Neutrophil development and function critically depend on Bruton tyrosine kinase in a mouse model of X-linked agammaglobulinemia

Abstract Bruton tyrosine kinase (Btk) is essential for B cell development and function and also appears to be important for myeloid cells. The bone marrow of Btk-deficient mice shows enhanced granulopoiesis compared with that of wild-type mice. In purified granulocyte-monocyte-progenitors (GMP) from Btk-deficient mice, the development of granulocytes is favored at the expense of monocytes. However, Btk-deficient neutrophils are impaired in maturation and function. Using bone marrow chimeras, we show that this defect is cell-intrinsic to neutrophils. In GMP and neutrophils, Btk plays a role in GM-CSF– and Toll-like receptor–induced differentiation. Molecular analyses revealed that expression of the lineage-determining transcription factors C/EBPα, C/EBPβ, and PU.1, depends on Btk. In addition, expression of several granule proteins, including myeloperoxidase, neutrophilic granule protein, gelatinase and neutrophil elastase, is Btk-dependent. In the Arthus reaction, an acute inflammatory response, neutrophil migration into tissues, edema formation, and hemorrhage are significantly reduced in Btk-deficient animals. Together, our findings implicate Btk as an important regulator of neutrophilic granulocyte maturation and function in vivo.

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The Vav binding site of the non–receptor tyrosine kinase Syk at Tyr 348 is critical for β2 integrin (CD11/CD18)–mediated neutrophil migration

Blood ◽

10.1182/blood-2005-12-030387 ◽

2006 ◽

Vol 108 (12) ◽

pp. 3919-3927 ◽

Cited By ~ 55

Author(s):

Jurgen Schymeinsky ◽

Anca Sindrilaru ◽

David Frommhold ◽

Markus Sperandio ◽

Ronald Gerstl ◽

...

Keyword(s):

Bone Marrow ◽

Tyrosine Kinase ◽

Binding Site ◽

Leukocyte Adhesion ◽

Neutrophil Migration ◽

Nucleotide Exchange ◽

Edema Formation ◽

Β2 Integrin

Abstract Leukocyte adhesion via β2 integrins (CD11/CD18) activates the tyrosine kinase Syk. We found that Syk was enriched at the lamellipodium during N-formyl-Met-Leu-Phe–induced migration of neutrophil-like differentiated HL-60 cells. Here, Syk colocalized with Vav, a guanine nucleotide exchange factor for Rac and Cdc42. The enrichment of Syk at the lamellipodium and its colocalization with Vav were absent upon expression of a Syk kinase-dead mutant (Syk K402R) or a Syk mutant lacking the binding site of Vav (Syk Y348F). Live cell imaging revealed that both mutations resulted in excessive lamellipodium formation and severely compromised migration compared with control cells. Similar results were obtained upon down-regulation of Syk by RNA interference (RNAi) technique as well as in Syk–/– neutrophils from wild-type mice reconstituted with Syk–/– bone marrow. A pivotal role of Syk in vivo was demonstrated in the Arthus reaction, where neutrophil extravasation, edema formation, and hemorrhage were profoundly diminished in Syk–/– bone marrow chimeras compared with those in control animals. In the inflamed cremaster muscle, Syk–/– neutrophils revealed a defect in adhesion and migration. These findings indicate that Syk is critical for β2 integrin–mediated neutrophil migration in vitro and plays a fundamental role in neutrophil recruitment during the inflammatory response in vivo.

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Mice lacking both macrophage- and granulocyte-macrophage colony- stimulating factor have macrophages and coexistent osteopetrosis and severe lung disease

Blood ◽

10.1182/blood.v84.1.27.27 ◽

1994 ◽

Vol 84 (1) ◽

pp. 27-35 ◽

Cited By ~ 92

Author(s):

GJ Lieschke ◽

E Stanley ◽

D Grail ◽

G Hodgson ◽

V Sinickas ◽

...

Keyword(s):

Lung Pathology ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Alveolar Proteinosis ◽

Macrophage Colony ◽

And Function ◽

Stimulating Factor ◽

Deficient Mice

Abstract Mice deficient in granulocyte-macrophage colony-stimulating factor (GM- CSF) and macrophage colony-stimulating factor (M-CSF, CSF-1) were generated by interbreeding GM-CSF-deficient mice generated by gene targeting (genotype GM-/-) with M-CSF-deficient osteopetrotic mice (genotype M-/-, op/op). Mice deficient in both GM-CSF and M-CSF (genotype GM-/-M-/-) are viable and have coexistent features corresponding to mice deficient in either factor alone. Like M-CSF- deficient mice, they have osteopetrosis and are toothless because of failure of incisor eruption. Like GM-CSF-deficient mice, they have a characteristic alveolar-proteinosis-like lung pathology, but it is more severe than that of GM-CSF-deficient mice and is often fatal. In particular, in GM-/-M-/- mice the accumulation of lipo-proteinaceous alveolar material is more marked, and bacterial pneumonic infections are more prevalent and more extensive, particularly involving Gram- negative bacteria. Neutrophilia consistently accompanies pulmonary infections, and some older GM-/-M-/- mice have polycythemia. Survival of GM-/-M-/- mice is significantly reduced compared with mice deficient in either factor alone, and all GM-/-M-/- mice have broncho- or lobar- pneumonia at death. These observations indicate that in vivo, M-CSF is involved in modulating the consequences of GM-CSF deficiency in the lung. Interestingly, GM-/-M-/- mice have circulating monocytes at levels comparable with those in M-CSF-deficient mice and the diseased lungs of all GM-/-M-/- mice contain numerous phagocytically active macrophages, indicating that in addition to GM-CSF and M-CSF, other factors can be used for macrophage production and function in vivo.

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Mice lacking both macrophage- and granulocyte-macrophage colony- stimulating factor have macrophages and coexistent osteopetrosis and severe lung disease

Blood ◽

10.1182/blood.v84.1.27.bloodjournal84127 ◽

1994 ◽

Vol 84 (1) ◽

pp. 27-35 ◽

Cited By ~ 6

Author(s):

GJ Lieschke ◽

E Stanley ◽

D Grail ◽

G Hodgson ◽

V Sinickas ◽

...

Keyword(s):

Lung Pathology ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Alveolar Proteinosis ◽

Macrophage Colony ◽

And Function ◽

Stimulating Factor ◽

Deficient Mice

Mice deficient in granulocyte-macrophage colony-stimulating factor (GM- CSF) and macrophage colony-stimulating factor (M-CSF, CSF-1) were generated by interbreeding GM-CSF-deficient mice generated by gene targeting (genotype GM-/-) with M-CSF-deficient osteopetrotic mice (genotype M-/-, op/op). Mice deficient in both GM-CSF and M-CSF (genotype GM-/-M-/-) are viable and have coexistent features corresponding to mice deficient in either factor alone. Like M-CSF- deficient mice, they have osteopetrosis and are toothless because of failure of incisor eruption. Like GM-CSF-deficient mice, they have a characteristic alveolar-proteinosis-like lung pathology, but it is more severe than that of GM-CSF-deficient mice and is often fatal. In particular, in GM-/-M-/- mice the accumulation of lipo-proteinaceous alveolar material is more marked, and bacterial pneumonic infections are more prevalent and more extensive, particularly involving Gram- negative bacteria. Neutrophilia consistently accompanies pulmonary infections, and some older GM-/-M-/- mice have polycythemia. Survival of GM-/-M-/- mice is significantly reduced compared with mice deficient in either factor alone, and all GM-/-M-/- mice have broncho- or lobar- pneumonia at death. These observations indicate that in vivo, M-CSF is involved in modulating the consequences of GM-CSF deficiency in the lung. Interestingly, GM-/-M-/- mice have circulating monocytes at levels comparable with those in M-CSF-deficient mice and the diseased lungs of all GM-/-M-/- mice contain numerous phagocytically active macrophages, indicating that in addition to GM-CSF and M-CSF, other factors can be used for macrophage production and function in vivo.

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The In Vivo Granulopoietic Response to Dexamethasone Injection Is Abolished in Perforin-Deficient Mutant Mice and Corrected by Lymphocyte Transfer from Nonsensitized Wild-Type Donors

Mediators of Inflammation ◽

10.1155/2015/495430 ◽

2015 ◽

Vol 2015 ◽

pp. 1-12 ◽

Cited By ~ 2

Author(s):

Pedro Xavier-Elsas ◽

Cássio Luiz Coutinho Almeida da Silva ◽

Bruno Marques Vieira ◽

Daniela Masid-de-Brito ◽

Túlio Queto ◽

...

Keyword(s):

Bone Marrow ◽

Colony Formation ◽

Allergic Sensitization ◽

Blood Eosinophil ◽

Neutrophil Granulocyte ◽

Splenic Lymphocytes ◽

Wild Type ◽

Gm Csf ◽

Deficient Mice

Exogenously administered glucocorticoids enhance eosinophil and neutrophil granulocyte production from murine bone-marrow. A hematological response dependent on endogenous glucocorticoids underlies bone-marrow eosinophilia induced by trauma or allergic sensitization/challenge. We detected a defect in granulopoiesis in nonsensitized, perforin-deficient mice. In steady-state conditions, perforin- (Pfp-) deficient mice showed significantly decreased bone-marrow and blood eosinophil and neutrophil counts, and colony formation in response to GM-CSF, relative to wild-type controls of comparable age and/or weight. By contrast, peripheral blood or spleen total cell and lymphocyte numbers were not affected by perforin deficiency. Dexamethasone enhanced colony formation by GM-CSF-stimulated progenitors from wild-type controls, but not Pfp mice. Dexamethasone injection increased bone-marrow eosinophil and neutrophil counts in wild-type controls, but not Pfp mice. Because perforin is expressed in effector lymphocytes, we examined whether this defect would be corrected by transferring wild-type lymphocytes into perforin-deficient recipients. Short-term reconstitution of the response to dexamethasone was separately achieved for eosinophils and neutrophils by transfer of distinct populations of splenic lymphocytes from nonsensitized wild-type donors. Transfer of the same amount of splenic lymphocytes from perforin-deficient donors was ineffective. This demonstrates that the perforin-dependent, granulopoietic response to dexamethasone can be restored by transfer of innate lymphocyte subpopulations.

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Macrophage Erythroblast Attacher (MAEA), but Not VCAM1, Is Required for the Bone Marrow Erythroblastic Niche

Blood ◽

10.1182/blood.v126.23.2128.2128 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2128-2128 ◽

Cited By ~ 2

Author(s):

Qiaozhi Wei ◽

Paul S. Frenette

Keyword(s):

Bone Marrow ◽

Adhesion Molecule ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Fold Reduction ◽

And Function ◽

Deficient Mice

Abstract Definitive mammalian erythropoiesis occurs in a specialized niche called erythroblastic island (EI), which is composed of a central macrophage surrounded by maturing erythroblasts. The attachment of the developing erythroblasts to the central macrophages within the islands has been suggested to be critical for the survival, proliferation and regulated differentiation of the developing erythrocytes both in vitro and in vivo. Several adhesion molecules have been suggested to mediate the interaction between the macrophage and erythroblasts in the EI niche. However, cell type-specific requirement of these molecules for EI formation and function in vivo hasn't been examined. We sought to identify the crucial adhesion molecule(s) responsible for the in vivo EI function using macrophage-specific conditional deletion mouse models. As Vascular Cell Adhesion Molecule-1 (VCAM1) has been suggested to play a key role in erythropoiesis, we deleted the gene selectively in macrophage by crossing Vcam1fl/fl mice with Csf1r-Cre transgenic mice. Our results revealed that macrophage VCAM1 was not required for steady-state erythropoiesis in vivo since bone marrow and spleen erythroblasts and hematocrit levels were not altered. Stress erythropoiesis induced by phenylhydrazine (PHZ) led to mild deficit in hematocrit recovery but no significant anemia, suggesting the contribution of other adhesion receptors. We next generated a conditional floxed allele of the Macrophage Erythroblast Attacher (Maea), which has also been suggested to be an important component of the erythroblastic niche (Soni et al. J. Biol. Chem, 2006). Efficient ablation ofMAEA (~2-fold protein level reduction on macrophages) using Csf1r-Cre resulted in a more than 3-fold reduction of leukocyte counts (p=0.003), but no significant anemia in peripheral blood. However, the cellularity in the bone marrow was significantly reduced in Maea -deficient mice, owing largely to > 2-fold reduction of erythroblasts (p=0.01). Analyses of erythroblast maturation by FACS revealed a significant increase in the proportion of the less mature erythroblasts at the expenses of terminal differentiation, indicating that MAEA regulates erythroblast maturation. Interestingly, BM macrophage numbers were also severely affected in Maea -deficient mice (~4-fold reduction, p=0.01), suggesting a role for MAEA in macrophage development. By contrast, the spleen of Maeafl/-; Csf1r-Cre mice did not exhibit any reduction in erythroblast numbers, indicating that the erythroblast-macrophage interaction may be differentially regulated in BM and spleen. Unexpectedly, Maea ablation impaired the engraftment ability of BM hematopoietic stem and progenitors (HSPCs) after transplantation, suggesting broader functions for this protein in hematopoiesis. These studies identify MAEA as a critical adhesion mediator between the erythroblasts and central macrophages in adult murine bone marrow. Ongoing studies will shed light on its involvement in stress and pathological erythropoiesis and HSPC regulation. Disclosures No relevant conflicts of interest to declare.

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Surfactant metabolism in transgenic mice after granulocyte macrophage-colony stimulating factor ablation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1996.270.4.l650 ◽

1996 ◽

Vol 270 (4) ◽

pp. L650-L658 ◽

Cited By ~ 39

Author(s):

M. Ikegami ◽

T. Ueda ◽

W. Hull ◽

J. A. Whitsett ◽

R. C. Mulligan ◽

...

Keyword(s):

Protein A ◽

Surfactant Protein ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

And Function ◽

Stimulating Factor ◽

Deficient Mice ◽

Surfactant Metabolism

Mice made granulocyte macrophage-colony stimulating factor (GM-CSF)-deficient by homologous recombination maintain normal steady-state hematopoiesis but have an alveolar accumulation of surfactant lipids and protein that is similar to pulmonary alveolar proteinosis in humans. We asked how GM-CSF deficiency alters surfactant metabolism and function in mice. Alveolar and lung tissue saturated phosphatidylcholine (Sat PC) were increased six- to eightfold in 7- to 9-wk-old GM-CSF-deficient mice relative to controls. Incorporation of radiolabeled palmitate and choline into Sat PC was higher in GM-CSF deficient mice than control mice, and no loss of labeled Sat PC occurred from the lungs of GM-CSF-deficient mice. Secretion of radiolabeled Sat PC to the alveolus was similar in GM-CSF-deficient and control mice. Labeled Sat PC and surfactant protein A (SP-A) given by tracheal instillation were cleared rapidly in control mice, but there was no measurable loss from the lungs of GM-CSF-deficient mice. The function of the surfactant from GM-CSF-deficient mice was normal when tested in preterm surfactant-deficient rabbits. GM-CSF deficiency results in a catabolic defect for Sat PC and SP-A.

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In vivo administration of cytokines in murine bone marrow chimeras

International Journal of Immunopharmacology ◽

10.1016/0192-0561(88)90419-5 ◽

1988 ◽

Vol 10 ◽

pp. 104

Author(s):

M. Imamura ◽

H. Fujimoto ◽

T. Fukuhara ◽

S. Hashino ◽

M. Kobayashi ◽

...

Keyword(s):

Bone Marrow ◽

Murine Bone Marrow ◽

Bone Marrow Chimeras ◽

In Vivo Administration

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The immunosuppressant rapamycin blocks in vitro responses to hematopoietic cytokines and inhibits recovering but not steady-state hematopoiesis in vivo

Blood ◽

10.1182/blood.v84.5.1543.1543 ◽

1994 ◽

Vol 84 (5) ◽

pp. 1543-1552 ◽

Cited By ~ 39

Author(s):

VF Quesniaux ◽

S Wehrli ◽

C Steiner ◽

J Joergensen ◽

HJ Schuurman ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

T Cell ◽

T Cell Response ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Hematopoietic Recovery ◽

Stimulating Factor

Abstract The immunosuppressive drug rapamycin suppresses T-cell activation by impairing the T-cell response to lymphokines such as interleukin-2 (IL- 2) and interleukin-4 (IL-4). In addition, rapamycin blocks the proliferative response of cell lines to a variety of hematopoietic growth factors, including interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage- colony stimulating factor (GM-CSF), and kit ligand (KL), suggesting that it should be a strong inhibitor of hematopoiesis. In this report, we studied the effects of rapamycin on different hematopoietic cell populations in vitro and in vivo. In vitro, rapamycin inhibited the proliferation of primary bone marrow cells induced by IL-3, GM-CSF, KL, or a complex mixture of factors present in cell-conditioned media. Rapamycin also inhibited the multiplication of colony-forming cells in suspension cultures containing IL-3 plus interleukin-1 (IL-1) or interleukin-11 (IL-11) plus KL. In vivo, treatment for 10 to 28 days with high doses of rapamycin (50 mg/kg/d, orally) had no effect on myelopoiesis in normal mice, as measured by bone marrow cellularity, proliferative capacity, and number of colony-forming progenitors. In contrast, the same treatment strongly suppressed the hematopoietic recovery normally seen 10 days after an injection of 5-fluorouracil (5- FU; 150 mg/kg, intravenously [i.v.]). Thus, rapamycin may be detrimental in myelocompromised individuals. In addition, the results suggest that the rapamycin-sensitive cytokine-driven pathways are essential for hematopoietic recovery after myelodepression, but not for steady-state hematopoiesis.

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Gene transfer into normal human hematopoietic cells using in vitro and in vivo assays

Blood ◽

10.1182/blood.v78.3.624.624 ◽

1991 ◽

Vol 78 (3) ◽

pp. 624-634 ◽

Cited By ~ 57

Author(s):

JE Dick ◽

S Kamel-Reid ◽

B Murdoch ◽

M Doedens

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Gene Transfer ◽

Hematopoietic Cells ◽

Human Stem Cells ◽

In Vivo Assays ◽

Genetically Manipulated ◽

Deficient Mice

Abstract The ability to transfer new genetic material into human hematopoietic cells provides the foundation for characterizing the organization and developmental program of human hematopoietic stem cells. It also provides a valuable model in which to test gene transfer and long-term expression in human hematopoietic cells as a prelude to human gene therapy. At the present time such studies are limited by the absence of in vivo assays for human stem cells, although recent descriptions of the engraftment of human hematopoietic cells in immune-deficient mice may provide the basis for such an assay. This study focuses on the establishment of conditions required for high efficiency retrovirus- mediated gene transfer into human hematopoietic progenitors that can be assayed in vitro in short-term colony assays and in vivo in immune- deficient mice. Here we report that a 24-hour preincubation of human bone marrow in 5637-conditioned medium, before infection, increases gene transfer efficiency into in vitro colony-forming cells by sixfold; interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) provide the same magnitude increase as 5637-conditioned medium. In contrast, incubation in recombinant growth factors IL-1, IL-3, and granulocyte- macrophage colony-stimulating factor increases gene transfer efficiency by 1.5- to 3-fold. Furthermore, preselection in high concentrations of G418 results in a population of cells significantly enriched for G418- resistant progenitors (up to 100%). These results, obtained using detailed survival curves based on colony formation in G418, have been substantiated by directly detecting the neo gene in individual colonies using the polymerase chain reaction. Using these optimized protocols, human bone marrow cells were genetically manipulated with a neo retrovirus vector and transplanted into immune-deficient bg/nu/xid mice. At 1 month and 4 months after the transplant, the hematopoietic tissues of these animals remained engrafted with genetically manipulated human cells. More importantly, G418-resistant progenitors that contained the neo gene were recovered from the bone marrow and spleen of engrafted animals after 4 months. These experiments establish the feasibility of characterizing human stem cells using the unique retrovirus integration site as a clonal marker, similar to techniques developed to elucidate the murine stem cell hierarchy.

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Leishmania donovani infection of bone marrow stromal macrophages selectively enhances myelopoiesis, by a mechanism involving GM-CSF and TNF-α

Blood ◽

10.1182/blood.v95.5.1642.005k10_1642_1651 ◽

2000 ◽

Vol 95 (5) ◽

pp. 1642-1651 ◽

Cited By ~ 45

Author(s):

Sara E. J. Cotterell ◽

Christian R. Engwerda ◽

Paul M. Kaye

Keyword(s):

Infectious Disease ◽

Bone Marrow ◽

Stromal Cell ◽

Leishmania Donovani ◽

Protozoan Parasite ◽

Gm Csf ◽

Tnf Α ◽

Macrophage Colony

Alterations in hematopoiesis are common in experimental infectious disease. However, few studies have addressed the mechanisms underlying changes in hematopoietic function or assessed the direct impact of infectious agents on the cells that regulate these processes. In experimental visceral leishmaniasis, caused by infection with the protozoan parasite Leishmania donovani, parasites persist in the spleen and bone marrow, and their expansion in these sites is associated with increases in local hematopoietic activity. The results of this study show that L donovani targets bone marrow stromal macrophages in vivo and can infect and multiply in stromal cell lines of macrophage, but not other lineages in vitro. Infection of stromal macrophages increases their capacity to support myelopoiesis in vitro, an effect mediated mainly through the induction of granulocyte macrophage-colony stimulating factor and tumor necrosis factor-. These data are the first to directly demonstrate that intracellular parasitism of a stromal cell population may modify its capacity to regulate hematopoiesis during infectious disease.

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