activity of producing M-CSF and G-CSF in different proportions, whereas no subclone produced G-CSF without producing M-CSF simultaneously. Recloning of the bipotential subclones again gave clonal derivatives producing two types of CSF in various proportions. The observed heterogeneity of the cloned cells seems to be an epigenetic phenomenon, because the cells resumed the G-CSF producing activity in the absence of cell proliferation. After equilibrium was achieved, all of the subclones produced both M-CSF and G-CSF nearly in equal proportions. Tumorigenic and leukocytosis-inducing activity of the cloned cells was nearly comparable with the activity of the original tumor cells.     

Effects of murine recombinant interleukin 1 alpha on the host response to bacterial infection

The effects of exogenously administered rIL-1 alpha on elimination of viable listeriae from the liver and spleen during the course of a primary Listeria monocytogenes infection was studied. Similar numbers of L. monocytogenes were recovered from rIL-1 alpha-treated and control mice at up to 24 h after infection; however, by 48 h after infection more than 1 log10 fewer viable L. monocytogenes were recovered from the spleens of rIL-1 alpha-treated mice than from Listeria-infected controls. The difference in bacterial burden between IL-1 alpha-treated and control mice increased with time; by 7 days after infection viable L. monocytogenes had been eliminated from most rIL-1 alpha-treated mice, whereas control mice still harbored 10(4) to 10(5) L. monocytogenes per spleen and liver. Histopathologic examination confirmed that rIL-1 alpha-treated mice suffered considerably less damage to the spleen, liver, lung, and brain than did control mice. To determine whether rIL-1 alpha-mediated protection indirectly by augmenting the release of other cytokines, we determined serum levels of colony-stimulating activity and IFN activity in rIL-1 alpha-treated and control Listeria-infected mice. Treatment with rIL-alpha elicited an early burst of serum colony-stimulating activity as compared with sera from Listeria-infected control mice. These data suggest that exogenous administration of rIL-1 initiates release of colony-stimulating activity, and perhaps other cytokines, that accelerate the protective response of the infected host. Prophylactic augmentation of antimicrobial resistance by administration of rIL-1 alpha may be worthy of further evaluation.     

Regulation of the host response to bacterial lipopolysaccharides

Bacterial endotoxins or lipopolysaccharides (LPS) are unique glycolipids present in the outer cell membrane of all gram-negative bacteria. It is now generally recognized that LPS is of primary importance in initiating the pathophysiological changes that often accompany gram-negative bacillary infections in humans including hypotensive shock, disseminated intravascular coagulation, and metabolic abnormalities. Although the biochemical mechanisms of these changes are not well understood, increasing emphasis has been placed on defining the biochemical response of the macrophage (M phi) to LPS. In this paper we describe two M phi-derived factors induced by LPS that may be important in the expression of endotoxic activity in the host. These are a procoagulant activity, which is present on the cell membrane of LPS-treated rabbit liver M phi and acts by directly activating coagulation factor X, and a factor released into the supernatant by LPS-treated peritoneal exudate M phi, which suppresses steroidogenesis in explanted adrenocortical cells. The potential role of the M phi in regulating the binding of LPS to high-density lipoproteins through the induction of acute phase proteins is also considered.