TABLE OF CONTENTS

Abbreviations ............................................................................... i-iii

1. Introduction................................................................. 1-5
2. Review ofliterature ...................................................... 6-51
I. NEUTROPHILS (PMNs) ..............................................................6
2.1) Neutrophil maturation ............................................................ 8
2.1.1) Stem cell to myeloblasts ...................................................... 9
2.1.2) Myeloblast ...................................................................... 11
2.1.3) Promyelocyte .................................................................... 12
2.1.4) Myelocyte ....................................................................... 12
2.1.5) Metamyelocyte/ Band cell ...................................................... 13
2.1.6) Mature Neutrophils ............................................................. 14
2.2) Cytoplasmic granules and their contents ..................................... 15
2.2.1) Azurophilic/primary granules ................................................. 16
2.2.2) Specific/secondary granules ................................................... 17
2.2.3) Gelatinase/tertiary granules .................................................... 17
2.2.4) Secretory vesicles ............................................................... 18
2.3) Bone marrow to circulation ...................................................... 19
2.4) Functions of neutrophils ......................................................... 20
2.4.1) Activation and extravasation .................................................. 20
2.4.2) Phagocytosis ..................................................................... 21
2.4.3) Neutrophil extracellular traps (NETs) ........................................ 22
2.4.4) Trans-differentiation into dendritic cells ..................................... 23
2.4.5) Antigen presentation ............................................................ 24
2.4.6) Neutrophil anti- inflammatory ................................................. 27
2.4.7) Cytokine synthesis and release ................................................ 27
2.4.8) Apoptosis ......................................................................... 29
II. NITRIC OXIDE ........................................................................ 33
2.5) Functions of N 0 .....................................................................34
2.6) NO mediated modulation of PMNs function ..................................37
2.7) Neutrophils and NO/NOS ........................................................42
2.7.1) Nitric oxide synthase (NOS) in neutrophil. ................................. .43
2.7.2) Nitric oxide in bone marrow .................................................... .45
2.7.3) Nitric oxide and hematopoiesis ............................................... .47
2.8) N eutrophils in Pathophysiology .................................................48
2.9) NO & cell proliferation ............................................................ 49

3. Materials and methods ................................................. 52-68

3.1) Chemicals .............................................................................. 52
3.2) Animals and human blood donors .................................................. 53
3.3) Isolation of human and rat PMNs ................................................... 54
3.4) Isolation of neutrophil precursor cells from rat bone marrow ............ 55
3.4.1) Cell viability...................................................................... 55
3.4.2) Immunolabeling of surface markers ............................................ 56
3.4.3) Cell sorting by using FACS- Aria TM ......................................... 56
3.5) NO level/ NOS activity assessment in neutrophW precursors ..............56
3.5.1) Nitrite estimation ................................................................. 56
3.5.2) Nitric oxide synthase activity .................................................. 56

3.5.3) NO generation by flow cytrometry ............................................. 57

3.6) Functional studies I activities ......................................................57
3.6.1) Free radical generation .......................................................... 57
3.6.2) Phagocytic activity ............................................................... 58
3.6.3) Myeloperoxidase activity ........................................................ 58
3.6.4) Elastase activity ................................................................... 58
3.6.5) Immunofluorescence assessment of NETs ................................... 59
3.7) Cell growth I proliferation studies ...............................................59
3.7.1) Cell culture ........................................................................ 59
3.7.2) Cell synchronization .............................................................. 59
3.7.3) Cell growth assessment ......................................................... 60
3.7.4) Cell cycle analysis ............................................................... 60
3.7.5) BrdU Incorporation .............................................................. 60
3.7.6) 3H- thymidine incorporation ................................................... 61
3.8) Apoptosis assay ...................................................................... 61
3.8.1) Annexin V-PI double staining by flow cytometer ........................... 61
3.8.2) Evaluation of mitochondrial membrane potential (~\jim) using JC-1. .... 61
3.8.3) TdT enzyme dependent dUTP nick end labelling (TUNEL) ............... 62
3.8.4) Fluorometric Caspase assay .................................................... 62
3.9) Determination of intracellular GSH ............................................. 62
3.10) Expression studies ................................................................. 63
3.10.1) Immunoprecipitation ........................................................63
3.10.2) Western blotting ............................................................... 63
3.10.3) RT-PCR analysis .............................................................. 64
3.10.3.1) RNA isolation .............................................................. 64
3.10.3.2) eDNA synthesis ............................................................ 64
3.10.3.3) Primers ...................................................................... 65
3.10.4) Immuno- cytochemical detection of nitro- tyrosine .................... 66
3.11) Cytometric bead array and Elisa ........... ~ ...................................67
3.12) Confocal microscopy ..............................................................67
3.13) Immunogold electron microscopy ............................................. 68
3.14) Statistical analysis ................................................................ 68

4. Results ...................................................................69-121
4.1. Status of NO/NOS in Neutrophils and its Precursor cells .......... 69
4.1.1) Isolation & characterization of rat neutrophils and its precursor cells
4.1.1.1) BMC characterization ....................................................... 69
4.1.1.2) Peroxidase staining..................................... . .................... 70
4.1.1.3) Granular marker proteins ..................................................... 70
4.1.1.4) Myeloperoxidase activity .................................................... 71
4.1.1.5) Nitrite level. .................................................................... 71
4.1.1.6) Nitric oxide ..................................................................... 71
4.1.1.7) Nitric oxide synthase activity ............................................... 71
4.1.1.8) Nitric oxide synthase proteins ................................................ 72
4.1.1.9) NOS m RNA expression ..................................................... 73
4.1.1.1 0) Immunolabelling of cells .................................................... 74
4.1.1.10.1) Flow Cytometer. ........................................................... 74
4.1.1.10.2) Confocal microscopy ...................................................... 74
4.1.2) Isolation and characterization of human neutrophil precursor cells ...77
4.1.2.1) Human neutrophil precursor cells ............................................. 77
4.1.2.2) Cell sorting ........................................................................ 77

4.1.2.3) Nitric oxide ..................................................................... 78

4.1.2.4) Nitric oxide synthase isoform proteins ...................................... 79
4.1.2.5) Immuno labelling .............................................................. 80
4.1.2.5.1) Confocal microscopy ..................................................... 80
4.1.2.5.2) Flow cytometer..................................... . ..................... 84
4.1.2.5.3) Co- localization of elastase and iN OS .................................. 85
4.1.2.5.4) Immuno- gold electron microscopy ..................................... 86

4.2. Functional analysis of neutrophil and precursor cells .................. 91

4.2.1) Free radical generation ..................................................... ... 91
4.2.1.1) DCF- 2DA ................................................................... 91
4.2.1.2) DHR-123 ...................................................................... 91
4.2.1.3) DHE ........................................................................... 92
4.2.1.4) NO mediated free radical generation ....................................... 93
4.2.2) Mitochondrial content ............................................................ 94
4.2.3) Phagocytic activity ................................................................ 94
4.2.4) Cell cycle analysis ................................................................. 95
4.2.5) Neutrophil extracellular trap (NETs) formation .............................. 97
4.2.6) Differentiation of promyelocytic cell line HL- 60 cells into neutrophils .. l 00

4.3. Nitric oxide and cell proliferation .......................................... 102

4.3 .1) Cell growth/ proliferation of HL-60 cells ..................................... 102
4.3 .2) Cell cycle modulation ............................................................ 103
4.3.3) NO dependent nitration/ nitrosylation ........................................ 105
4.3.4) Regulation ofCyclins/CDKs ..................................................... 107

4.4. Nitric oxide and cell death (Apoptosis) ..................................... 109

4.4.1) Annexin V-PI staining ............................................................ 109
4.4.2) Mitochondrial membrane potential (~'I'm) .................................. 109
4.4.3) TUNEL assay ...................................................................... 110
4.4 .4) Caspase- 3 activity. . . . . . . . . . . . . . . . . . . . . . ......................................... 111
4.4.5) Cytokines profile following NO treatment. .................................... 112

4.5. Nitrite and cell proliferation ................................................... 113

4.5.1) Cell cycle modulation ............................................................ 113
4.5.2) Nitrite and Cyclin/ CDKs ....................................................... 114
4.5.3) Effect of nitrite on Synchronized cells ......................................... 115
4.5.4) Effect on cell granularity........................................................ 116
4.5.5) NO release from nitrite and ROS generation ................................. .117
4.5.6) Cytokine release ................................................................... 120

5. Discussion .............................................................. 122-138

6. Summary ............................................................... 139-142
7. Bibliography ........................................................... 143-158