Cells Of Myelomonocytic Lineage Research Articles

Abstract LB363: Patient-derived iPSCs faithfully represent the genetic diversity and cellular architecture of human acute myeloid leukemia

Abstract The reprogramming of malignant cells to pluripotency has presented considerable challenges, hindering the application of induced pluripotent stem cell (iPSC) modeling technology to human cancers. In addition, how well iPSC-derived cells resemble their primary counterparts is currently largely unknown. We developed a reprogramming method tailored to human malignancies, “Complete Capture of Mutational Burden” (CCoMB), that combines comprehensive genetic characterization of the starting sample and inference of its clonal architecture with large-scale screening of clonally reprogrammed colonies. Using this method we were able to generate a panel of iPSC lines representing all major genetic groups of acute myeloid leukemia (AML). Specifically, we derived iPSC lines from 15 patients representing all major genetic groups of AML - PML-RARA; chromatin-spliceosome; TP53-mutated/aneuploidy; AML1-ETO, MLL-rearranged; NPM1-mutated and others - collectively capturing 21 distinct genotypes and 24 driver genetic lesions (mutations, translocations, deletions). Matched normal iPSCs were derived from 7 of these patients. Reprogramming to iPSCs captured both preleukemic (CH/initiating mutation only) and fully leukemic clones (baring the full set of patient mutations). In almost all cases, reprogramming informed reconstruction of the evolutionary hierarchy of the AML, with unexpected hierarchies unveiled in 4 of the cases. These AML-iPSCs retain genetic fidelity and, upon in vitro hematopoietic differentiation, produce hematopoietic cells with hallmark phenotypic leukemic features, including serial engraftment of a lethal myeloid leukemia into immunocompromised mice and extended self-renewal in vitro. Transplantation of cells derived from iPSCs representing two distinct AML clones from each of 3 patients revealed that these mimic the clonal dynamics in the patients, with more advanced clones showing increased representation in the xenografts, compared to the earlier clones. To compare the iPSC-derived to the primary leukemias, we performed single-cell transcriptomics analyses in patient-matched iPSC-derived and primary leukemic cells from 3 patients, both ex vivo/in vitro and after transplantation into NSGS mice. Clustering analyses identified cell types corresponding to primitive hematopoietic stem cell (HSC)/multipotent progenitor (MPP), hematopoietic progenitor cells (HPCs) and more mature myelomonocytic lineage cells in all samples from all patients, at varying frequencies. Leukemias derived through in vitro differentiation from iPSC lines exhibited both similarities, as well as differences, in their cellular composition and transcriptome, compared to the patient-matched ex vivo leukemias. However, upon transplantation of the same leukemias into NSGS mice, iPSC-derived xenografts were strikingly similar to the patient-derived xenografts. iPSC-derived leukemic cells exhibited a more stem/progenitor cell phenotype in vitro, with progressive maturation along the myeloid lineage upon primary and, even more, upon secondary transplantation, mimicking primary xenografts. In summary, our results reveal very few true biological barriers to the reprogramming of AML cells and show that AML-iPSC-derived leukemias faithfully mimic the primary patient leukemias upon xenotransplantation. Citation Format: Andriana Kotini, Saul Carcamo, Nataly Cruz-Rodriguez, Malgorzata Olszewska, Dan Hasson, Eirini Papapetrou. Patient-derived iPSCs faithfully represent the genetic diversity and cellular architecture of human acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB363.

Patient-Derived iPSCs Faithfully Represent the Genetic Diversity and Cellular Architecture of Human Acute Myeloid Leukemia

The reprogramming of malignant cells to pluripotency has presented considerable challenges, hindering the application of induced pluripotent stem cell (iPSC) modeling technology to human leukemia. In addition, how well iPSC-derived cells resemble their primary counterparts is currently largely unknown. We developed a reprogramming method tailored to human malignancies, "Complete Capture of Mutational Burden" (CCoMB), that combines comprehensive genetic characterization of the starting sample and inference of its clonal architecture with large-scale screening of clonally reprogrammed colonies. Using this method we were able to generate a panel of iPSC lines for the modeling of human acute myeloid leukemia (AML) representing all major genetic groups of AML. Specifically, we derived iPSC lines from 15 patients representing all major genetic groups of AML - PML-RARA; chromatin-spliceosome; TP53-mutated/aneuploidy; AML1-ETO, MLL-rearranged; NPM1-mutated and others - collectively capturing 21 distinct genotypes and 24 driver genetic lesions (mutations, translocations, deletions). Matched normal iPSCs were derived from 7 of these patients. Reprogramming to iPSCs captured both preleukemic (CH/initiating mutation only) and fully leukemic clones (baring the full set of patient mutations). In almost all cases, reprogramming informed reconstruction of the evolutionary hierarchy of the AML, with unexpected hierarchies unveiled in 4 of the cases. These AML-iPSCs retain genetic fidelity and, upon in vitro hematopoietic differentiation, produce hematopoietic cells with hallmark phenotypic leukemic features, including serial engraftment of a lethal myeloid leukemia into immunocompromised mice and extended self-renewal in vitro. Transplantation of cells derived from iPSCs representing two distinct AML clones from each of 3 patients revealed that these mimic the clonal dynamics in the patients, with more advanced clones showing increased representation in the xenografts, compared to the earlier clones. To compare the iPSC-derived to the primary leukemias, we performed single-cell transcriptomics analyses in patient-matched iPSC-derived and primary leukemic cells from 3 patients, both ex vivo/in vitro and after transplantation into NSGS mice. Clustering analyses identified cell types corresponding to primitive hematopoietic stem cell (HSC)/ multipotent progenitor (MPP), hematopoietic progenitor cells (HPCs) and more mature myelomonocytic lineage cells in all samples from all patients, at varying frequencies. Leukemias derived through in vitro differentiation from iPSC lines exhibited both similarities, as well as differences, in their cellular composition and transcriptome, compared to the patient-matched ex vivo leukemias. However, upon transplantation of the same leukemias into NSG-3GS mice, iPSC-derived xenografts were strikingly similar to the patient-derived xenografts. iPSC-derived leukemic cells exhibited a more stem/progenitor cell phenotype in vitro, with progressive maturation along the myeloid lineage upon primary and, even more, upon secondary transplantation, mimicking primary xenografts. In summary, our results reveal very few true biological barriers to the reprogramming of AML cells (with NPM1 mutations the most notable) and show that AML-iPSC-derived leukemias faithfully mimic the primary patient leukemias upon xenotransplantation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Morpho-functional variation and response pattern of microglia through rodent ontogeny showing infant microglia as stable and adaptive than matured.

Microglia, myelo‐monocytic lineage cells, that enter in the developing brain at early embryonic stages and integrate in CNS, are involved in almost all neuroinflammatory conditions. We studied how microglia change their responses through the development and maturation of brain in normal physiological conditions using an ex situ model to delineate their age‐specific morpho‐functional responsiveness. Rapidly isolated microglia from different age‐matched rats were characterized with Iba1+/CD11b/c+/MHCclassII+, cultured, studied for cell‐cycle/proliferative potency, ROS generation and phagocytosis, viability and morphological analysis induced with GMCSF, MCSF, IL‐4, IL‐6, IL‐10, and IFN‐γ. The study showed marked differences in cellular properties, stability, and viability of microglia through ontogeny with specific patterns in their studied functions which were coherent with their in situ morpho‐functional attributes. Phagocytic behavior showed a notable shift from ROS independence to dependence toward maturation. Perinatal microglia were found persistent in ex situ environment and neonatal microglia qualified as the most potent and versatile responders for morpho‐functional variations under cytokine induced conditions. The study identified that microglia from infants were the most stable, adaptive, and better responders, which can perform as an ex situ model system to study microglial biology.

Metabolic Effects of Selective Deletion of Group VIA Phospholipase A2 from Macrophages or Pancreatic Islet Beta-Cells.

To examine the role of group VIA phospholipase A2 (iPLA2β) in specific cell lineages in insulin secretion and insulin action, we prepared mice with a selective iPLA2β deficiency in cells of myelomonocytic lineage, including macrophages (MØ-iPLA2β-KO), or in insulin-secreting β-cells (β-Cell-iPLA2β-KO), respectively. MØ-iPLA2β-KO mice exhibited normal glucose tolerance when fed standard chow and better glucose tolerance than floxed-iPLA2β control mice after consuming a high-fat diet (HFD). MØ-iPLA2β-KO mice exhibited normal glucose-stimulated insulin secretion (GSIS) in vivo and from isolated islets ex vivo compared to controls. Male MØ-iPLA2β-KO mice exhibited enhanced insulin responsivity vs. controls after a prolonged HFD. In contrast, β-cell-iPLA2β-KO mice exhibited impaired glucose tolerance when fed standard chow, and glucose tolerance deteriorated further when introduced to a HFD. β-Cell-iPLA2β-KO mice exhibited impaired GSIS in vivo and from isolated islets ex vivo vs. controls. β-Cell-iPLA2β-KO mice also exhibited an enhanced insulin responsivity compared to controls. These findings suggest that MØ iPLA2β participates in HFD-induced deterioration in glucose tolerance and that this mainly reflects an effect on insulin responsivity rather than on insulin secretion. In contrast, β-cell iPLA2β plays a role in GSIS and also appears to confer some protection against deterioration in β-cell functions induced by a HFD.

Overexpression of myeloid angiotensin-converting enzyme (ACE) reduces atherosclerosis

BackgroundMacrophages are ubiquitous in all stages of atherosclerosis, exerting tremendous impact on lesion progression and plaque stability. Because macrophages in atherosclerotic plaques express angiotensin-converting enzyme (ACE), current dogma posits that local myeloid-mediated effects worsen the disease. In contrast, we previously reported that myeloid ACE overexpression augments macrophage resistance to various immune challenges, including tumors, bacterial infection and Alzheimer’s plaque deposition. Here, we sought to assess the impact of myeloid ACE on atherosclerosis. MethodsA mouse model in which ACE is overexpressed in myelomonocytic lineage cells, called ACE10, was generated and sequentially crossed with ApoE-deficient mice to create ACE10/10ApoE−/− (ACE10/ApoE). Control mice were ACEWT/WTApoE−/− (WT/ApoE). Atherosclerosis was induced using an atherogenic diet alone, or in combination with unilateral nephrectomy plus deoxycorticosterone acetate (DOCA) salt for eight weeks. ResultsWith an atherogenic diet alone or in combination with DOCA, the ACE10/ApoE mice showed significantly less atherosclerotic plaques compared to their WT/ApoE counterparts (p < 0.01). When recipient ApoE−/− mice were reconstituted with ACE10/10 bone marrow, these mice showed significantly reduced lesion areas compared to recipients reconstituted with wild type bone marrow. Furthermore, transfer of ACE-deficient bone marrow had no impact on lesion area. ConclusionOur data indicate that while myeloid ACE may not be required for atherosclerosis, enhanced ACE expression paradoxically reduced disease progression.

Immunomodulatory activity of extracellular Hsp70 mediated via paired receptors Siglec-5 andSiglec-14.

The intracellular chaperone heat-shock protein 70 (Hsp70) can be secreted from cells, but its extracellular role is unclear, as the protein has been reported to both activate and suppress the innate immune response. Potential immunomodulatory receptors on myelomonocytic lineage cells that bind extracellular Hsp70 are not well defined. Siglecs are Ig-superfamily lectins on mammalian leukocytes that recognize sialic acid-bearing glycans and thereby modulate immune responses. Siglec-5 and Siglec-14, expressed onmonocytes and neutrophils, share identical ligand-binding domains but have opposing signaling functions. Based on phylogenetic analyses of these receptors, we predicted that endogenous sialic acid-independent ligands should exist. An unbiased screen revealed Hsp70 as a ligand for Siglec-5 and Siglec-14. Hsp70 stimulation through Siglec-5 delivers an anti-inflammatory signal, while stimulation through Siglec-14 is pro-inflammatory. The functional consequences of this interaction are also addressed in relation to a SIGLEC14 polymorphism found in humans. Our results demonstrate that an endogenous non-sialic acid-bearing molecule can be either a danger-associated or self-associated signal through paired Siglecs, and may explain seemingly contradictory prior reports on extracellular Hsp70 action.

Aminopeptidase N (CD13) Is Involved in Phagocytic Processes in Human Dendritic Cells and Macrophages

Aminopeptidase N (APN or CD13) is a membrane ectopeptidase expressed by many cell types, including myelomonocytic lineage cells: monocytes, macrophages, and dendritic cells. CD13 is known to regulate the biological activity of various peptides by proteolysis, and it has been proposed that CD13 also participates in several functions such as angiogenesis, cell adhesion, metastasis, and tumor invasion. We had previously reported that, in human monocytes and macrophages, CD13 modulates the phagocytosis mediated by receptors for the Fc portion of IgG antibodies (FcγRs). In this work, we analyzed the possible interaction of CD13 with other phagocytic receptors. We found out that the cross-linking of CD13 positively modulates the phagocytosis mediated by receptors of the innate immune system, since a significant increase in the phagocytosis of zymosan particles or heat-killed E. coli was observed when CD13 was cross-linked using anti-CD13 antibodies, in both macrophages and dendritic cells. Also, we observed that, during the phagocytosis of zymosan, CD13 redistributes and is internalized into the phagosome. These findings suggest that, besides its known functions, CD13 participates in phagocytic processes in dendritic cells and macrophages.

Disease Modeling and Drug Discovery Using Induced Pluripotent Stem Cells From CINCA Syndrome Patients

AbstractAbstract 4680Chronic infantile neurologic cutaneous and articular syndrome (CINCA syndrome; MIM #607715) is a dominantly inherited autoinflammatory disease characterized by systemic inflammation with an urticaria-like rash, neurological manifestations, and arthropathy. NLRP3 mutation is the first and so far the only identified mutation that is responsible for CINCA syndrome. NLRP3 is expressed mainly in myelomonocytic lineage cells and chondrocytes, and acts as an intracellular sensor of danger signals from various cellular insults. In normal macrophages, a first stimulus, such as lipopolysaccharide (LPS), induces the synthesis of NLRP3 and the biologically inactive proIL-1β. A second stimulus, such as ATP, enhances the assembly of a protein complex called the NLRP3-inflammasome. The inflammasome contains caspase1, which executes the proteolytic maturation and secretion of IL-1β. While normal monocytes/macrophages show no or limited IL-1β secretion in response to LPS stimulation alone, CINCA patients’ cells exhibit robust IL-1β secretion, because the mutant NLRP3-inflammasome is auto-activated without the need for any second stimulus.While approximately half of CINCA patients carry heterozygous gain-of-function mutations of the NLRP3 gene, 30 to 40% of all patients have mutations in NLRP3 in only a small number of somatic cells. Since the population of mutant cells is relatively small (4.2–35.8% in blood cells), it remains controversial whether the small fraction of NLRP3-mutated cells actually causes the strong autoinflammation observed in CINCA patients, or whether the NLRP3 mutations found in mosaic patients are just a bystander, with all cells carrying an unknown mutation of another gene that causes the disease. The pathogenesis of CINCA syndrome patients who carry NLRP3mutations as somatic mosaicism has not been precisely described because of the difficulty in separating live cells based on the presence or absence of the mutation.It is believed that most of the manifestations of CINCA syndrome are caused by the excessive secretion of the proinflammatory cytokine, IL-1β, and this concept is supported by the efficacy of an IL-1 receptor antagonist (IL-1Ra) for decreasing most of the symptoms. However, an NLRP3-targeted therapeutic approach would be attractive because 1) the progressive arthropathy despite anti-IL-1 therapy indicates that the presence of additional proteins processed by the inflammasome is also involved in the pathogenesis of CINCA syndrome 2) specific inhibition of the NLRP3-inflammasome can avoid unfavorable suppression of other IL-1β processing pathways in response to various triggers, and 3) these drugs may be also effective for various other NLRP3-related chronic inflammatory conditions, such as Alzheimer’s disease, diabetes, severe gout and atherosclerosis.Here, we report the generation of NLRP3-mutant and non-mutant induced pluripotent stem cell (iPSC) lines from two CINCA syndrome patients with somatic mosaicism, and describe their differentiation into macrophages (iPS-MPs). We found that mutant cells are predominantly responsible for the pathogenesis in these mosaic patients because only mutant iPS-MPs showed the disease relevant phenotype of abnormal IL-1β secretion.Next, after confirming that the existing anti-inflammatory compounds inhibited the abnormal IL-1β secretion from iPS-MPs, we started drug screening for CINCA syndrome and other NLRP3-related inflammatory conditions using a recently-established feeder-free differentiation protocol. Among the 3,939 chemically-potent low molecule compounds, 127 candidates inhibited IL-1β secretion by more than 30%. We then excluded compounds that also inhibited IL-6 secretion and selected 23 candidate compounds. We are now validating the potency of these compounds.Our results illustrate that patient-derived iPSCs are useful for dissecting somatic mosaicism, and that NLRP3-mutant iPSCs can provide a valuable platform for drug discovery for multiple NLRP3-related disorders. Disclosures:Yamanaka:iPierian: Membership on an entity's Board of Directors or advisory committees.

Angiotensin-converting Enzyme Overexpression in Mouse Myelomonocytic Cells Augments Resistance to Listeria and Methicillin-resistant Staphylococcus aureus

Gene targeting in ES cells was used to substitute control of angiotensin converting enzyme (ACE) expression from the endogenous promoter to the mouse c-fms promoter. The result is an animal model called ACE 10/10 in which ACE is overexpressed by monocytes, macrophages, and other myelomonocytic lineage cells. To study the immune response of these mice to bacterial infection, we challenged them with Listeria monocytogenes or methicillin-resistant Staphylococcus aureus (MRSA). ACE 10/10 mice have a significantly enhanced immune response to both bacteria in vivo and in vitro. For example, 5 days after Listeria infection, the spleen and liver of ACE 10/10 mice had 8.0- and 5.2-fold less bacteria than wild type mice (WT). In a model of MRSA skin infection, ACE 10/10 mice had 50-fold less bacteria than WT mice. Histologic examination showed a prominent infiltrate of ACE-positive mononuclear cells in the skin lesions from ACE 10/10. Increased bacterial resistance in ACE 10/10 is directly due to overexpression of ACE, as it is eliminated by an ACE inhibitor. Critical to increased immunity in ACE 10/10 is the overexpression of iNOS and reactive nitrogen intermediates, as inhibition of iNOS by the inhibitor 1400W eliminated all in vitro and in vivo differences in innate bacterial resistance between ACE 10/10 and WT mice. Increased resistance to MRSA was transferable by bone marrow transplantation. The overexpression of ACE and iNOS by myelomonocytic cells substantially boosts innate immunity and may represent a new means to address serious bacterial infections.

CD33 Detection by Immunohistochemistry in Paraffin-Embedded Tissues: A New Antibody Shows Excellent Specificity and Sensitivity for Cells of Myelomonocytic Lineage

CD33 Detection by Immunohistochemistry in Paraffin-Embedded Tissues: A New Antibody Shows Excellent Specificity and Sensitivity for Cells of Myelomonocytic Lineage

CD33 Detection by Immunohistochemistry in Paraffin-Embedded Tissues: A New Antibody Shows Excellent Specificity and Sensitivity for Cells of Myelomonocytic Lineage.

CD33 Detection by Immunohistochemistry in Paraffin-Embedded Tissues: A New Antibody Shows Excellent Specificity and Sensitivity for Cells of Myelomonocytic Lineage.

Elevated synovial expression of triggering receptor expressed on myeloid cells 1 in patients with septic arthritis or rheumatoid arthritis

Objective:To determine whether synovial expression of triggering receptor expressed on myeloid cells 1 (TREM-1) is upregulated in patients with distinct types of inflammatory or non-inflammatory arthritis.Methods:Synovial fluid (SF) samples were...

Hypersensitivity Reaction of Gemtuzumab Ozogamicin (Mylotarg™) Associated with Platelet Transfusion: Two Case Reports.

IntroductionGemtuzumab ozogamicin (Mylotarg™) is a monoclonal antibody developed for the treatment of myeloid leukemias. Mylotarg is composed of a recombinant humanized IgG4 kappa antibody conjugated with a cytotoxic antitumor antibiotic, calicheamicin. It binds specifically to the CD33 antigen, which is expressed on the surface of leukemic myeloblasts and immature normal cells of myelomonocytic lineage. It is approved for the treatment of CD33 positive AML in first relapse in patients >= 60 years of age and are not considered candidates for cytotoxic chemotherapy. Hypersensitivity reactions, including anaphylactoid reactions, anaphylactic shock, dyspnea, and severe hypotension, have been reported during clinical use of gemtuzumab ozogamicin. Infrequently, hypersensitivity reactions have been fatal. It is unknown if the risk of hypersensitivity reaction increases with administration of blood products in the same day of Mylotarg infusion. We are reporting 2 cases of severe hypersensitivity reaction to Mylotarg on 2 patients who received platelete transfusion in the same day.Case reports:Case number 1: A 75 year old male diagosed with AML with normal cytogenetics in April 2005. Relapse was noted in March 2006. He was admitted for Mylotarg infusion at a dose of 9mg/m2 which was started at 11:00 a.m. after receiving the appropriate premedications and finished at 1:00 p.m. without any signs of distress or reaction. He received 6 units of platelets at 6:40 p.m. At 1:00 a.m. the next morning he went into severe respiratory distress and died shortly after that in the ICU.Case number 2: A 68 y/o male with the history of NHL in 1991, received CHOP chemotherapy in 1991 and 1992, now has secondary AML (diagnosed in October 2005 and underwent chemo in November 2005, and relapsed after induction and consolidation. He was admitted for Mylotarg infusion. Initially he received 6 units of platelets at 8:30a.m. Mylotarg was started at 10:15a.m. and finished at 12:10p.m.No signs of distress or reaction post infusion. At 2:30 p.m. the patient started having chills, rigors with fever, tachycardia, hypertension and hypoxia (pO2 86%). The patient was treated with Solu-medrol, Benadryl, Tylenol and Demerol. The reaction ceased with no further adverse eventsConclusion:Mylotarg is known to cause hypersensitivity reaction, but it is unknown if the risk of this reaction increases with blood products transfusion around the time of Mylotarg transfusion. We are reporting these 2 cases to draw attention to this serious observation.

Expression of an anti-CD33 single-chain antibody by Pichia pastoris

CD33 is a cell surface glycoprotein expressed on cells of myelomonocytic lineage, leukaemic cells, but not haematopoietic stem cells. By virtue of its expression pattern, CD33 has become a popular target for new immunotherapeutic approaches to treat acute myeloid leukaemia. The methylotrophic yeast Pichia pastoris strain KM71H was used to produce an anti-CD33 single chain variable fragment (scFv), with the intention of conjugation to a radioisotope, for therapeutic use. To direct secreted expression of the anti-CD33-scFv the α-mating factor secretory signal sequence (α-MF) was used, with constructs containing a complete (CS) and incomplete (INCS) cleavage site to accommodate the potential outcomes of dibasic endopeptidase, Kex2, and dipeptidyl amino peptidase, Ste13, processing. The anti-CD33-scFv was expressed in BMMY cultures using both constructs, with a final yield of 48 mg/l (CS) and 11 mg/l (INCS). N-terminal sequencing showed that the CS-scFv had not been cleaved by Ste13, leaving amino acids EAEA at the N-terminus. The INCS-scFv construct produced a mixture of 50% authentic scFv and 50% with 11 amino acids from the α-MF remaining at the N-terminus. Despite the aberrations in α-MF processing, the anti-CD33-scFv's produced from both constructs were found to be functional. Flow cytometry and Biacore analysis demonstrated binding to target antigen CD33 on the surface of human leukaemic cell line HL-60, and to recombinant soluble CD33 respectively.

Mutant N- ras preferentially drives human CD34 + hematopoietic progenitor cells into myeloid differentiation and proliferation both in vitro and in the NOD/SCID mouse

Ras oncogene mutations are the most frequently observed genetic abnormality (20-40% of patients) in acute myeloid leukemia (AML), and in the preleukemic conditions myelodysplastic syndrome (MDS) and myeloproliferative disorder (MPD). We have previously shown that mutant N-ras (N-rasm) can induce myeloproliferative disorders and apoptosis in a murine reconstitution system. In the present study we investigated the effect of N-rasm in human primary hematopoietic progenitor cells (HPC). Cord blood CD34+ hematopoietic progenitor cells (HPC) were transduced with retroviral vectors containing green fluorescence protein (GFP) alone, or in combination with N-rasm. Cells were then cultured in vitro with a cytokine supplement or cocultured with murine stroma MS-5 cells. The in vivo behavior of transduced cells was examined in the NOD/SCID mouse model. N-rasm-transduced cells exhibited greater proliferative capacity; a higher frequency of granulocyte-macrophage colony-forming unit (CFU-GM); and an increase in myelomonocytic lineage cells with a concomitant decrease in lymphoid and erythroid cells. Analysis of transduced HPC in NOD/SCID mice revealed higher bone marrow engraftment by N-rasm HPC and increased numbers of myeloid lineage cells. The results demonstrate that N-rasm in HPC induces myeloproliferation both in vitro and in the NOD/SCID mouse model as a primary event that does not appear to be dependent on cooperating transforming events.

Physical and Functional Interaction between Hck Tyrosine Kinase and Guanine Nucleotide Exchange Factor C3G Results in Apoptosis, Which Is Independent of C3G Catalytic Domain

The hematopoietic cell kinase Hck is a Src family tyrosine kinase expressed in cells of myelomonocytic lineage, B lymphocytes, and embryonic stem cells. To study its role in signaling pathways we used the Hck-SH3 domain in protein interaction cloning and identified C3G, the guanine nucleotide exchange factor for Rap1 and R-Ras, as a protein that associated with Hck. This interaction was direct and was mediated partly through the proline-rich region of C3G. C3G could be co-immunoprecipitated with Hck from Cos-1 cells transfected with Hck and C3G. C3G was phosphorylated on tyrosine 504 in cells when coexpressed with Hck but not with a catalytically inactive mutant of Hck. Phosphorylation of endogenous C3G at Tyr-504 was increased by treatment of human myelomonocytic THP-1 cells with mercuric chloride, which is known to activate Hck tyrosine kinase specifically. Coexpression of Hck with C3G induced a high level of apoptosis in many cell lines by 30-42 h of transfection. Induction of apoptosis was not dependent on Tyr-504 phosphorylation or the catalytic domain of C3G but required the catalytic activity of Hck. Using dominant negative constructs of caspases we found that caspase-1, -8, and -9 are involved in this apoptotic pathway. These results suggest that C3G and Hck interact physically and functionally in vivo to activate kinase-dependent and caspase-mediated apoptosis, which is independent of catalytic domain of C3G.

T lymphocytes promote the development of bone marrow-derived APC in the central nervous system.

Certain cells within the CNS, microglial cells and perivascular macrophages, develop from hemopoietic myelomonocytic lineage progenitors in the bone marrow (BM). Such BM-derived cells function as CNS APC during the development of T cell-mediated paralytic inflammation in diseases such as experimental autoimmune encephalomyelitis and multiple sclerosis. We used a novel, interspecies, rat-into-mouse T cell and/or BM cell-transfer method to examine the development and function of BM-derived APC in the CNS. Activated rat T cells, specific for either myelin or nonmyelin Ag, entered the SCID mouse CNS within 3-5 days of cell transfer and caused an accelerated recruitment of BM-derived APC into the CNS. Rat APC in the mouse CNS developed from transferred rat BM within an 8-day period and were entirely sufficient for induction of CNS inflammation and paralysis mediated by myelin-specific rat T cells. The results demonstrate that T cells modulate the development of BM-derived CNS APC in an Ag-independent fashion. This previously unrecognized regulatory pathway, governing the presence of functional APC in the CNS, may be relevant to pathogenesis in experimental autoimmune encephalomyelitis, multiple sclerosis, and/or other CNS diseases involving myelomonocytic lineage cells.

Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages

AbstractWe investigated the functions of adiponectin, an adipocyte-specific secretory protein and a new member of the family of soluble defense collagens, in hematopoiesis and immune responses. Adiponectin suppressed colony formation from colony-forming units (CFU)—granulocyte-macrophage, CFU-macrophage, and CFU-granulocyte, whereas it had no effect on that of burst-forming units—erythroid or mixed erythroid-myeloid CFU. In addition, adiponectin inhibited proliferation of 4 of 9 myeloid cell lines but did not suppress proliferation of erythroid or lymphoid cell lines except for one cell line. These results suggest that adiponectin predominantly inhibits proliferation of myelomonocytic lineage cells. At least one mechanism of the growth inhibition is induction of apoptosis because treatment of acute myelomonocytic leukemia lines with adiponectin induced the appearance of subdiploid peaks and oligonucleosomal DNA fragmentation. Aside from inhibiting growth of myelomonocytic progenitors, adiponectin suppressed mature macrophage functions. Treatment of cultured macrophages with adiponectin significantly inhibited their phagocytic activity and their lipopolysaccharide-induced production of tumor necrosis factor α. Suppression of phagocytosis by adiponectin is mediated by one of the complement C1q receptors, C1qRp, because this function was completely abrogated by the addition of an anti-C1qRp monoclonal antibody. These observations suggest that adiponectin is an important negative regulator in hematopoiesis and immune systems and raise the possibility that it may be involved in ending inflammatory responses through its inhibitory functions.

Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages

We investigated the functions of adiponectin, an adipocyte-specific secretory protein and a new member of the family of soluble defense collagens, in hematopoiesis and immune responses. Adiponectin suppressed colony formation from colony-forming units (CFU)—granulocyte-macrophage, CFU-macrophage, and CFU-granulocyte, whereas it had no effect on that of burst-forming units—erythroid or mixed erythroid-myeloid CFU. In addition, adiponectin inhibited proliferation of 4 of 9 myeloid cell lines but did not suppress proliferation of erythroid or lymphoid cell lines except for one cell line. These results suggest that adiponectin predominantly inhibits proliferation of myelomonocytic lineage cells. At least one mechanism of the growth inhibition is induction of apoptosis because treatment of acute myelomonocytic leukemia lines with adiponectin induced the appearance of subdiploid peaks and oligonucleosomal DNA fragmentation. Aside from inhibiting growth of myelomonocytic progenitors, adiponectin suppressed mature macrophage functions. Treatment of cultured macrophages with adiponectin significantly inhibited their phagocytic activity and their lipopolysaccharide-induced production of tumor necrosis factor α. Suppression of phagocytosis by adiponectin is mediated by one of the complement C1q receptors, C1qRp, because this function was completely abrogated by the addition of an anti-C1qRp monoclonal antibody. These observations suggest that adiponectin is an important negative regulator in hematopoiesis and immune systems and raise the possibility that it may be involved in ending inflammatory responses through its inhibitory functions.

The sialoadhesin CD33 is a myeloid-specific inhibitory receptor

Activating and inhibitory receptors act in concert to regulate cellular activation. Inhibitory receptors are characterized by the presence of a characteristic sequence known as an immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic tail. Phosphorylated ITIM serve as docking sites for the SH2-containing phosphatases which then inhibit signal transduction. CD33 is a member of the immunoglobulin superfamily and contains two immunoglobulin-like domains, a transmembrane region and a cytoplasmic tail that has two potential ITIM sequences. CD33 expression is restricted to cells of myelomonocytic lineage. The precise function of CD33 is unknown although it is a lectin that binds sialic acid residues in N- and O-glycans on cell surfaces. Co-immunoprecipitation studies demonstrate that CD33 associates with the SH2-containing tyrosine phosphatase SHP-1 in monocytes. The proximal ITIM is necessary and sufficient for SHP-1 binding which is mediated by the aminoterminal SH2 domain. Treatment of SHP-1 with a phosphopeptide representing the proximal CD33 ITIM results in increased SHP-1 enzymatic activity. CD33 exerts an inhibitory effect on tyrosine phosphorylation and Ca2+ mobilization when co-engaged with the activating FcγRI receptor. This data indicates that CD33 is an inhibitory receptor that may regulate FcγRI signal transduction.