GM-CSF Transcripts Research Articles

Salt-inducible kinases are required for the IL-33–dependent secretion of cytokines and chemokines in mast cells

Cytokines and chemokines are important regulators of airway hyper-responsiveness, immune cell infiltration, and inflammation and are produced when mast cells are stimulated with interleukin-33 (IL-33). Here, we establish that the salt-inducible kinases (SIKs) are required for the IL-33-stimulated transcription of il13, gm-csf and tnf and hence the production of these cytokines. The IL-33–stimulated secretion of IL-13, granulocyte-macrophage colony stimulating factor, and tumor necrosis factor was strongly reduced in fetal liver–derived mast cells from mice expressing a kinase-inactive mutant of SIK3 and abolished in cells expressing kinase-inactive mutants of SIK2 and SIK3. The IL-33–dependent secretion of these cytokines and several chemokines was also abolished in SIK2/3 double knock-out bone marrow–derived mast cells (BMMC), reduced in SIK3 KO cells but little affected in BMMC expressing kinase-inactive mutants of SIK1 and SIK2 or lacking SIK2 expression. In SIK2 knock-out BMMC, the expression of SIK3 was greatly increased. Our studies identify essential roles for SIK2 and SIK3 in producing inflammatory mediators that trigger airway inflammation. The effects of SIKs were independent of IκB kinase β, IκB kinase β-mediated NF-κB-dependent gene transcription, and activation of the mitogen-activated protein kinase family members p38α and c-jun N-terminal kinases. Our results suggest that dual inhibitors of SIK2 and SIK3 may have therapeutic potential for the treatment of mast cell–driven diseases.

Arctigenin Attenuates Breast Cancer Progression through Decreasing GM-CSF/TSLP/STAT3/β-Catenin Signaling.

Invasive breast cancer is highly regulated by tumor-derived cytokines in tumor microenvironment. The development of drugs that specifically target cytokines are promising in breast cancer treatment. In this study, we reported that arctigenin, a bioactive compound from Arctium lappa L., could decrease tumor-promoting cytokines GM-CSF, MMP-3, MMP-9 and TSLP in breast cancer cells. Arctigenin not only inhibited the proliferation, but also the invasion and stemness of breast cancer cells via decreasing GM-CSF and TSLP. Mechanistically, arctigenin decreased the promoter activities of GM-CSF and TSLP via reducing the nuclear translocation of NF-κB p65 which is crucial for the transcription of GM-CSF and TSLP. Furthermore, arctigenin-induced depletion of GM-CSF and TSLP inhibited STAT3 phosphorylation and β-catenin signaling resulting in decreased proliferation, invasion and stemness of breast cancer cells in vitro and in vivo. Our findings provide new insights into the mechanism by which tumor-promoting cytokines regulate breast cancer progression and suggest that arctigenin is a promising candidate for cytokine-targeted breast cancer therapy.

Single Cell Immune Profiling of Colitogenic T Cells during Acute Graft Versus Host Disease Reveals Two Novel Transcriptionally Distinct CD4+ GM-CSF+ Lineages

Gastrointestinal (GI) tract involvement is the major cause of morbidity and mortality in acute graft versus host disease (GVHD) and pathological damage is largely attributable to inflammatory cytokine production. Recently, we and others identified GM-CSF as a cytokine that is produced primarily by donor-derived CD4+ T cells and mediates inflammation in the GI tract. However, the precise mechanism by which GM-CSF induces pathological damage and the transcriptional profile of this novel colitogenic CD4+ GM-CSF+ T cell population have not been defined. To address these questions, we employed a well-defined murine model of GVHD [C57BL/6 (H-2b)→Balb/c (H-2d)] and demonstrated that GM-CSF induces inflammation by enhancing the activation of donor-derived dendritic cells in the colon as evidenced by increased expression of costimulatory molecules (i.e. CD80 and CD86) and the production of IL-23. In addition, GM-CSF linked adaptive to innate immunity by promoting indirect alloantigen presentation in the mesenteric lymph nodes which was IL-23 dependent and characterized by an increased number of CD103+ CD11b+ dendritic cells and donor CD4+ T cells with a proinflammatory cytokine phenotype. Unexpectedly, we observed two distinct CD4+ GM-CSF+ populations in the GI tract that were distinguishable by the presence or absence of IFN-γ production by intracellular cytokine staining (i.e. CD4+ GM-CSF+ IFN-γ+ and CD4+ GM-CSF+ IFN-γ-). Notably, CD4+ GM-CSF+ IFN-γ- cells were largely absent from other target organs (e.g. liver, lung), suggesting that this population had a unique role in the biology of GVHD in the GI tract. To determine whether these two populations represented transcriptionally distinct lineages or reflected TH1-biased lineage plasticity, we performed single cell RNA sequencing and immunological profiling on donor-derived sort-purified T cells from the colons of GVHD mice one and three weeks post-transplant using the 10X Genomics platform. After selecting only high quality reads, we recovered 6315 unique barcodes corresponding to individual cells and identified several transcriptionally distinct cell clusters that spatially segregated following Seurat and UMAP analysis. Colonic T cells obtained on days 7 and 21 post transplantation completely separated, indicating that the transcriptional profile of these cells changes dramatically between early and later time points. Detectable transcription of GM-CSF was observed in two distinct populations of CD4+ T cells only at the 21-day timepoint. Notably, only one of these clusters co-expressed IFN-γ, confirming our flow-based results, and indicating that CD4+ GM-CSF+ IFN-γ+ and GM-CSF+ IFN-γ- T cells represented distinct populations. Further analysis revealed that CD4+ GM-CSF+ IFN-γ+ T cells were T-bet+ and differentially expressed high levels of costimulatory molecules (CD137, OX40, and CD81) and PD-1, indicative of an activated T cell phenotype. In contrast, CD4+ GM-CSF+ IFN-γ- T cells were distinguishable by the co-expression of T-bet and Gata-3, which is a TH2-defining transcription factor, as well as by the IL-7R and a series of interferon stimulated genes (IFITM1, IFITM2, and IFITM3), supporting the premise that these cells constitute a discrete TH cell lineage. To further characterize these CD4+ T cell populations, we examined the T cell repertoire (TCR) using a targeted sequencing analysis approach of our barcoded cDNA library. We identified 444 unique clonotypes among CD4+ GM-CSF+ T cells based on sequencing of CDR3 regions of TCR alpha and beta chains. Notably, only 5 clonotypes were shared between CD4+ GM-CSF+ IFN-γ+ and CD4+ GM-CSF+ IFN-γ-T cells, representing 58 of 1154 (~5%) of the total cells in both clusters. Thus, this minimal overlap suggested that these T cells were responding to non-overlapping antigens within the GI tract. Analysis of V beta TCR gene usage revealed that CD4+ GM-CSF+ IFN-γ- cells had a highly-biased repertoire with approximately half of cells utilizing a single V beta gene, Vbeta3. In contrast, CD4+ GM-CSF+ IFN-γ+ T cells had a much more evenly distributed Vbeta receptor profile with no predominant Vbeta usage. Collectively, these studies demonstrate the existence of two transcriptionally distinct CD4+ GM-CSF+ T cell populations that accumulate within the GI tract, possess non-overlapping T cell repertoires, promote indirect alloantigen presentation, and mediate pathological damage during GVHD. Disclosures No relevant conflicts of interest to declare.

Consequences of Hypoxia for the Pulmonary Alveolar Epithelial Cell Innate Immune Response.

Pulmonary innate immune responses involve a highly regulated multicellular network to defend the enormous surface area of the lung. Disruption of these responses renders the host susceptible to pneumonia. Alveolar epithelial cells (AEC) are a critical source of innate immune molecules such as GM-CSF, which determine the functional maturation of alveolar macrophages. In many pulmonary diseases, heterogeneous ventilation leads to regional hypoxia in the lung. The effect of hypoxia on AEC innate immune function is unknown. We now report that exposure of primary murine AEC to hypoxia (1% oxygen) for 24 h results in significant suppression of key innate immune molecules, including GM-CSF, CCL2, and IL-6. This exposure did not cause toxicity but did induce stabilization of hypoxia-inducible factor 1α protein (HIF-1α) and shift to glycolytic metabolism. Focusing on GM-CSF, we found that hypoxia greatly decreased the rate of GM-CSF transcription. Hypoxia both decreased NF-κB signaling in AEC and induced chromosomal changes, resulting in decreased accessibility in the GM-CSF proximal promoter of target sequences for NF-κB binding. In mice exposed to hypoxia in vivo (12% oxygen for 2 d), lung GM-CSF protein expression was reduced. In vivo phagocytosis of fluorescent beads by alveolar macrophages was also suppressed, but this effect was reversed by treatment with GM-CSF. These studies suggest that in critically ill patients, local hypoxia may contribute to the susceptibility of poorly ventilated lung units to infection through complementary effects on several pathways, reducing AEC expression of GM-CSF and other key innate immune molecules.

Neuroimmune Regulation of JC Virus by Intracellular and Extracellular Agnoprotein.

JC virus (JCV) is a human polyomavirus and the etiologic agent of the demyelinating disease progressive multifocal leukoencephalopathy (PML). PML is observed in patients with underlying immunocompromising conditions, suggesting that neuro-immune interactions between peripheral immune cells and neuro-glia play an important role in controlling viral reactivation in the brain. There is little known about the immunobiology of JCV reactivation in glial cells and the role of immune, glial, and viral players in this regulation. We have previously showed that agnoprotein, a small JCV regulatory protein, is released from infected cells and internalized by neighboring bystander cells. Here we have investigated the possible role of extracellular and intracellular agnoprotein in the neuroimmune response to JC virus. Our findings suggest that glial cells exposed to agnoprotein secrete significantly less GM-CSF, which is mediated by agnoprotein induced suppression of GM-CSF transcription. Likewise, monocytes treated with agnoprotein showed altered differentiation and maturation. In addition, monocytes and microglial cells exposed to agnoprotein showed a significant reduction in their phagocytic activities. Moreover, when an in vitro blood-brain barrier model was used, agnoprotein treatment resulted in decreased monocyte migration through the endothelial cell layer in response to activated astrocytes. All together, these results have revealed a novel immunomodulatory function of agnoprotein during JCV infection within theCNS and open a new avenue of research to better understand the mechanisms associated with JCV reactivation in patients who are at risk of developing PML.

Activated microglia trigger inflammasome activation and lysosomal destabilization in human RPE cells

Activation of the innate immune system plays a major role in retinal degenerative diseases including age-related macular degeneration (AMD). In this study, we investigated whether reactive microglia trigger and sustain NLRP3 inflammasome activation in human retinal pigment epithelium (ARPE-19) cells. Specifically, we analyzed the potential of cell culture supernatants from lipopolysaccharide (LPS)-stimulated human microglia in combination with the lysosomal destabilization agent Leu-Leu-O-Me (LLOMe) to activate the inflammasome in ARPE-19 cells. We found disorganization of ARPE-19 cytoskeletal structure after incubation with conditioned media of LPS-stimulated microglia and LLOMe and accumulation of lipid deposits in these cells using Nile Red staining. LC3-II, the active form of the autophagy marker microtubule-associated protein 1 light chain 3 beta (LC3B), was also elevated in ARPE-19 cells after inducing inflammasome activation. Finally, a significant increase of transcripts for IL-6, IL-8, IL-1ß, GM-CSF and CCL-2 was detected in ARPE-19 stimulated with both microglia-conditioned medium and LLOMe. Our findings highlight a potential role of microglia in RPE inflammasome activation.

Selective VIP Receptor Agonists Facilitate Immune Transformation for Dopaminergic Neuroprotection in MPTP-Intoxicated Mice.

Vasoactive intestinal peptide receptor 2 can elicit immune transformation in a model of Parkinson's disease (PD). Such immunomodulatory capabilities can lead to neuroprotection by attenuating microglial activation and by slowing degradation of neuronal cell bodies and termini in MPTP-intoxicated mice. The protective mechanism arises from altering a Th1/Th2 immune cytokine response into an anti-inflammatory and neuronal sparing profile. These results are directly applicable for the development of novel PD therapies.

Gene transcription of pro-inflammatory cytokines and chemokines induced by IL-17A in canine keratinocytes.

Pro-inflammatory cytokines and chemokines produced by activated keratinocytes play an important role in the pathogenesis of canine atopic dermatitis (AD) as well as human AD. Recent studies suggest that keratinocytes activated by IL-17A are involved in the pathogenesis of human AD. However, the role of IL-17A in canine keratinocytes is poorly understood. Interleukin-17A would induce the transcription of pro-inflammatory cytokines and chemokines in canine keratinocytes. The transcription levels of pro-inflammatory cytokines and chemokines were quantified in a canine keratinocyte cell line stimulated with recombinant canine (rc) IL-17A. The transcription of GM-CSF, S100A8, IL-8 and IL-19 in cultured keratinocytes was significantly enhanced at 24 h after stimulation with rcIL-17A. Keratinocytes activated by IL-17A have the ability to produce various pro-inflammatory cytokines and chemokines, suggesting that IL-17A may play a central role of the development of Th2-associated inflammation in canine AD.

Autocrine GM-CSF transcription in the leukemic progenitor cell line KG1a is mediated by the transcription factor ETS1 and is negatively regulated through SECTM1 mediated ligation of CD7

BackgroundCD7 expression is found on ~30% of acute myeloblastic leukemias (AML). The leukemic progenitor cell line KG1a (CD7+) constitutively expresses GM-CSF while the parental KG1 (CD7-) cell line does not. This study focuses on the molecular basis of CD7 mediated GM-CSF regulation. MethodsKG1a cells were treated with recombinant SECTM1-Fc protein, the PI3K kinase inhibitors wortmannin, LY292004, or PI4K activator spermine. Stable KG1-CD7+, KG1a-shCD7, KG1a-shETS1 as well as KG1a-GFP, KG1a-PKCβII-GFP cell lines were generated and the levels of CD7, GM-CSF and ETS-1 mRNA and protein were compared by real-time-PCR, western blotting, flow cytometry and ELISA. ResultsSECTM1 is expressed in Human Bone Marrow Endothelial Cells (HBMEC) and its expression can be upregulated by both IFN-γ. KG1a cells demonstrated high expression levels of CD7 and ETS-1 allowing a constitutative signaling through the PI3K/Atk pathway to promote GM-CSF expression, while KG1 cells with low expression of CD7 and ETS-1 showed low GM-CSF expression. On KG1a cells GM-CSF expression could be negatively regulated by PI3K inhibitors or by recombinant SECTM1-Fc. Overexpression of CD7 in KG1 cells was insufficient to promote GM-CSF expression, while silencing of CD7 or ETS-1 resulted in reduced GM-CSF expression levels. Differentiation capable KG1a cells overexpressing PKCβII illustrated complete loss of CD7, but maintained normal levels of both ETS-1 and GM-CSF expression. ConclusionThese findings add an additional layer to the previously described autocrine/paracrine signaling between leukemic progenitor cells and the bone marrow microenvironment and highlight a role for SECTM1 in both normal and malignant hematopoiesis. General SignificanceThis work shows that SECTM1 secreted from bone marrow stromal cells may interact with CD7 to influence GM-CSF expression in leukemic cells.

Th17 cell LFA-1 engagement-mediated IL-17A and GM-CSF mRNA stabilization is HuR-dependent (54.14)

Abstract Recent studies demonstrate that the β2 integrin LFA-1 is required for entry of encephalitogenic Th17 cells into the brain parenchyma during murine experimental autoimmune encephalomyelitis (EAE). We previously showed that LFA-1 engagement by its ligand, intracellular adhesion molecule-1 (ICAM-1), in human T cells results in translocation of the RNA-binding protein HuR and stabilization of TNFα and IFNγ mRNAs. The role of HuR in the regulation of Th17 cell-mediated inflammation is unknown. We hypothesize that LFA-1 engagement in Th17 cells leads to increased neuropathology-relevant cytokine expression at inflammatory sites through HuR-mediated mRNA stabilization. LFA-1 engagement led to HuR nuclear-to-cytoplasmic translocation and stabilization of both IL-17A and GM-CSF transcripts in mouse EL4 T cells, in vitro differentiated mouse Th17 cells and human primary T cells. EL4 and human primary T cell adhesion to ICAM-1 led to increased levels of IL-17A and GM-CSF mRNA in HuR immunoprecipitates. LFA-1 engagement-stimulated IL-17A and GM-CSF mRNA stabilization was lost in HuR-deficient Th17 cells generated from RORcγt-Cre/HuRflox/flox mice. We conclude that LFA-1 engagement induces HuR-dependent stabilization of the otherwise labile GM-CSF and IL-17A transcripts in Th17 cells, and that this may be highly relevant in Th17 cell-induced CNS pathology. Future experiments will focus on the role of Th17 cell HuR in the EAE model.

SHARPIN is essential for cytokine production, NF-κB signaling, and induction of Th1 differentiation by dendritic cells.

Spontaneous mutations of the Sharpin (SHANK-associated RH domain-interacting protein, other aliases: Rbckl1, Sipl1) gene in mice result in systemic inflammation that is characterized by chronic proliferative dermatitis and dysregulated secretion of T helper1 (Th1) and Th2 cytokines. The cellular and molecular mechanisms underlying this inflammatory phenotype remain elusive. Dendritic cells may contribute to the initiation and progression of the phenotype of SHARPIN-deficient mice because of their pivotal role in innate and adaptive immunity. Here we show by flow cytometry that SHARPIN- deficiency did not alter the distribution of different DC subtypes in the spleen. In response to TOLL-like receptor (TLR) agonists LPS and poly I:C, cultured bone marrow-derived dendritic cells (BMDC) from WT and mutant mice exhibited similar increases in expression of co-stimulatory molecules CD40, CD80, and CD86. However, stimulated SHARPIN-deficient BMDC had reduced transcription and secretion of pro-inflammatory mediators IL6, IL12P70, GMCSF, and nitric oxide. Mutant BMDC had defective activation of NF-κB signaling, whereas the MAPK1/3 (ERK1/2) and MAPK11/12/13/14 (p38 MAP kinase isoforms) and TBK1 signaling pathways were intact. A mixed lymphocyte reaction showed that mutant BMDC only induced a weak Th1 immune response but stimulated increased Th2 cytokine production from allogeneic naïve CD4+ T cells. In conclusion, loss of Sharpin in mice significantly affects the immune function of DC and this may partially account for the systemic inflammation and Th2-biased immune response.

NF-κB and BRG1 bind a distal regulatory element in the IL-3/GM-CSF locus

We investigated gene regulation at the IL-3/GM-CSF gene cluster. We found BRG1, a SWI/SNF remodeling ATPase, bound a distal element, CNSa. BRG1 binding was strongest in differentiated, stimulated T helper cells, paralleling IL-3 and GM-CSF expression. Depletion of BRG1 reduced IL-3 and GM-CSF transcription. BAF-specific SWI/SNF subunits bound to this locus and regulated IL-3 expression. CNSa was in closed chromatin in fibroblasts, open chromatin in differentiated T helper cells, and moderately open chromatin in naïve (undifferentiated) T helper cells; BRG1 was required for the most open state. CNSa increased transcription of a reporter in an episomal expression system, in a BRG1-dependent manner. The NF-κB subunit RelA/p65 bound CNSa in activated T helper cells. Inhibition of NF-κB blocked BRG1 binding to CNSa, chromatin opening at CNSa, and activation of IL-3 and GM-CSF. Together, these findings suggest CNSa is a distal enhancer that binds BRG1 and NF-κB.

Systematic review of toxicities of lineage-specific hematopoietic growth factors toxicities: Implications for biosimilar erythropoietins, thromopoietic receptor agonists, and continuous erythropoietin receptor agonists

e20733 Background: Erythropoiesis stimulating agents (ESAs) and the granulocyte and granulocyte macrophage colony stimulating factors (G-CSF/GM-CSF) have been widely used for the past two decades. Recently, thrombopoietin receptor agonists, romiplostim and eltrombopag, biosimilar erythropoietins, and a continuous erythropoietin receptor agonist were licensed. Although lineage specific hematopoietic growth factors play important roles in the treatment of cancer and chronic kidney disease, recent basic and clinical research studies have identified toxicities. Herein, we compared toxicities of ESAs, thrombopoietins and G-CSF/CSFs and the implications of these findings. Methods: Meta-analyses, reviews, clinical guidelines, and FDA transcripts for G-CSF, GM-CSF, and ESAs were reviewed. For MGDF, romiplostim, and eltrombopag, clinical trial reports, reviews, and FDA transcripts were reviewed. The study period was 2000 to 2008. Results: See Table . Conclusions: Commonalities in toxicities exist across lineage specific hematopoietic growth factors. Delayed recognition of adverse effects with ESAs and G-CSF highlights the importance of anticipating toxicities with biosimilars and thrombopoietin receptor agonists. [Table: see text] No significant financial relationships to disclose.

Early stress response of human nasal respiratory epithelia after exposure to 1-methoxypropanol-2

To evaluate the impact of 1-methoxypropanol-2 (MEP) for the stimulation of an inflammatory response in human respiratory mucosa, we exposed 22 primary cell cultures of nasal respiratory epithelia of healthy individuals to MEP concentrations at the level of the German MAK-value (100 ppm) and to the 10-fold concentration (1000 ppm). After 4 and 24 h we analyzed the transcription of TNF-α, IL-1β, IL-6, IL-8, MCP-1, GMCSF, Cox-1 and Cox-2 by quantitative PCR as well as the release of the respective cytokines by ELISA. At both MEP concentrations we observed a significant increase of TNF-α-, IL-1β-, IL-6- and Cox-2-transcripts after 4 h. After 24 h cytokine transcription of TNF-α, IL-1β and IL-6 was normalized, but Cox-2 remained elevated. On the protein level IL-1β as well as granulocyte macrophages colony stimulating factor (GM-CSF) were decreased after 4 h or 24 h and uniquely IL-8 levels were increased after 4 h. Our data suggest that MEP induces the transcription of genes encoding proinflammatory cytokines and mediators but largely not translation of those. Considering these in vitro data, existing exposure limits seem to be safe with respect to inflammatory responses of the upper respiratory tract. However, the effects of long-term exposures to MEP should be watched closely.

Bovine recombinant IFNγ induces endothelial cell gene transcription of immunoregulatory molecules and upregulates PMN and PBMC adhesion on bovine endothelial cells

Interferon gamma (IFNgamma) is an important modulator of immune responses acting on multiple cell types, such as lymphocytes, macrophages or endothelial cells. We investigated the effects of recombinant bovine IFNgamma on bovine umbilical vein endothelial cells (BUVEC) for the level of polymorphonuclear neutrophil cell (PMN)- and peripheral blood mononuclear cell (PBMC)-adhesion as well as the gene transcription of endothelial cell-derived adhesion molecules (E-selectin, P-selectin, VCAM-1, ICAM-1), chemokines (CXCL1, CXCL8, CXCL10, CCL2, CCL5), GM-CSF, iNOS and COX-2 in comparison to TNFalpha-stimulation. IFNgamma strongly induced PMN and PBMC adhesion on BUVEC involving CD4(+), CD8(+) and gammadelta-TCR(+) (WC1(+)) lymphocytes. Furthermore, IFNgamma-stimulation led to a strong upregulation in the transcription of VCAM-1, ICAM-1, CXCL10 and CCL2 genes and to a low to moderate increase in the E- and P-selectin, CXCL1, CXCL8, CCL5, COX-2 and iNOS gene transcripts, but failed to enhance GM-CSF gene transcription. These results indicate that IFNgamma can be considered an important activator of endothelial cells in the bovine system, most probably by influencing the outcome of inflammatory responses through selective upregulation of immunoregulatory molecules.

Murine mammary carcinoma 4T1 induces a leukemoid reaction with splenomegaly: Association with tumor-derived growth factors

A leukemoid reaction with granulocytosis and splenomegaly has been observed in animals and humans with a variety of tumors. We have employed four color flow cytometry to characterize the leukemoid reaction induced by the transplantable mouse mammary carcinoma 4T1 in female BALB/c mice. Gr-1 + myeloid cells with the morphology of granulocytes increased in peripheral blood from < 15% pre-transplant to nearly 80% of total CD45 + leukocytes at four weeks post-transplant. Though the granulocyte:lymphocyte ratio increased markedly, the absolute numbers of CD19 + B lymphocytes, CD4 + and CD8 + T lymphocytes, and the CD4/CD8 ratio in peripheral blood did not change significantly. Femurs from tumor-bearing mice showed myeloid hyperplasia of the fatty marrow. There was a notable increase in cells with a Gr-1 dim/CD11b bright immature granulocyte phenotype, and these cells were also found in peripheral blood and spleen. Spleen weights had increased 8.5-fold by four weeks post-tumor transplant, mainly due to granulocytic hyperplasia. Cultured 4T1 tumor cells constitutively expressed mRNA for the myeloid colony-stimulating factors G-CSF and GM-CSF, and IFN-γ-inducible M-CSF transcripts were also detected. Tumors excised from mice had transcripts for G-CSF and GM-CSF, but only G-CSF protein was found in high levels in serum of tumor-bearing mice. These data demonstrate that 4T1 tumor-bearing mice exhibit a leukemoid reaction that apparently is caused by the production of colony-stimulating factors produced by the tumor. The 4T1 tumor may serve as an excellent model for the study of this reaction.

S100A8 Chemotactic Protein Is Abundantly Increased, but Only a Minor Contributor to LPS-Induced, Steroid Resistant Neutrophilic Lung Inflammation in Vivo

Neutrophilic lung inflammation is an essential component of host defense against diverse eukaryotic and prokaryotic pathogens, but in chronic inflammatory lung diseases, such as chronic obstructive lung disease (COPD), severe asthma, cystic fibrosis, and bronchiolitis, it may damage the host. Glucocorticosteroids are widely used in these conditions and in their infectious exacerbations; however, the clinical efficacy of steroids is disputed. In this study, we used a proteomic approach to identify molecules contributing to neutrophilic inflammation induced by transnasal administration of lipopolysaccharide (LPS) that were also resistant to the potent glucocorticosteroid dexamethasone (Dex). We confirmed that Dex was biologically active at both the transcript (suppression of GM-CSF and TNFalphatranscripts) and protein levels (induction of lipocortin) and used 2D-PAGE/MALDI-TOF to generate global expression profiles, identifying six LPS-induced proteins that were Dex resistant. Of these, S100A8, a candidate neutrophil chemotactic factor, was profiled in detail. Steroid refractory S100A8 expression was highly abundant, transcriptionally regulated, secreted into lung lavage fluid and immunohistochemically localized to tissue infiltrating neutrophils. However, in marked contrast to other vascular beds, neutralizing antibodies to S100A8 had only a weak anti-neutrophil recruitment effect and antibodies against the related S100A9 were ineffective. These data highlight the need for extensive in vivo profiling of proteomically identified candidate molecules and demonstrates that S100A8, despite its abundance, resistance to steroids and known chemotactic activity, is unlikely to be an important determinant of LPS-induced neutrophilic lung inflammation in vivo.

Glycosylinositolphosphate soluble variant surface glycoprotein inhibits IFN-gamma-induced nitric oxide production via reduction in STAT1 phosphorylation in African trypanosomiasis.

Macrophages are centrally involved in the host immune response to infection with Trypanosoma brucei rhodesiense, a protozoan parasite responsible for human sleeping sickness in Africa. During trypanosome infections, the host is exposed to parasite-derived molecules that mediate macrophage activation, specifically GPI anchor substituents associated with the shed variant surface glycoprotein (VSG), plus the host-activating agent IFN-gamma, which is derived from activated T cells and is essential for resistance to trypanosomes. In this study, we demonstrate that the level and timing of exposure of macrophages to IFN-gamma vs GPI ultimately determine the macrophage response at the level of induced gene expression. Treatment of macrophages with IFN-gamma followed by GIP-sVSG (the soluble form of VSG containing the glycosylinositolphosphate substituent that is released by parasites) stimulated the induction of gene expression, including transcription of TNF-alpha, IL-6, GM-CSF, and IL-12p40. In contrast, treatment of macrophages with GIP-sVSG before IFN-gamma stimulation resulted in a marked reduction of IFN-gamma-induced responses, including transcription of inducible NO synthase and secretion of NO. Additional experiments revealed that the inhibitory activity of GIP-sVSG was associated with reduction in the level of STAT1 phosphorylation, an event required for IFN-gamma-induced macrophage activation. These results suggest that modulation of specific aspects of the IFN-gamma response may be one mechanism by which trypanosomes overcome host resistance during African trypanosomiasis.

The role of CD28-B7 costimulation in allergen-induced cytokine release by bronchial mucosa from patients with moderately severe asthma

Background: T cells play an important role in airway inflammation in asthma through the release of TH2 cytokines. Optimal T-cell activation by antigen-presenting cells requires co-stimulatory signaling, such as the interaction of CD80, CD86, or both with CD28. In patients with mild allergic asthma, the fusion protein cytotoxic T-lymphocyte antigen 4Ig (CTLA-4Ig), which inhibits CD28-mediated signaling, blocks the release of IL-5 and IL-13 from bronchial explant cultures exposed to the allergen Dermatophagoides pteronyssinus . Objectives: To assess costimulation in more severe forms of atopic asthma, we have compared the ability of CTLA-4Ig to block allergen-induced cytokine responses of bronchial explants and PBMCs from patients with moderately severe asthma. Methods: Bronchial explants and PBMCs were cultured in vitro, and cytokine expression was measured by means of quantitative RT-PCR and ELISA. Results: Constitutive mRNA transcripts for IL-5, IL-13, and GM-CSF were detected in the tissue explants, but only IL-5 mRNA increased significantly with allergen stimulation. Consistent with increased transcription, allergen-stimulated IL-5 protein release into explant supernatants, but this was not blocked by CTLA-4Ig. Allergen did not induce GM-CSF release, and IL-13 protein could not be detected in the explant supernatants under any condition. In contrast, allergen enhanced production of IL-5 and IL-13 by PBMC cultures from the same subjects, and this was inhibited effectively by CTLA-4Ig. Conclusions: These data suggest that IL-5 production in the airways of subjects with moderately severe asthma is largely independent of CD28-mediated costimulation. The different requirements for CD28-mediated costimulation in PBMC cultures and bronchial tissue cultures emphasizes the importance of the tissue microenvironment in pulmonary inflammatory responses in severe asthma. (J Allergy Clin Immunol 2001;108:976-81.)

CYTOKINE PRODUCTION BY IN VITRO PROCESSED AND UNPROCESSED HAEMATOPOIETIC CELLS

The studies described here demonstrate that in vitro processing of cells before extraction of RNA has a major effect on the number and type of cytokine transcripts present within MDS and leukemia cells. Transcripts for GM-CSF, a cytokine whose production by leukemia cells is believed to play an important role in the pathogenesis of leukemia, was not detectable in 12/13 unprocessed AML specimens, in 12/12 MDS specimens, or in 7/7 CML specimens but once detected in many specimens after processing. These data strongly suggest that leukemia cell production of GMCSF rarely occurs in vivo.