Csf2ra deletion attenuates acute lung injuries induced by intratracheal inoculation of aerosolized ricin in mice.

Specific therapeutics are not available for acute lung injury (ALI) induced by ricin toxin (RT). Inhibiting the host immune response in the course of pulmonary ricinosis is hypothesized to be of benefit and can be achieved by impairing granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, thereby reducing the pro-inflammatory response to exogenous foreign body invasion. However, it is unknown whether mice with impaired GM-CSF signaling can survive after RT inhalation. To test this, colony stimulating factor 2 receptor alpha (Csf2ra) knockout (KO) mice that lack GM-CSF signaling and wild-type (WT) mice models of intratracheal exposure to a lethal dose (2× LD50) of RT were established. Survival was greater in Csf2ra KO mice 21 days after RT inhalation compared with WT mice. Highly co-expressed genes that probably attenuated the pro-inflammatory response in the lung of Csf2ra KO mice were identified. Bioinformatics analysis revealed that transcriptome changes involved mostly inflammation-related genes after RT exposure in both Csf2ra KO mice and WT mice. However, the activity levels of pro-inflammatory pathways, such as the TNF signaling pathway and NF-κB signaling pathway, in Csf2ra KO mice were significantly decreased and the degree of neutrophil chemotaxis and recruitment inhibited after RT-exposure relative to WT mice. RT-qPCR and flow cytometry validated results of RNA-Seq analysis. This work provides potential avenues for host-directed therapeutic applications that can mitigate the severity of ALI-induced by RT.

The Role of Zinc in GM-CSF-Induced Signaling in Human Polymorphonuclear Leukocytes.

SCOPE: Zinc is suggested to be necessary for functional signaling induced by certain growth factors. The granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key factor for differentiation and activation of myeloid cells. This report analyses the impact of different zinc concentrations on GM-CSF-induced signaling in mature polymorphonuclear leukocytes (PMN). METHODS AND RESULTS: As measured by flow cytometry, zinc increases surface GM-CSF receptor (GM-CSFR) in PMN, whereas monocytes respond with decreased GM-CSFR surface expression. Since total cellular GM-CSFR expression remains unaffected, the observed zinc-induced GM-CSFR surface dynamics may be explained by receptor redistribution. In PMN, zinc enhanced phosphorylation of mitogen-activated protein kinases (MAPK) in a dose-dependent manner as found in western blot. Zinc-induced MAPK phosphorylation is additionally augmented by moderate GM-CSF stimulation. CONCLUSION: The present study demonstrates the opposing influence of zinc on GM-CSFR surface expression in monocytes and PMN. Zinc and GM-CSF, use in optimized concentrations, augment MAPK signaling, and increase expression of MAPK-induced myeloid cell leukemia-1 (Mcl-1) in PMN. Thus, this study concludes that zinc strengthens growth factor-induced signaling. Hence, the study provides a basis for further in vivo studies, focusing on the therapeutic value of zinc in patients with a disturbed GM-CSF signaling.

A murine model of hereditary pulmonary alveolar proteinosis caused by homozygous Csf2ra gene disruption.

Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder caused by recessive mutations in GM-CSF receptor subunit α/ß genes (CSF2RA/CSF2RB, respectively) characterized by impaired GM-CSF-dependent surfactant clearance by alveolar macrophages (AMs) resulting in alveolar surfactant accumulation and hypoxemic respiratory failure. Because hPAP is caused by CSF2RA mutations in most patients, we created an animal model of hPAP caused by Csf2ra gene disruption (Csf2ra-/- mice) and evaluated the effects on AMs and lungs. Macrophages from Csf2ra-/- mice were unable to bind and clear GM-CSF, did not exhibit GM-CSF signaling, and had functional defects in phagocytosis, cholesterol clearance, and surfactant clearance. Csf2ra-/- mice developed a time-dependent, progressive lung disease similar to hPAP in children caused by CSF2RA mutations with respect to the clinical, physiological, histopathological, biochemical abnormalities, biomarkers of PAP lung disease, and clinical course. In contrast, Csf2ra+/- mice had functionally normal AMs and no lung disease. Pulmonary macrophage transplantation (PMT) without myeloablation resulted in long-term engraftment, restoration of GM-CSF responsiveness to AMs, and a safe and durable treatment effect that lasted for the duration of the experiment (6 mo). Results demonstrate that homozygous (but not heterozygous) Csf2ra gene ablation caused hPAP identical to hPAP in children with CSF2RA mutations, identified AMs as the cellular site of hPAP pathogenesis in Csf2ra-/- mice, and have implications for preclinical studies supporting the translation of PMT as therapy of hPAP in humans.

Mutated GM-CSF-based CAR-T cells targeting CD116/CD131 complexes exhibit enhanced anti-tumor effects against acute myeloid leukaemia.

OBJECTIVES: As the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)-T cells for AML are more challenging than those targeting CD19 in B-cell malignancies. We recently developed piggyBac-modified ligand-based CAR-T cells that target CD116/CD131 complexes, also known as the GM-CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. METHODS: To further improve the efficacy of the original GMR CAR-T cells, we have developed novel GMR CAR vectors incorporating a mutated GM-CSF for the antigen-binding domain and G4S spacer. All GMR CAR-T cells were generated using a piggyBac-based gene transfer system. The anti-tumor effect of GMR CAR-T cells was tested in mouse AML xenograft models. RESULTS: Nearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. Furthermore, GMR CAR-T cells incorporating a G4S spacer significantly improved long-term in vitro and in vivo anti-tumor effects. By employing a mutated GM-CSF at residue 21 (E21K), the anti-tumor effects of GMR CAR-T cells were also improved especially in long-term in vitro settings. Although GMR CAR-T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal. CONCLUSIONS: GMR CAR-T cell therapy represents a promising strategy for CD116+ R/R AML.

Ssu72 regulates alveolar macrophage development and allergic airway inflammation by fine-tuning of GM-CSF receptor signaling.

BACKGROUND: Fine-tuning of immune receptor signaling is critical for the development and functioning of immune cells. Moreover, GM-CSF receptor (GM-CSFR) signaling plays an essential role in the development of certain myeloid lineage cells, including alveolar macrophages (AMs). However, the significance of fine-tuning of GM-CSFR signaling in AMs and its relevance in allergic inflammation have not been reported. OBJECTIVE: Our aim was to explore whether phosphatase Ssu72, originally identified as a regulator of RNA polymerase II activity, regulates AM development and allergic airway inflammation by regulating GM-CSF signaling. METHODS: To address these issues, we generated LysM-CreSsu72fl/fl and Cd11c-CreSsu72fl/fl mice and used ovalbumin- or house dust mite-induced allergic asthma models. RESULTS: Following GM-CSF stimulation, Ssu72 directly bound to the GM-CSFR ß-chain in AMs, preventing phosphorylation. Consistently, mature Ssu72-deficient AMs showed higher phosphorylation of the GM-CSFR ß-chain and downstream molecules, which resulted in greater dysregulation of cell cycle, cell death, cell turnover, mitochondria-related metabolism, and LPS responsiveness in AMs than in mature wild-type AMs. The dysregulation was restored by using a Janus kinase 2 inhibitor, which reduced GM-CSFR ß-chain phosphorylation. LysM-CreSsu72fl/fl mice exhibited deficits in development and maturation of AMs, which were also seen postnatally in Cd11c-CreSsu72fl/fl mice. Furthermore, LysM-CreSsu72fl/fl mice were less responsive to ovalbumin- or house dust mite-induced allergic asthma models than the control mice were; however, their responsiveness was restored by adoptive transfer of JAK2 inhibitor-pretreated mature Ssu72-deficient AMs. CONCLUSION: Our results demonstrate that Ssu72 fine-tunes GM-CSFR signaling by both binding to and reducing phosphorylation of GM-CSFR ß-chain, thereby regulating the development, maturation, and mitochondrial functions of AMs and allergic airway inflammation.

Granulocyte Macrophage-Colony Stimulating Factor Produces a Splenic Subset of Monocyte-Derived Dendritic Cells That Efficiently Polarize T Helper Type 2 Cells in Response to Blood-Borne Antigen.

Dendritic cells (DCs) are key antigen-presenting cells that prime naive T cells and initiate adaptive immunity. Although the genetic deficiency and transgenic overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) signaling were reported to influence the homeostasis of DCs, the in vivo development of DC subsets following injection of GM-CSF has not been analyzed in detail. Among the treatment of mice with different hematopoietic cytokines, only GM-CSF generates a distinct subset of XCR1-33D1- DCs which make up the majority of DCs in the spleen after three daily injections. These GM-CSF-induced DCs (GMiDCs) are distinguished from classical DCs (cDCs) in the spleen by their expression of CD115 and CD301b and by their superior ability to present blood-borne antigen and thus to stimulate CD4+ T cells. Unlike cDCs in the spleen, GMiDCs are exceptionally effective to polarize and expand T helper type 2 (Th2) cells and able to induce allergic sensitization in response to blood-borne antigen. Single-cell RNA sequencing analysis and adoptive cell transfer assay reveal the sequential differentiation of classical monocytes into pre-GMiDCs and GMiDCs. Interestingly, mixed bone marrow chimeric mice of Csf2rb+/+ and Csf2rb-/- demonstrate that the generation of GMiDCs necessitates the cis expression of GM-CSF receptor. Besides the spleen, GMiDCs are generated in the CCR7-independent resident DCs of the LNs and in some peripheral tissues with GM-CSF treatment. Also, small but significant numbers of GMiDCs are generated in the spleen and other tissues during chronic allergic inflammation. Collectively, our present study identifies a splenic subset of CD115hiCD301b+ GMiDCs that possess a strong capacity to promote Th2 polarization and allergic sensitization against blood-borne antigen.

CCL2/MCP-1 signaling drives extracellular matrix turnover by diverse macrophage subsets.

Macrophage plasticity, cellular origin, and phenotypic heterogeneity are perpetual challenges for studies addressing the biology of this pivotal immune cell in development, homeostasis, and tissue remodeling/repair. Consequently, a myriad of macrophage subtypes has been described in these contexts. To facilitate the identification of functional macrophage subtypes in vivo, here we used a flow cytometry-based assay that allows for detailed phenotyping of macrophages engaged in extracellular matrix (ECM) degradation. Of the five macrophage subtypes identified in the remodeling dermis by using this assay, collagen degradation was primarily executed by Ly6C - CCR2 + and Ly6C - CCR2 low macrophages via mannose receptor-dependent collagen endocytosis, while Ly6C + CCR2 + macrophages were the dominant fibrin-endocytosing cells. Unexpectedly, the CCL2/MCP1-CCR2 signaling axis was critical for both collagen and fibrin degradation, while collagen degradation was independent of IL-4Ra signaling. Furthermore, the cytokine GM-CSF selectively enhanced collagen degradation by Ly6C + CCR2 + macrophages. This study reveals distinct subsets of macrophages engaged in ECM turnover and identifies novel wound healing-associated functions for CCL2 and GM-CSF inflammatory cytokines.

Engineered cell migration to lesions linked to autoimmune disease.

The damaging and degenerative effects in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and Crohn's disease often manifests as the formation of lesions that feature a high local concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF along with other pro-inflammatory factors form a positive feedback loop that ultimately perpetuate the lesions. Hence, to engineer chemotaxis to GM-CSF, we created a new chimeric GM-CSF receptor alpha subunit (GMRchi) that was coupled with a previously engineered Ca2+ -activated RhoA. When these proteins were expressed in mammalian cells, it allowed migration to chemical and cellular sources of GM-CSF. As a possible therapeutic intervention, we further implemented the mechanism of cell-cell membrane fusion and subsequent death. Since the microenvironment of lesions is more than just GM-CSF secretion, the further ability to recognize a combination of other features such as tissue markers will be needed for greater specificity. Nonetheless, this work represents a first step to enable cell-based therapy of autoimmune lesions.

Elderly-onset hereditary pulmonary alveolar proteinosis and its cytokine profile.

BACKGROUND: Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by surfactant accumulation, and is caused by disruption of granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. Abnormalities in CSF2 receptor alpha (CSF2RA) were reported to cause pediatric hereditary PAP. We report here the first case of CSF2RA-mutated, elderly-onset hereditary (h) PAP. CASE PRESENTATION: The patient developed dyspnea on exertion, and was diagnosed with PAP at the age of 77 years, based on findings from chest computed tomography scan and bronchoalveolar lavage. She tested negative for GM-CSF autoantibodies, with no underlying disease. Her serum GM-CSF level was elevated (91.3 pg/mL), indicating GM-CSF signaling impairment and genetic defects in the GM-CSF receptor. GM-CSF-stimulated phosphorylation in signal transducer and activator of transcription 5 (STAT5) was not observed, and GM-CSF-Rα expression was defective in her blood cells. Genetic screening revealed a homozygous, single-base C > T mutation at nt 508-a nonsense mutation that yields a stop codon (Q170X)-in exon 7 of CSF2RA. High-resolution analysis of single nucleotide polymorphism array confirmed a 22.8-Mb loss of heterozygosity region in Xp22.33p22.11, encompassing the CSF2RA gene. She was successfully treated with whole lung lavage (WLL), which reduced the serum levels of interleukin (IL)-2, IL-5, and IL-17, although IL-3 and M-CSF levels remained high. CONCLUSIONS: This is the first known report of elderly-onset hPAP associated with a CSF2RA mutation, which caused defective GM-CSF-Rα expression and impaired signaling. The analyses of serum cytokine levels during WLL suggested that GM-CSF signaling might be compensated by other signaling pathways, leading to elderly-onset PAP.

Targeting granulocyte-macrophage colony-stimulating factor in epithelial and vascular remodeling in experimental eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic antigen-mediated clinicopathologic disease of the esophagus characterized by an eosinophil-predominant inflammatory infiltrate. A clinical hallmark is extensive tissue remodeling including basal zone hyperplasia, fibrosis, and angiogenesis. However, the cellular mechanisms responsible for these processes are not fully defined. We hypothesized that targeting granulocyte-macrophage colony-stimulating factor (GM-CSF; an agonist cytokine linked with eosinophil survival and activation) would be protective in a preclinical model of EoE. METHODS: Eosinophilic esophagitis-like esophageal inflammation was induced in the L2-IL5OXA EoE mouse model, and GM-CSF production was assessed by mRNA and protein analyses. Granulocyte-macrophage colony-stimulating factor-receptor-alpha expression patterns were examined by flow cytometric and immunofluorescence analysis. L2-IL5OXA EoE mice were treated with anti-GM-CSF neutralizing antibody or isotype control and assessed for histopathological indices of eosinophilia, epithelial hyperplasia, and angiogenesis by immunohistochemistry and RT-PCR. RESULTS: Significantly increased levels of esophageal GM-CSF expression was detected in the L2-IL5OXA mouse EoE model during active inflammation. Granulocyte-macrophage colony-stimulating factor-receptor-alpha was predominantly expressed on esophageal eosinophils during EoE, in addition to select cells within the lamina propria. Anti-GM-CSF neutralization in L2-IL5OXA EoE mice resulted in a significant diminution of epithelial eosinophilia in addition to basal cell hyperplasia and vascular remodeling. This treatment response was independent of effects on esophageal eosinophil maturation or activation. CONCLUSION: Granulocyte-macrophage colony-stimulating factor is a potential therapeutic target to reduce esophageal eosinophilia and remodeling.

Frontline Science: Coincidental null mutation of Csf2rα in a colony of PI3Kγ-/- mice causes alveolar macrophage deficiency and fatal respiratory viral infection.

PI3Ks have been identified as key signaling proteins involved in many basic biologic processes in health and disease. Transgenic animals have been essential tools to study the underlying molecular mechanisms in this context and therefore, have been widely used to elucidate the role of these factors in many different settings. More specifically, PI3Kγ, a subunit highly expressed in the hematopoietic system, has been implicated to play an important role in many inflammatory diseases as well as cancer. Here, we report identification of multiple, additional, previously unknown mutations in the genome of a widely used PI3Kγ-deficient (PI3Kγ-/-) mouse colony. These include a STOP mutation in the GM-CSFRα chain, leading to a complete and specific deficiency in GM-CSF signaling. PI3Kγ-/- animals consequently lacked alveolar macrophages (AMs) and succumbed rapidly to influenza virus infection. Furthermore, PI3Kγ-/- mice carried an additional mutation that affects mucin 2 (Muc2) transcripts. This protein is strongly involved in the regulation of colorectal cancer, and indeed, conflicting reports have indicated that PI3Kγ-/- animals spontaneously develop colorectal tumors. Thus, we uncover previously unknown, confounding factors present in a strain of PI3Kγ-/- mice, leading to additional deficiencies in important signaling pathways with potentially wide-ranging implications for the interpretation of previous studies. By separating the mutations, we established a unique Csf2ra-/- mouse model that allows us to study the role of cell intrinsic GM-CSFR signaling in vivo without confounding variables introduced by defective IL-5R and IL-3R signaling in mice lacking the common ß chain (Csf2rb).

Pulmonary pharmacodynamics of an anti-GM-CSFRα antibody enables therapeutic dosing that limits exposure in the lung.

Pulmonary alveolar proteinosis is associated with impaired alveolar macrophage differentiation due to genetic defects in the granulocyte macrophage colony-stimulating factor (GM-CSF) axis or autoantibody blockade of GM-CSF. The anti-GM-CSFRα antibody mavrilimumab has shown clinical benefit in patients with rheumatoid arthritis, but with no accompanying pulmonary pathology observed to date. We aimed to model systemic versus pulmonary pharmacodynamics of an anti-GM-CSFRα antibody to understand the pharmacology that contributes to this therapeutic margin. Mice were dosed intraperitoneal with anti-GM-CSFRα antibody, and pharmacodynamics bioassays for GM-CSFRα inhibition performed on blood and bronchoalveolar lavage (BAL) cells to quantify coverage in the circulation and lung, respectively. A single dose of 3 mg/kg of the anti-GM-CSFRα antibody saturated the systemic cellular pool, but dosing up to 10 times higher had no effect on the responsiveness of BAL cells to GM-CSF. Continued administration of this dose of anti-GM-CSFRα antibody for 7 consecutive days also had no inhibitory effect on these cells. Partial inhibition of GM-CSFRα function on cells from the BAL was only observed after dosing for 5 or 7 consecutive days at 30 mg/kg, 10-fold higher than the proposed therapeutic dose. In conclusion, dosing with anti-GM-CSFRα antibody using regimes that saturate circulating cells, and have been shown to be efficacious in inflammatory arthritis models, did not lead to complete blockade of the alveolar macrophages response to GM-CSF. This suggests a significant therapeutic window is possible with GM-CSF axis inhibition.

Anti-proliferative effects of T cells expressing a ligand-based chimeric antigen receptor against CD116 on CD34(+) cells of juvenile myelomonocytic leukemia.

BACKGROUND: Juvenile myelomonocytic leukemia (JMML) is a fatal, myelodysplastic/myeloproliferative neoplasm of early childhood. Patients with JMML have mutually exclusive genetic abnormalities in granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR, CD116) signaling pathway. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option for JMML; however, disease recurrence is a major cause of treatment failure. We investigated adoptive immunotherapy using GMR-targeted chimeric antigen receptor (CAR) for JMML. METHODS: We constructed a novel CAR capable of binding to GMR via its ligand, GM-CSF, and generated piggyBac transposon-based GMR CAR-modified T cells from three healthy donors and two patients with JMML. We further evaluated the anti-proliferative potential of GMR CAR T cells on leukemic CD34(+) cells from six patients with JMML (two NRAS mutations, three PTPN11 mutations, and one monosomy 7), and normal CD34(+) cells. RESULTS: GMR CAR T cells from healthy donors suppressed the cytokine-dependent growth of MO7e cells, but not the growth of K562 and Daudi cells. Co-culture of healthy GMR CAR T cells with CD34(+) cells of five patients with JMML at effector to target ratios of 1:1 and 1:4 for 2 days significantly decreased total colony growth, regardless of genetic abnormality. Furthermore, GMR CAR T cells from a non-transplanted patient and a transplanted patient inhibited the proliferation of respective JMML CD34(+) cells at onset to a degree comparable to healthy GMR CAR T cells. Seven-day co-culture of GMR CAR T cells resulted in a marked suppression of JMML CD34(+) cell proliferation, particularly CD34(+)CD38(-) cell proliferation stimulated with stem cell factor and thrombopoietin on AGM-S3 cells. Meanwhile, GMR CAR T cells exerted no effects on normal CD34(+) cell colony growth. CONCLUSIONS: Ligand-based GMR CAR T cells may have anti-proliferative effects on stem and progenitor cells in JMML.

European protocols for the diagnosis and initial treatment of interstitial lung disease in children.

Interstitial lung disease in children (chILD) is rare, and most centres will only see a few cases/year. There are numerous possible underlying diagnoses, with specific and non-specific treatment possibilities. The chILD-EU collaboration has brought together centres from across Europe to advance understanding of these considerations, and as part of this process, has created standard operating procedures and protocols for the investigation of chILD. Where established consensus documents exist already, for example, for the performance of bronchoalveolar lavage and processing of lung biopsies, these have been adopted. This manuscript reports our proposals for a staged investigation of chILD, starting from when the condition is suspected to defining the diagnosis, using pathways dependent on the clinical condition and the degree of illness of the child. These include the performance of genetic testing, echocardiography, high-resolution CT, bronchoscopy when appropriate and the definitive investigation of lung biopsy, in order to establish a precise diagnosis. Since no randomised controlled trials of treatment have ever been performed, we also report a Delphi consensus process to try to harmonise treatment protocols such as the use of intravenous and oral corticosteroids, and add-on therapies such as hydroxychloroquine and azithromycin. The aim is not to dictate to clinicians when a therapeutic trial should be performed, but to offer the possibility to collaborators of having a unified approach when a decision to treat has been made.

GM-CSF as a therapeutic target in inflammatory diseases.

GM-CSF is a well-known haemopoietic growth factor that is used in the clinic to correct neutropaenia, usually as a result of chemotherapy. GM-CSF also has many pro-inflammatory functions and recent data implicates GM-CSF as a key factor in Th17 driven autoimmune inflammatory conditions. In this review we summarize the findings that have led to the development of GM-CSF antagonists for the treatment of autoimmune diseases like rheumatoid arthritis (RA) and discuss some results of recent clinical trials of these agents.

Positive influence of Methotrexate-Hydroxychloroquine combination on the expression of GM-CSF receptor on neutrophils of synovial fluid in rheumatoid arthritis.

Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) has been inducted as a mediator of inflammation in rheumatoid arthritis. Methotrexate combination therapy forms an important component of the treatment regimen in rheumatoid arthritis. The present study was undertaken to evaluate the influence of Methotrexate-Hydroxychloroquine (MTX-HCQ) combination and Sulfsalazine- Hydroxychloroquine (SSZ-HCQ) combination on the expression GM-CSFR in neutrophils isolated from synovial fluids. 15 cases of confirmed rheumatoid arthritis patients who presented at the hospital for surgical correction of joint deformities were selected for the study. Neutrophils isolated from the synovial fluids were used as the source of the receptor for quantitation on an enzyme immunoassay (EIA). The EIA was developed and standardized in our laboratory for quantification of the GM-CSF R. The findings are suggestive of the fact that the administration of MTX-HCQ combination has positive influence on the expression of the GM-CSF R on neutrophils as against SSZ-HCQ combination. The physiological basis of this increase needs further investigation.

Plasma G-CSF and GM-CSF Concentration and Amount of Their Receptors on the Granulocyte in Kawasaki Disease

PURPOSE: This study aimed to demonstrate the possible pathogenesis of granulopoiesis in patients of Kawasaki disease(KD) using quantitative analysis of G-CSF, GM-CSF and their CSFr. METHODS: The plasma levels of G-CSF, GM-CSF, G-CSFr and GM-CSFr were studied in 14 patients in the acute phase of KD; 13 children with normal peripheral white blood cell counts were used as the normal control group. The plasma concentration of G-CSF, GM-CSF were analyzed by ELISA. The G-CSFr and GM-CSFr on the peripheral granulocytes were analyzed by a quantitative flow cytometric assay and QuantiBRITE, and the quantitative changes of receptors which did not combine with G-CSF and GM-CSF were measured. RESULTS: The total number of leukocytes in KD was similar to normal control group, but the leukocytes increased according to the number of neutrophils. The plasma concentration of G-CSF were decreased similar to normal control group(P=0.133), but that of GM-CSF decreased more than the normal control group(P=0.227). The quantity of G-CSFr, GM-CSFr were revealed to be no less than the normal control(P=0.721, P=0.912). After incubation with excessive G-CSF, the expressed G-CSFr on the neutrophils were decreased in both groups(P=0.554). The quantities of expressions of GM- CSFr on the neutrophil after incubation with the excessive GM-CSF were always increased in both groups(P=0.255). The amount of GM-CSFr of neutrophils are in proportion to total white blood cells (r=0.788, P=0.035), but it wasn't in the case of KD(P=0.644). CONCLUSION: The leukocytosis in KD that mediated by increasing neutrophil was not correlated with the plasma concentrations of G-CSF and GM-CSF, and the amount of expression of G-CSFr and GM-CSFr on granulocyte. It is possible that the reduction of concentration of GM-CSF results by increasing the active GM-CSFr.

Plasma G-CSF and GM-CSF Concentrations and Expression of their Receptors on the Granulocyte in Children with Leukocytosis

PURPOSE: Granulocyte-colony stimulating factor(G-CSF) and granulocyte macrophage-colony stimulating factor(GM-CSF) are principal cytokines in granulopoiesis and their physiologic effects are mediated through binding to specific cell surface receptors. Although it is known that the level of serum G-CSF and GM-CSF, and presentation of the receptors are increased in infectious diseases, there have been no studies to find the correlation between the granulopoiesis and leukocytosis. This study was designed to measure G-CSF and GM-CSF in leukocytosis and in control and to demonstrate the possible pathogenesis of granulopoiesis in leukocytosis using quantitative analysis of G- CSF, GM-CSF and their CSFr. METHODS: The plasma levels of G-CSF, GM-CSF of 13 children without leukocytosis and 14 children with leukocytosis were measured. Counts of cell surface G-CSFr and GM-CSFr were measured by combining anti G-CSFr and anti GM-CSFr monoclonal antibodies to their respective receptors by using quantitative flow cytometric assay. RESULTS: There was no significant difference betweeen the plasma concentration of G-CSF and GM-CSF in acute leukocytosis and in the control group. However, levels of G-CSFr in acute leukocytosis decreased significantly compared to the control(P=0.012) and the levels of GM-CSFr in both groups revealed no significant difference. CONCLUSION: Increase in the number of leukocyte in leukocytosis was mediated by increasing the number of neutrophil, and increased plasma concentration of G-CSF may be the cause of neutrophilia. But GM-CSF did not have any influence on leukocytosis.

Quantities of Receptor Molecules for Colony Stimulating Factors on Leukocytes in Measles

We analyzed the comparative amounts of granulocyte-colony stimulating factor (G-CSFr) and granulocyte macrophage CSF (GM-CSFr) receptors expressed on neutrophils and monocytes in measles patients to investigate the role of these CSFrs in the development of leukopenia including neutropenia and monocytopenia in measles. EDTA-anticoagulated peripheral blood of 19 measles patients, 10 children with other infections showing leukopenia and 16 children with normal complete blood cell counts (CBC)s were analyzed using flow cytometry and QuantiBRITE. The leukocyte (5260 +/- 2030/uL vs. 9900 + 2680/uL, p=0.000), neutrophil (2580 +/- 960/uL vs. 4250 +/- 2750/uL, p=0.024) and the lymphocyte counts of measles patients (1810 +/- 1430/uL vs. 4530 +/- 3450/uL, p= 0.006) were lower than in the normal controls. The neutrophils of measles patients expressed similar amounts of G- CSFr (1858 +/- 355) as normal children (1764 +/- 477, p= 0.564) and leukopenic patients (1773 +/- 673, p=0.713), but lower levels of GM-CSFr (535 +/- 118) than normal children (957 +/- 344, p=0.000) and leukopenic patients (832 +/- 294, p=0.002). The monocytes of measles patients expressed similar amounts of G-CSFr (916 +/- 336) and GM-CSFr (3718 +/- 906) as normal children (1013 +/- 391 and 4125 (2645, p > 0.05) but less than leukopenic patients (1454 +/- 398 and 5388 +/- 806, p > 0.05). The neutrophil and monocyte counts of measles patients did not correlate with the amount of G-CSFr or GM-CSFr expressed on neutrophils or monocytes (p > 0.05), but in the normal children, the monocyte count correlated with the levels of GM-CSFr on monocytes (r=0.951, p=0.049). In conclusion, neutropenia is one of the more important characteristics of measles patients, which could be due to the decreased GM-CSFr expression on neutrophils. However, the monocytopenia found in measles patients is not due to the decreased expression of CSFr on the monocytes.