09_review of literature

Review of Literature

"If you do not know history, you don't know anything You

are a leaf that doesn't know its part of the tree"

-Michael Crichton

Review of literature

I.) NEUTROPHILS (PMNs)

Elic Metchnikoff's (1883) micro-phagocytes, popularly known as

polymorphonuclear leukocytes (PMNs/ Neutrophils) have gone through a paradigm

shift with time (more than a century) from an innate phagocyte to initiator of acquired

immunity. PMNs, the most abundant leukocyte, about 60-70% of circulating WBCs

present in circulation, are the first cells to respond to inflammatory stimuli, ready to

attack any microorganism or fungal assault (Segal, 2005). Circulating neutrophils

were thought to be terminally differentiated and post mitotic phagocytes, those kill the

invading pathogens by their ability to recognize, chemotaxis, phagocytosis,

production of highly reactive oxygen species and microbicidal proteases. The latter

function is associated with more or less collateral tissue damage in inflammatory

conditions (Nathan, 2006).

The ability of these cells to engulf and degrade bacteria was logically assumed

to indicate a killing function. A microbicidal function was ascribed to the contents of

their abundant cytoplasmic granules that were discharged into the phagocytic vacuole

containing the microbe (Cohn and Hirsch, 1960; Hirsch and Cohn, 1960). Attention

was then directed toward the characterization of the granules by electron microscopy,

fractionation and biochemical analysis (Bainton et al., 1971). Several of the purified

granule proteins were shown to kill microbes (Borregaard et al., 2007). Parallel with

studies into microbicidal activity of the granule contents, investigations were

undertaken into the metabolism of phagocytosing neutrophils. The neutrophils

demonstrated a significant "extra respiration of phagocytosis" (Bladridge and Gerard,

1932) which was non-mitochondrial and was associated with a dramatic increase in

turnover of the hexose monophosphate (HMP) shunt (Kamovsky, 1962) and the

production of large amounts of H20 2• Soon after its discovery in 1969 (McCord and

Fridovich, 1969), superoxide dismutase was used to show that activated neutrophils

generate superoxide and this leukocyte oxidase activity was lacking in chronic

granulomatous disease (CGD) (Baehner and Nathan, 1967). In addition,

myeloperoxidase (MPO)-mediated halogenation, which is microbicidal in the test

tube, was also defective in CGD patients (Klebanoff, 1968; Klebanoff and White,

1969). The remarkable research which led to paradigm shift in neutrophil biology is

summarized in table 1.

Role ofnitric oxide in neutrophil maturation and function 6


Table 1: Landmark researches in the field of neutrophil biology

1884

1932

1956

1960

1967

1968

197 1

by

Lioyd and Oppenheim postulation that neutrophils also participated in

adaptive immunity (Lloyd and Oppenheim, 1992) led to paradigm shift in neutrophil

biology with the following research. Despite phagocytic killing, neutrophils also

express class I MHC (Neuman et al., 1992), class II MHC (Gosselin et al. , 1993), T

Role oj'nitric oxide in neutrophil maturation and f unction 7


cell co stimulatory molecules [CD80 (87-1), CD86 (B7-2)] (Sandilands et al., 2005),

instruct recruitment and activation of dendritic cells (Bennouna et al., 2003) and

efficiently prime naive T cells (Beauvillain et al., 2007). PMNs can be reverted in

their functional maturation program and driven to acquire DC features (Oehler et al.,

1998). Recently a new concept has emerged in neutrophil biology, that neutrophils

can make spider web trap like structure to capture and kill the pathogens known as

neutrophil extracelluar traps (NETs) (Brinkmann et al., 2004) which is a topic of

intense research. Moreover with recent advancements in techniques, last decade has

revolutionized the neutrophils research from high metabolic, phagocytic and post

mitotic cells to cells with multiple functions like antigen presentation, neutrophil

extracellular traps formation and transdifferentiation to dendritic cell, early anti

inflammatory ectosomes release and cross-priming of T -cells thus a key player to

uphold the homeostasis from innate to specific immunity.

Neutrophils are highly specialized, non-dividing, terminally differentiated

cells with a short life span. The bulk of its life cycle is spent in the marrow, where it

proliferates, differentiates, and is stored for a few days. The mature cells, then

released into the blood and circulates briefly before migrating into the tissues where it

functions as a mobile phagocyte (Bainton et al., 1971).

2.1) Neutrophil maturation

Neutrophils are produced in the bone marrow, released into blood, circulate briefly,

and migrate into tissue spaces or on to epithelial surfaces such as those in the

respiratory, digestive, or urogenital tracts. Neutrophils have a small life span of only 4

-10 hours in circulation and one to two days in the tissue. Production is continuous in

order to provide the continual demand of neutrophils in the tissues and maintain the

circulating pool in the blood. The daily turnover of neutrophils production is 1010-1 0 11 per human body. Transit time for generation of neutrophils in marrow is

approximately 10-14 days and the marrow maintains a five-day supply of mature

neutrophils in storage (Bainton et al., 1971).

Principle processes during maturation:

1) Modest reduction in size

2) increasing darkening and lobation of nucleus

3) accumulation of specific granules

Role of nitric oxide in neutrophil maturation andfimction 8


PMN development in the bone marrow has classically been divided into six

stages myeloblasts (MBs), promyelocytes (PMs), myelocytes (MCs), metamyelocytes

(MMs), band cells (BCs), segmented neutrophil on the basis of cell size, nuclear

morphology, and granule content (Borregaard and Cowland, 1997). In addition to the

appearance of cytoplasmic granules, the maturation process is characterized by a

decrease in cell size, development of nuclear lobulation, a decrease in cytoplasmic

basophilia and in the number of mitochondria. Mitoses occur only during the first

three stages; most take place during the myelocyte stage (Bainton et al., 1971;

Theilgaard-Monch et al., 2006). Neutrophils cell division is brought to an end after

the myelocytes-metamyelocyte stage. Progression of the cell from Go/G1 to S phage

and finally to G2/M phage is determined by the sequential expression of various

cyclins (cell cycle regulating proteins), and cyclin dependent kinases (CDKs)

(Theilgaard-Monch et al., 2006). Expression profiles of these cyclins in myeloid

series of cells from human and rat bone marrow demonstrate down regulation of

CDKs from MC/MM stages onward and complete cell cycle arrest in BCs/SCs and

mature neutrophils (Bogdan, 2001) . . 2.1.1) Stem cell to myeloblasts

Granulopoiesis is highly controlled process of granulocytes formation from

pluripotent hematopoietic stem cells (HSC), which engage with orchestration of

different transcription factors, growth factors, cytokines and cell cycle regulators

(Fig.l) (Zhu and Emerson, 2002). The cell lineage commitment and their number

depend on proliferative and self-renewal capacity and differentiation of HSCs. Stem

cells niche plays critical role in the determination of their self-renewal or

differentiation choices as stromal cells secretes low level of growth factors and

cytokines (Friedman, 2002).

Hematopoietic stem cells originate from mesenchymal tissue in the yolk sac

during very early embryonic life. In the human, at some point during the first six

weeks of gestation, primitive blood islands appear in the yolk sac, which represent the

earliest sites of stem cells proliferation and differentiation. At about week six, the

stem cells migrate from the yolk sac into the substance of the embryo where they

proliferate first in the liver (from about the sixth through the tenth week of gestation),

next in the spleen (weeks 1 0-15), and finally in the bone marrow, where they take up

residence by about the 15th week of fetal life. From about the fifteenth week of

gestation through the death of the individual, the bone marrow is the primary site of

Role of nitric oxide in neutrophil maturation and fimction 9


blood cell production. In healthy persons, the manow is for practical purposes the

only site of hematopoiesis. However, during periods of hematopoietic stress, the liver

and spleen may revert to their fetal role and produce blood cells in the adult. This

event may be seen, for example, when the manow is replaced by metastatic breast

cancer or by fibrosis.

monocyte

neutrophil

• PU.1 & GATA-1 C/EBP~ . GATA-1

eosinophil

GATA -1/FOG GATA -1 • erythrocyte

megakaryocyte

Fig I: Transcriptional regulation of common mye lo id precursor (CMP) commi tment CMPs differentiate into either common precursors for granulocytic and monocytic lineages (GM Ps) or common precursors for both erythroid and megakaryocytic lineages (EM Ps) (Zh u and Emerson, 2002).

The cunent model of hematopoiesis proposes that early in their differentiation,

long-term hematopoietic stem cells (HSCs) lose their capacity for self-renewal,

differentiating first into short-term HSCs and subsequently to multipotent progenitors

(MPPs) (Kondo et al. , 1997). MPPs generate common myeloid progenitors (CMPs)

that are able to differentiate into the erythromyeloid lineages, and common lymphoid

progenitors that produce B, T, and natural killer cells. CMPs may form two more

restricted cell types, the granulocyte-macrophage progenitors (GMPs) and

megakaryocyte-erythrocyte progenitors. A potential CMP population is with the

phenotype IL-7K/LinT-kit/Sca-1' populations (0.2% ofBM cells). This subset was

further divided into three subpopulations by the differential expression of CD34 and

FcgR: (1) CD34+ FcgR10, (2) CD34-FcgR10 and (3) CD34+FcgR+. CD34+FcgR+ cells

exclusively produced granulocytes/monocytes (Akashi et al. , 2000). Neutrophils

develop from GMPs through the myeloblast, the promyelocyte, the myelocyte, and

the metamyelocyte stages.

Role of nitric oxide in neutrophil maturation and.fimction 10


2.1.2) Myeloblast

The earliest cell of the neutrophilic series a relatively small ( - 1 0!-t) undifferentiated

cell with a high nuclear (large ovoid nucleus): cytoplasmic ratio (strongly basophilic

cytoplasm) and prominent nucleoli. The general organization of the cytoplasm: there

are abundant free ribosomes, only a few isolated cisternae of the rough-surfaced

endoplasmic reticulum, a small centrosphere or Golgi region located near the nucleus,

and numerous mitochondria which are frequently aggregated at the cytoplasmic pole

opposite the Golgi zone (Bainton et al. , 1971 ). The neutrophil maturation overview in

bone marrow is depicted in Fig. 2.

-Azurophilic I prim:uy granules

Myeloblast (0.5%) Promyelocyte(S%)

Tissue 1-2 Days

"'-- Blood . -= 10 Hrs

Mitotic (7 days, 3-4 Division)

Post Mitotic (6 days)

Geb.tin.,.e I terti:uy granules .......,.,....,.....,.,......,

Mature Neutrophil (20%) Band cell

\ Myelocyte(12%)

Specific- Gelatirutse Granules

Metamyelocyte

MB/ BC(22%)

Fig 2: Diagrammatic representation of neutrophil maturation in bone marrow

The nucleus usually contains several nucleoli and has a relatively light

background density, except peripherally where the chromatin is concentrated along

the nuclear membrane . Centrioles are not seen. The first indication that a myeloblast

is beginning to differentiate into a progranulocyte is the appearance in the Golgi zone

of vacuoles with a dense core, which represent early stages in the formation of

azurophil granules. Very early promyelocytes resemble the myeloblast, but contain a

few azurophil granules and a larger Golgi complex.

Role o..f nitric oxide in neutrophil maturation and jimction 11


2.1.3) Promyelocyte

The promyelocyte nucleus is less regularly round, an indentation being frequently

seen near the centrospheres region. The promyelocyte can be recognized by its large

size ( ~ 15 fl ), rounded nucleus, and Increase in the amount of rough-surfaced

endoplasmic reticulum, enlargement of the Golgi complex, and especially to the

accumulation of azurophil granules. Cell size and granule content differ widely

depending on whether the cell is an early or late promyelocyte. Fully developed, more

heavily granulated promyelocytes measure up to 16 fl, and are the largest cells of the

PMN series; they have a large centrosphere region (Bainton et al., 1971).

All granules of the promyelocyte represent a single population of azurophilic

granules. Mature azurophil granules appear ovoid, irregularly spherical, or slightly

angular. The diameter ( ~ 900 rnlz) of this immature form is greater than that of the

more condensed, mature azurophil granule. Two main shapes can be identified, round

and football-shaped. The predominant form is round (diameter~ 500 nm); Football

shaped forms (300 X 900 nm) are less common. Production of peroxidase-positive

azurophil granules ends in the promyelocyte, and the beginning of the myelocytes

stage is marked by the production of a second population of granules which are

peroxidase negative. Morphological characteristics of neutrophils and precursor cells

under light and electron microscope are discussed in table 2.

2.1.4) Myelocyte

The myelocyte is distinguished from the promyelocyte by the more variable shape of

its indented nucleus, smaller cell size (10 to 12 f.l) and particularly by its content of

two types of granules; in addition to azurophil granules, there are variable numbers of

smaller ( ~ 500 rnlz), less dense, peroxidase-negative specific granules (Borregaard

and Cowland, 1997). The nucleus appears more distinctly indented and its chromatin

is more condensed than that of the promyelocyte. In the cytoplasm, ribosomes are

numerous, but there is a marked decrease in the amount of rough-surfaced

endoplasmic reticulum and mitochondria compared to the promyelocyte. Golgi

complex is similar to that of the pro granulocyte; it is noteworthy that specific granules

are apparently formed along the distal face of the Golgi complex whereas azurophil

granules are formed along its proximal face, and that no azurophil granules are

produced during the myelocyte stage.

Table 2: Characteristics of neutrophil precursors under light and electron microscope



CELL SIZE NUCLEUS& NUCL- CYTOPLASM GRANULES ELECTRON

!!!m2 MITOSIS EOLI MICROSCOPY

Myelo- 10-12 Round, raddish 2-3 Blue clumps in None RER, small blast blue chromatin pale blue Golgi, many (MB) networks fine, background, mitochondria and

mitosis cytoplasmic polysomes blebs at cell periphery

Pro- 12-16 Round to oval, 1-2 Bluish Azurophilic I RER, Large myeocyte reddish blue; cytoplasm, no primary Golgi, many (PM) chromatin cytoplasmic granules mitochondria and

networks coarse, blebs at cell lysosomes mitosis periphery

Myelocyte 10-12 Flattened, 0-1 Pale blue Azurophilic I RER, Large (MC) acentric cytoplasm primary and Golgi, many

chromatin; specific mitochondria and chromatin granules lysosomes network coarse , (.5J.!m) and mitosis specific granules

(.J J.!m)

Meta- 10-12 Kidney shape None Pale blue Azurophilic, Organelles no. Myelocyte dense, cytoplasm specific and reduced, granules (MM) chromatin gelatinase similar

network coarse, granules no mitosis

Band cell 9-12 Horseshoe None Pale bluish pink Azurophilic, Same (BC) shaped, cytoplasm specific and

chromatin gelatinase network very granules coarse, no mitosis

Neutro- 9-12 Multilobulated, None Pale bluish pink Azurophilic, Same phil chromatin cytoplasm specific, (PMN) network very gelatinase

coarse, no granules & mitosis Secretary

vesicles

2.1.5) Metamyelocyte/ Band cells

When the nucleus appears distinctly indented and cell s1ze is reduced, the cell is

designated a metamyelocytes and with marked nuclear indentation, it is called a band

cell. The metamyelocyte, band, and mature PMN are nondividing, nonsecretory stages

which are identified by their nuclear morphology, mixed granule population with

tertiary/gelatinase granules and secretary vesicles, inactive Golgi region and

accumulation of glycogen particles. The number of granules present at these stages is

quite large. Nuclear shape as determined in smears of whole cells is frequently

difficult. In addition to progressive nuclear indentation and reduction in cell size,

these two stages are characterized by a predominance of specific granules and a

decline in cytoplasmic organelles (ribosomes, rough-surfaced endoplasmic reticulum,

Role o.lnitric oxide in neutrophil maturation and fimction 13


mitochondria, and Golgi complex); the nuclear chromatin is more condensed and

nucleoli are not seen. Specific and gelatinase granules invariably predominate and

relatively few azurophil granules are present. The cells are smaller than the preceding

myelocytes. Only a few short elongate cisternae of rough endoplasmic reticulum can

be found. The Golgi-to-cytoplasm ratio is almost half of that in the myelocyte. Most

cells show only 1 or 2 stacks ofGolgi cisternae (Bainton et al., 1971).

2.1.6) Mature neutrophils

The mature cell differs from preceding stages in its smaller size (8-10~-t), multi

lobulated nucleus, darker and more condensed cytoplasm. The latter contains

particulate glycogen and granules of all four types but lacks significant quantities of

most other cell organelles (i.e. mitochondria, microtubules, ribosomes and

endoplasmic reticulum). The nucleus usually appears as one or more profiles of

seemingly isolated lobes, for the connections between the lobes are so thin ( ~ 40 nm)

that they are seldom included in the plane of section.

Golgi complex is small and rudimentary only 2 to 3 short cisternae, which

have lost their circular orientation around the centriole, are present. There is a further

increase of chromatin condensation and nucleoli are no longer seen. The mature

neutrophil is characterized by huge numbers of granules. On average, more than 200

granules were counted per cell per cross-section.

Furthermore, Surface antigens undergo several changes during neutrophilic

maturation to accommodate the cell's function. Surface antigens may appear with

neutrophilic maturation, such as CD16b, CD35, and CDlO; disappear with

maturation, such as CD49d and CD64; be maintained during maturation, such as

CD32, CD59, and CD82; or disappear with maturation but reappear after neutrophilic

extravasation, such as CD49b and extensively reviewed by Elghetany (Elghetany,

2002). Table 3 represents some of important surface marker during neutrophil

maturation.



Table 3: Surface marker expression during neutrophil maturation.

Cell Stage CDllb CD15 CD16 CD24 CD35/ CDlO CD64 CD83

Myeloblasts + + (MBs) Promyelocytes ++ ++ (PMs) Myelocytes + ++ ++ ++ (MCs) Metamyelocytes ++ ++ + ++ ++ (MMs)

Band Cells ++ ++ ++ ++ ++

Segmented ++ ++ +++ ++ ++ ++ PMNs

2.2) Cytoplasmic granules and their contents

Neutrophils are double edged sword of immune system with high reservoir of toxic

proteases and antimicrobial agents. These damaging agents are arranged in granules

containing an abundant matrix composed of strongly negatively charged sulphated

proteoglycans to keep them in dormant state. The various subsets of granules

contained within the neutrophil constitute an important reservoir not only of

antimicrobial proteins, proteases and components of the respiratory burst oxidase, but

also of a wide range of membrane-bound receptors for endothelial adhesion molecules

and extracellular matrix proteins. Additionally, the regulated exocytosis of granules

enables the neutrophil to deliver its arsenal of potentially cytotoxic granule proteins in

a targeted manner, thus preventing widespread damage to host tissue in most

situations. There are four predominant types of granules, azurophil, specific,

gelatinase and secretory vesicles.

Neutrophil granules are formed sequentially during myeloid cell

differentiation. Formation of granules is initiated in early promyelocytes, the early

appearing granules were originally defined by their high content of myeloperoxidase

(MPO) and consequently named "peroxidase-positive granules", but they are also

referred to as "azurophil granules" due to their affinity for the basic dye azure A or

simply designated "primary granules"(Borregaard and Cowland, 1997). The

production of MPO ceases at the promyelocyte/myelocyte transition. Accordingly,

granules formed at later stages of myelopoiesis are peroxidase negative (Faurschou

Role o._fnitric oxide in neutrophil maturation andfimction 15


and Borregaard, 2003). Peroxidase-negative granules can be subdivided into specific

(secondary) and gelatinase (tertiary) granules, based on their time of appearance and

content of granule matrix proteins (Le Cabec et al., 1996). Specific granules are

formed in myelocytes and metamyelocytes and have a high content of lactoferrin and

a low content of gelatinase, while gelatinase granules form in band cells and

segmented neutrophils and are low in lactoferrin but high in gelatinase. Secretory

vesicles, like granules, are regulated exocytic vesicles that appear in segmented

neutrophils. The fact that these vesicles contain plasma proteins suggests that

secretory vesicles form by endocytosis (Segal, 2005). The overview of neutrophil

granules content is given in table 4.

2.2.1.) Azurophilic/Primary granules

These granules were identified on the basis of myeloperoxidase (MPO) and

consequently named "peroxidase-positive granules". These granules are packaged

with acidic hydrolases and antimicrobial proteins and display great heterogeneity in

size and shape. Azurophil granules undergo limited exocytosis in response to

stimulation and they are believed to contribute primarily to the killing and degradation

of engulfed microorganisms that take place in the phagolysosome. The defining

protein of peroxidase-positive granules, myeloperoxidase is a 150-kDa microbicidal

heme protein. MPO reacts with H20 2, formed by the NADPH oxidase, and increases

the toxic potential of this oxidant. Through oxidation of chloride, tyrosine and nitrite,

the H20 2-MPO system induces formation of hypochlorous acid (HOCl) (Borregaard

and Cowland, 1997; Klebanoff, 2005). Defensins are small (~3.5 kDa) cationic,

antimicrobial and cytotoxic peptides major constituents of azurophil granules, at least

5% of the protein content of neutrophils. They exert their antimicrobial effect through

the formation of multimeric transmembrane pores (Borregaard et al., 2007).

Bactericidal/permeability increasing protein (BPI) is another highly cationic,

~50-kDa antimicrobial peptide of azurophil granules. BPI binds to negatively charged

residues of lipopolysaccharide (LPS) in the outer membrane of Gram-negative

bacteria via its antibacterial N-terminal region. Azurophil granules contain three

structurally related serprocidins (serine proteases with microbicidal activity):

proteinase-3, cathepsin G and elastase. The serprocidins are cationic polypeptides of

25-29 kDa, which display proteolytic activity against a variety of extra-cellular

matrix components, such as elastin, fibronectin, laminin, type IV collagen, and

Role ofnitric oxide in neutrophil maturation andfimction 16


vitronectin. Furthermore, they induce activation of endothelial and epithelial cells,

macrophages, lymphocytes and platelets, and possess antimicrobial properties.

Azurocidin is a 29-kDa antimicrobial serine protease homologue found in

azurophil granules. Azurocidin is chemotactic for monocytes, fibroblasts and T cells,

and increases vascular permeability during neutrophil extravasation (Borregaard and

Cowland, 1997). About one third of the total lysozyme is found in these granules.

These granules contain an abundant matrix composed of strongly negatively charged

sulphated proteoglycans. This matrix strongly binds almost all the peptides and

proteins other than lysozyme, which are strongly cationic. This sequestration together

with the acidic pH at which the granule interior is maintained keeps these enzymes in

a quiescent, inactivated state (Segal, 2005).

2.2.2) Specific/Secondary granules

Specific granules contain unsaturated lactoferrin, which binds and sequesters iron and

copper. Lactoferrin is a 78-kDa antimicrobial glycoprotein against a broad spectrum

of Gram-positive and Gram-negative bacteria. The protein is a member of the

transferrin family of iron-binding proteins and impairs bacterial growth by

sequestration of iron polymers of bacterial cell walls. hCAP-18 is a 19-kDa

cathelicidin group antimicrobial peptides. Transcobalamin II, which binds

cyanocobalamin, Lipocalin 25-kDa antimicrobial peptide, lipocalin neutrophil

gelatinase-associated lipocalin (NGAL) (Borregaard et al., 2007). It was originally

identified as a protein covalently bound to neutrophil gelatinase. Lysozyme is a

cationic antimicrobial peptide of 14 kDa. In agreement with its biosynthetic profile,

lysozyme is present in all granule subsets, with peak concentrations about two thirds

in specific granules, arginase 1 and a number of membrane proteins are also present in

the plasma membrane, including flavocytochrome b558 of the NADPH oxidase

(Borregaard and Cowland, 1997).

2.2.3) Gelatinase/Tertiary granules

The designation of granules as "gelatinase granule" refers to granules that contain

gelatinase and matrix metalloproteinases (MMPs) but not lactoferrin, zinc-dependent

endopeptidases (Faurschou and Borregaard, 2003). Collectively they are capable of

degrading all kinds of extracellular matrix proteins, cell surface receptors, release of

apoptotic ligands (such as the FAS ligand), and chemokine in/activation, cell

proliferation, migration (adhesion/dispersion), differentiation, angiogenesis, apoptosis



and in host defense, morphogenesis, angiogenesis, tissue repair, arthritis and

metastasis.

Table 4: Content of neutrophil granules adapted from Borregaard et al., (2007)

AZUROPHILIC SPECIFIC GELATINASE SECRETORY GRANULES GRANULES GRANULES VESICLES

Membrane proteins

CD63 CD11b/CD18, CD11b/CD18, Alkaline phosphatase CD68 CD15, CD66, CD67, Gp91phox/p22phox, CD10, CDllb/CD18, Presenilin 1 Gp91phox/p22phox, fMLP-R, CD13, CD14, CD16, Stoma tin fMLP-R, Leukolysin, CD45, CR1, C1q-R, V-type-H+ ATPase Fibronectin-R, SNAP-23, Gp91phox/p22phox,

Rap1, 2 VAl\IIP-2, fMLP-R, SCAMP, SNAP-23, V-type-H+ ATPase, Leukolysin, Stomas tin TNF-R, VAMP-2, Thrombospondin-R MMP 25 TNF-R, TNF-R, V-type-H+ ATPase, VAMP-2, Albumin Vitronectin-R

Matrix proteins

Mucopolysacharide, 132-Micro globulin, Acetyl transferase, Plasma protein Azurocidin, Collagenase, 132-Microglobulin, BPI, MPO hCAP-18, Gelatinase, 13glycerophosphatase Histaminase, Lysozyme, 13-glucuronidase, Heparanase, Arginase-1 Cathepsins, Lactoferin, Defensins, Lysozyme, Elastase, NGAL, Lysozyme, Transcobalamin-1 Proteinase-3, Sialidase

Gelatinase granules are more easily exocytosed than specific granules

(Sengelov et al., 1993). The MMPs are stored as inactive proforms that undergo

proteolytic activation following exocytosis. These characteristics reflect that

gelatinase granules are important primarily as a reservoir of matrix degrading

enzymes and membrane receptors needed during neutrophil extravasation and

diapedesis.

2.2.4) Secretory vesicles

These endocytic vesicles constitute a reservoir of membrane-associated receptors

needed at the earliest phases of the neutrophil-mediated inflammatory response and

serum albumin. The membranes of secretory vesicles are rich in the P2-integrin

CD11b/CD18 (Mac-1), the complement receptor 1 (CR1), flavocytochrome b558,

receptors for formylated bacterial peptides (formyl methionyl-leucyl phenylalanine

(fMLP)-receptors), the LPS/ lipoteichoic acid-receptor CD14, the Felli receptor



CD16 and the metalloprotease leukolysin, all of which are incorporated in the plasma

membrane after exocytosis (Faurschou and Borregaard, 2003). Exocytosis of granules

is a consequence of granule membrane with the plasma membrane. In this way,

membrane proteins located to the membrane of granules translocate to the surface

membrane and furnish the cell with new receptors and other functional proteins. The

neutrophil plasma membrane contains several membrane channels, adhesive proteins,

receptors for various ligands, ion pumps, and ectoenzymes. Neutrophils contain a

complex cytoskeleton, which is responsible for chemotaxis, phagocytosis and

exocytosis.

2.3) Bone marrow to circulation

Release mechanisms of hematopoietic stem cells, myeloid progenitors and

granulocytes from the bone marrow have been studied extensively under normal and

emergency conditions (Christopher and Link, 2007; von Vietinghoff and Ley, 2008).

The interaction of SDFl (stromal derived factor-1; CXCL12) with the chemokine

receptor CXCR4 is important for neutrophil retention in the bone marrow. CXCR4

deficiency results in decreased bone marrow but increased peripheral neutrophils as

identified by the marker Gr-1 (Ma et al., 1999; von Vietinghoff and Ley, 2008).

CXCR4 mRNA is constitutively expressed in almost all types of leukocytes, CXCR4

is expressed on the neutrophil surface and its level is altered on neutrophils of

different stages of maturation and activation as surface expression of CXCR4 was

diminished in peripheral neutrophils compared with marrow neutrophils and further

reduced in peritoneal exudate neutrophils (Suratt et al., 2004). In contrast Nagase et

al.,(2002) found that cultured peripheral neutrophils apparently increased CXCR4

expression over 48 hours (Nagase et al., 2002). Moreover, CXCR4 and CXCL12 are

down-regulated by G-CSF (Kim et al., 2006; Suratt et al., 2004), but neutrophil

mobilization can also be induced by anti-CXCR4 Abs and a number of peptide

antagonists (Levesque et al., 2004).

G protein-coupled receptor kinases are essential for desensitization of CXCR4

and subsequent neutrophil release from the bone marrow. There was no evidence for

altered neutrophil mobilization in selectin-deficient or ~2 integrin- deficient mice

(Forlow et al., 2001). Neutrophil serine protease expression correlates with neutrophil

release from the bone marrow. Cathepsin G, neutrophil elastase and matrix

metalloproteinase 9 were increased by G-CSF treatment, and inhibition by a-1-



antitrypsin inhibited neutrophil release from the bone marrow (Winkler et al., 2005).

However, neither deficiencies in both cathepsin G and neutrophil elastase nor a mouse

model lacking the serine proteinase activator dipeptidyl peptidase I showed altered

neutrophil mobilization, thus challenging the role of serine proteases in neutrophil

liberation (Levesque et al., 2004).

2.4) Functions of neutrophils

2.4.1) Activation and extravasation

Leukocyte recruitment to sites of injury or infection known as extravasation, involves

sequential interactions with endothelium and extravascular tissue components. This

process involved several steps as chemoattraction, rolling, adhesion and

transmigration. The migrating neutrophils need to establish transient and dynamic

adhesive contacts with extracellular matrix proteins. Integrin receptors expressed on

the leukocyte surface play a central role in these interactions, mediating linkages

between the cytoskeleton and the external environment.

Mobilization of leukocytes and plasma proteins from the postcapillary venule

to extravascular tissue space are major characteristics of the inflammatory response.

Neutrophils are the first leukocytes, hours before monocytes or lymphocytes, to

migrate to the inflammation site (Seely et al., 2003). Chemotactic stimulation of

PMN s induces a cascade of events which include actin reorganization, shape changes,

development of polarity and reversible adhesion (Berton and Lowell, 1999),

culminating in chemotaxis. Different chemoattractant such as N-formylmethyl

leucyl-phenylalanine (fMLP), TNFa and chemokines (IL-l, C5a, leukotriene B4,

GRO-b and IL-8) are known to attract neutrophils to site of infection. IL-l and TNFa

cause the endothelial cells of blood vessels near the site of infection to express

cellular adhesion molecules (ICAMl & 2 and VCAMl), including selectins (E

selectin, P-selectin) and produces platelet activating factor (P AF) and IL-8 (Witko

Sarsat et al., 2000). The rolling step is mediated by neutrophil L-selectin and by E

and P-selectins newly expressed on inflamed endothelial cells with marginal affinity

and sheds L selectin. In the activated state, integrins (LFAl, Macl & VLA4) bind

tightly to complementary receptors (ICAMl & 2 and VCAMl) expressed on

endothelial cells with high affinity (Seely et al., 2003). The cytoskeletons of the

leukocytes are reorganized in such a way that the neutrophils are spread out over the

endothelial cells and transmigrate. Exocytosis of gelatinase and partial exocytosis of



specific and azurophil granules mobilize receptors for extracellular matrix

components and liberate collagenolytic metalloproteases, matrix-degrading enzymes

during neutrophil extravasation to perforate the vascular basement, allowing them to

escape the blood vessel through diapedesis.

2.4.2) Phagocytosis

After neutrophils migrate to the site of infection, they engulf the external pathogens,

kill them inside the phagolysosomal vesicles and instruct the other immune cells and

extensively reviewed by Lee et al. (2003) and Segal (2005). Opsonizing factors on the

microbes enable recognition of the target while complement receptor, Fe receptor,

mannose receptor, P-glucan receptor, scavenger receptor and Toll-like receptor (TLR)

present on the surface of neutrophils play important roles in the trapping of infective

pathogens (Ishikawa and Miyazaki, 2005). Complement fragment C3bi is recognized

by the activated P2 integrin MAC1 (CD1lb/CD18). Once formed, the vacuole

undergoes a rapid series of remodeling events that alter its composition, conferring

onto it the ability to kill pathogens and dispose of debris (Lee et al., 2003). The

phagosome in neutrophils acquires its antimicrobial effects through fusion with

secretory vesicles and granules. Moreover, elevated cytosolic free calcium is known

to accompany particle ingestion during phagocytosis (Stendahl et al., 1994). This

changes in the level of free cytosolic calcium are required for granule secretion and

granular fusion with phagosomes in neutrophils (Sengelov et al., 1993). The

subsequent rise in cytosolic calcium activates calpain and release P2 integrin from its

tethers, thus allowing more P2 integrin to diffuse to the phagocytic cup that in tum

expedites phagocytosis (Dewitt and Hallett, 2002). A heavily opsonized particle is

taken up into the phagocytic vacuole within 20 s and killing is almost immediate.

NADPH oxidase elevates the pH to about 7.8-8.0 in the first 3 min after

phagocytosis, after which it gradually falls to about 7.0 after 10-15 min (Segal, 2005).

Neutrophils kill bacteria through both oxygen-dependent (including myeloperoxidase

with the subsequent generation of superoxide, hydrogen peroxide and hypohalous

acids), and oxygen-independent mechanisms (involving bactericidal proteins such as

lysozyme and lactoferrin and proteases such as elastase) inside phagosomes (Roos

and Winterboum, 2002; Segal, 2005). Recent development in Neutrophils functions

are summarised in Fig.3. T-H _ ( 6. 4l /. 6tl·67Gf . ~ j<.q605 R0



2.4.3) Neutrophil extracellular traps (NETs)

A novel mechanism, formation of neutrophil extracellular traps (NETs), to eliminate

invading pathogens has been reported recently (Brinkmann et al., 2004). NETs

considered as beneficial suicide (Brinkmann and Zychlinsky, 2007) of neutrophils

that binds microorganisms, prevents them from spreading and ensures a high local

concentration of antimicrobial agents. In vivo NETs contents are expectedly abundant

at the site of infection and acute inflammation (Beiter et al., 2006; Brinkmann et al.,

2004; Buchanan et al., 2006; Clark et al., 2007; Gupta et al., 2005). NETs formation

by the addition of PMA or IL-8, indicated that in addition to bacteria, cytokines or

PKC activation also induce NETs release (Brinkmann et al., 2004; Clark et al., 2007).

Platelet TLR4 mediated neutrophil activation and NETs formation has been reported

in severe sepsis (Clark et al., 2007). Since chronic granulomatous disease (CGD)

patients did not form NETs, it was delineated that NADPH oxidase dependent

generation of reactive oxygen species (ROS) mediate NETs release (Fuchs et al.,

2007). Identification of new mechanisms and mediators involved in NETs formation

is thus an area of intense research. The nucleus and cytoplasmic granular content

undergo a series of changes during NET formation following a particular pattern that

is initiated by the loss of nuclear segregation into eu- and heterochromatin.

Simultaneously, the nucleus looses its lobular characteristic, nuclear envelope

disintegrates and homogenization of nuclear, cytoplasm, and granular components.

The extrusion of homogenized nuclear and cytoplasmic content mediates the NETs

formation (Brinkmann and Zychlinsky, 2007). The mechanism of NET formation is

clearly distinct from apoptosis (Fadeel et al., 1998) because there is no DNA

fragmentation, phosphatidyl serine (PS) is not exposed before cell death, caspases

involvement, and time required for NET formation (1 0 minutes of stimulation) is too

short to be apoptosis (Fuchs et al., 2007). There are several reports about apoptosis in

activated neutrophils (Fadeel et al., 1998; Hampton et al., 2002; Lundqvist-Gustafsson

and Bengtsson, 1999). ROS-dependent apoptosis were performed with neutrophils in

suspension and the presence of high concentrations of serum, which inhibits NET

formation. Furthermore, the cells with lost membrane integrity were not considered

(Hampton et al., 2002; Lundqvist-Gustafsson and Bengtsson, 1999) might excluded

the potential NETs forming neutrophils.

The most apparent difference (Between NET and Necrosis) is the

morphological change of the nucleus preceding the formation of NETs. While in

Role of nitric oxide in neutrophil maturation and function 22


necrosis, the nuclear envelope remains intact, whereas prior to NETs release, the

nuclear membranes disintegrate into vesicles and requirement for specific cellular

activation (ROS production) in the case of NET formation (Fuchs et al., 2007). This

novel form of cell death is coined as "Netosis" (Brinkmann and Zychlinsky, 2007).

DNA (15-17nm) forms the backbone of NETs, in which the histones and

granular proteins (25nm) are decorated (Brinkmann et al., 2004). Other species

neutrophils, Fish and chicken hetrophils have also been shown to cast NETs

(Chuammitri et al., 2009; Palic et al., 2007). High circulating levels of DNA have

been assigned to NETs in malaria and sepsis patients (Baker et al., 2008; Margraf et

al., 2008). Initially it was opined that agents known to delay neutrophil apoptosis, led

to NETs release, however subsequent research have evidenced that both anti

apoptotic (IL-8, LPS & IFN-y) and pro-apoptotic (PMA, bacteria, ionomycin) agents

initiate NETs formation (Clark et al., 2007; Fuchs et al., 2007; Gupta et al., 2005;

Martinelli et al., 2004; Palic et al., 2007). Recently, a novel innate immune deficiency

of Impaired neutrophil extracellular traps (NETs) formation in human neonates have

demonstrated with glucose oxidase (Yost et al., 2009) and suggest the other

modulators of NETs formation except ROS.

2.4.4) Trans differentiation into dendritic cells

Transdifferentiation takes place when already committed progenitor cell transforms

into a different type of cell. Transdifferentiation is a type of metaplasia, which

includes all cell fate switches, including the interconversion of stem cells. Highly

purified lactoferrin-positive immediate precursors of end-stage neutrophilic PMN

(PMNp) have been reverted in their functional maturation program and driven to

acquire characteristic DC features. Upon culture with GM-CSF plus IL-4 plus TNFa,

they develop DC morphology and acquire molecular features including neo

expression of the DC-associated surface molecules CD 1 a, CD 1 b, CD 1 c, human

leukocyte antigen (HLA)-DR, HLA-DQ, CD80, CD86, CD40, CD54, and CDS, while

down regulation of CD15 and CD65s. The neutrophil-turned DCs are 10,000 times

more efficient as presenting soluble antigen to autologous T cells when compared to

freshly isolated monocytes (Oehler et al., 1998).

Transdifferentiation of polymorphonuclear neutrophils to dendritic-like cells

as fluid (SF) PMN from patients with RA undergo major alterations, including trans

differentiation to cells with dendritic-like characteristics, probably induced by T cell

derived cytokines. Because MHC class II positive PMN are known to activate T cells,



the mutual activation of PMN and T cells might contribute to the perpetuation of the

local inflammatory process, and eventually to the destructive process in RA (lking

Konet1 et a!. , 2005). Cultivating PMN of healthy donors, with either IFNy,

granulocyte/macrophage colony stimulating factor (GM-CSF/M-CSF) or a

combination there of, escaped from apoptosis, and protein synthesis was induced,

notably of the maJor histocompatibility complex (MHC) class II antigens, CD 14,

CD80, CD83 and CD86. Typical markers of PMN, including CD66b,

CD11 a/CD11b/CDllc, CD15. and CD18 were preserved (Iking-Konert et a!., 2001).

O:• l•duct-d bde12r1<tl ph.;gocy t Q·;. i ~ Uf maC!OphaqE' ·:0. Use antr .microhi.a l protease of PMN s ·H.11P.Gro -•. IUl & t.1CP Che rn o anra ct;mt ·>Removal of Senescetll Neu tro11 hils

l' r. • \ 1\0 ·, __ _

-~ / ) '--

• ~ "1 (/ "'Transdifferenti ati on into DC

/\ ( ··D irect interactio n DC. SI GN

• • ?· \ \ .. ~ DC Maturation

\/ \; "'Antigen Presentation to D'/ • • J • ••

DC • •• •• • Anti .in Oam atory ~icr opar1icle re lease • •

NETs ' To Kill extracell ular pathogen ,Homogenizati on of r~utleus and cytoplasmic. granules > Localfzed th e in11 ammatory enviro nment

• Induces TGF -b, At111ex in 1 • • • Redu ces DC ma t ration, T cell proUerat ion •4 • 1nhihit recruitme n of PMNs •

Micro-particle

• An1igen Presentation to T cells .o Th1 Jrola ri z-alion by IFN -v ·• Oefensi n s as c:hern o attr actants •r.11P -1a, t.11P-1h, I.TAC

Apoptosis

vApopto sis - Homeostasis ·:-Resolution of in0amma1ion .:. Granulopoieses fed back signal

Fig 3: Overview of neut rophil functions. Neutrophils interact with macrophage, dendritic cells, T cells and platelets in a bidirectional manner. Through cell-cell contact and secreted products, neutrophils recruit and activate monocytes, dendritic cells (DCs) and lymphocytes. Besides phagocytosis, neutrophils recently have shown to release anti-inflammatory micro-particles. Tissue macrophages ingest apoptotic neutrophils and used their anti -microbial components. Neutrophils can trans-differentiate into DC and work as APC. NETs formation takes place in high load of intruders and maintain anti-microbial milieu

2.4.5) Antigen presentation

In order to function as an APC, a cell needs to collect and cleave antigens, to generate

antigenic determinants, to subsequent present with MHC class I or class II molecules,

Role a,( nitric oxide in neutrophil maturation am/f unction 24


to express co-stimulatory molecules and to secrete cytokines, creating a milieu

conducive for T -cell differentiation. In the last decade it has been argued that

neutrophils satisfy all these criteria sufficiently to be regarded as APCs.

Complement receptor, Fe receptor, mannose receptor, ~-glucan receptor,

scavenger receptor, and Toll- -like receptor (TLR) present on the surface of

neutrophils play important roles in the trapping of infective pathogens (Ishikawa and

Miyazaki, 2005). PMNs actively acquire antigens, transcribe and express the genes

for MHC class I and class II molecules, costimulatory molecules and several

functionally diverse cytokines and chemokines that induce T -cell migration and

differentiation. Resting neutrophils expressed MHC class I molecules (Neuman et al.,

1992), while MHC class II molecules and costimulatory molecules such as CD80 and

CD86 exist intracellularly, and their induction by cytokines such as interferon (IFN-y)

and GM-CSF, IL-l, IL-6, and TNF-a on pure neutrophil culture (Gosselin et al.,

1993). PMNs cultured with autologous serum, IFN-y and GM-CSF, expressed MHC

class II, CD80 and CD86 and require de novo protein synthesis. These PMNs induced

proliferation ofTT-specific T cells in a MHC class II-restricted manner (Radsak et al.,

2000).

Because the neutrophils are short-lived and metabolically highly active, it is

unlikely that a pathogen will make a neutrophil its home. Therefore, despite MHC

class I expression, presentation of foreign antigens through the cytosolic MHC class I

pathway seems improbable for neutrophils. The paradox was solved when neutrophils

were shown to process exogenous bacteria and particulate antigens through an

alternate MHC class I processing pathway for presentation of peptides to T cells

(Potter and Harding, 2001). Sandilands et al 2005 have found Cross-linking of

neutrophil CDllb results in rapid cell surface expression of molecules CD80 (B7-1)

and CD86 (B7-2) and DR antigen required for antigen presentation and T-cell

activation. They do not constitutively express the cell surface molecules considered

necessary for antigen presentation and subsequent T -cell activation i.e. MHC class II

(DR) antigen (Sandilands et al., 2005). Several studies have however, shown that

following in vivo and/or in vitro activation by cytokines, neutrophils do appear to

express these molecules on the cell surface. Peripheral blood PMNs constitutively

express a B7-l-like molecule that interacts with CD28, regulating T-cell function

(Windhagen et al., 1999). By contrast, human neutrophils augment IFN-y secretion

from T cells (Venuprasad et al., 2003).

Role o.(nitric oxide in neutrophil maturation andfimction 25


Oehler L et al. have been demonstrated lactoferrin-positive immediate

precursors of end-stage neutrophilic PMN (PMNp) can be acquire characteristic DC

features (Oehler et al., 1998). Furthermore Iking-Konert et al. 2005 have been shown

Transdifferentiation of polymorphonuclear neutrophils to dendritic-like cells at the

site of inflammation in rheumatoid arthritis (Iking-Konert et al., 2005). Thus PMNs

actively acquire antigens, transcribe and express the genes for MHC class I and class

II molecules, costimulatory molecules and several functionally diverse cytokines and

chemokines that induce T -cell migration and differentiation.

The neutrophil subsets might also differ in their APC functions as described

for the other professional APCs. For example, activated but not naive resting B cells

can tum on virgin T cells (Ashtekar and Saba, 2003); resting macrophages and IFN

gactivated macrophages differ in their APC functions. Likewise, it has been observed

that only, 47% of human peripheral blood neutrophils express CD28 (Venuprasad et

al., 2001) and that CD28 signaling induces IFN-y. IFN-y might result in augmentation

of MHC class II expression in an autocrine manner. Therefore, it is possible that the

CD28+ neutrophils are better APCs than the CD28- subset. However these two subsets

really differ in terms of MHC class II expression remains to be tested. The other

factor, which modulates MHC class II expression in neutrophils, is GM-CSF. It is

observed that 13-72% ofthe GM-CSF treated neutrophils can be induced to express

MHC class II molecules (Potter and Harding, 2001). In primary proliferative

polycythaemia (PPP), CD14+CD64+ PMNs demonstrate higher phagocytic activity

(Ashtekar and Saha, 2003).

Being active phagocytes and expressing the Fe receptor (FeR) on their surface,

neutrophils can expeditiously collect antigens by phagocytosis and FeR-mediated

internalization (Chang, 1981; van Spriel et al., 1999). N eutrophils expressed TLR 1, 2,

4, 5, 6, 7, 8, 9, and 10-all the TLRs except TLR3. (Hayashi et al., 2003; Sabroe et al.,

2005). Hypothetically, neutrophils may directly present peptide to effector T cells in

vivo at sites of inflammation, inducing cytokine production, whereas dendritic cells in

receipt of neutrophil-derived antigenic peptides may migrate to lymphoid organs to

initiate T cell responses (Potter and Harding, 2001). The resting neutrophils activate

only memory T cells, whereas activated neutrophils stimulate natve T cells.

Neutrophils have been shown to efficiently cross-prime naive T cells in vivo

(Beauvillain et al., 2007).



2.4.6) Neutrophil anti- inflammatory

Neutrophils are known to be major culprit of tissue damage at inflammatory sites. But

recent data suggest some initial anti-inflamatory signaling from neutrophils during

activation. At the time of degranulation, activated PMNs release small microvesicles

(ectosomes 50 -200 nm by ectocytosis) directly from the cell surface membrane.

Ectosomes from platelets, endothelial cells and monocytes have been associated with

procoagulant and proinflammatory effects (Scapini et al., 2003). Recently Gasser and

Schifferli have been shown PMN-derived ectosomes, unexpectedly attribute

immunosuppressive/ anti-inflammatory functions. Neutrophil ectosomes have no

proinflammatory activity on human macrophages as assessed by the release of IL-8

and TNFa. On the contrary, ectosomes increase the release of anti-inflammatory

transforming growth factor ~ 1 (TGF~ 1) in vitro (Gasser and Schifferli, 2004). This

work was substantiated by exposing immature Monocyte derived dendritic cells

(MoDCs) to PMN-Ect, which modified their morphology, reduced their phagocytic

activity, and increased the release of TGF-~1. When immature MoDCs were co

cultured with PMN-Ect and stimulated with LPS, the maturation was partially

inhibited confirmed by reduced expression of surface markers (CD40, CD80, CD83,

CD86 and HLA-DP DQ DR), inhibition of cytokine-release (IL-8, IL-10, IL-12 and

TNF-a.), and a reduced capacity to induce T cell proliferation (Eken et al., 2008).

PMN-derived microparticles display inhibitory properties on target cells as assessed

in vitro; this phenomenon have been shown through the endogenous anti

inflammatory protein annexin 1 (AnxA1), present in PMN-derived microparticles

(Dalli et al., 2008). Furthermore, Kobayashi demonstrated that PMN s down-regulate

proinflammatory capacity at the level of gene expression during induction of

apoptosis (Kobayashi et al., 2003b ).

2.4.7) Cytokine synthesis and release

Neutrophils were long considered to be devoid oftranscriptional activity and capable

of performing no or little protein synthesis whose major role is to destroy intruders to

the body. The production of cytokines by activated neutrophils is striking in its

diversity. Neutrophils have been considered for long a phagocytic cell with a short

life-span. Toll receptors and anti-infectious factors such as defensin, perforin and

granzymes are newly discovered mechanisms used by neutrophils for the first line of

defense against invaders. Moreover, subpopulations of neutrophils share specific

functions like the synthesis of certain cytokines and chemokines as well as the



expressiOn of immunoreceptors like the T cell receptor (Ishikawa and Miyazaki,

2005). A primary consequence of inflammation on neutrophils is a delay in their

spontaneous programmed cell death. Neutrophils have the capacity to degrade and

process antigens as well as efficiently present antigenic peptides to lymphocytes.

Neutrophil interactions with immune cells, in particular dendritic cells, lead to the

formation of IL-12 and TNF-a deviating the immune response towards a Thl

phenotype.

Neutrophils are exquisite targets of proinflammatory cytokines, e.g. IL-l and

TNF-a, IL-8 and growth factors such as granulocyte/ monocyte colony stimulating

factor (G-CSF and GM-CSF). Convincing molecular evidence has now been afforded

that neutrophils either constitutively or in an inducible manner can synthesize and

release a wide range of proinflammatory, anti-inflammatory cytokines, other

chemokines and growth factors. However, it remains much lower in its degree than

that produced by the mononuclear phagocytes, namely the monocytes (Cassatella,

1995). Number of circulating neutrophils is almost 20 times higher than that of

monocytes and at the site of inflammation, neutrophils are the first to be recruited and

largely predominate over monocytes. TNF-a is also a priming agent for neutrophils

that notably increases their phagocytosis, degranulation and oxidative responses.

TNF-a itself, IL-1b, GM-CSF, and IL-2, are also potent inducers of TNF-a mRNA

expression and secretion by neutrophils. The release of cytokines from neutrophils

modulates the T cell responses, such as chemotaxis and cytokine secretion (Ashtekar

and Saha, 2003). IL-8 and GRO-a are chemo-attractive for neutrophils, while MIP-1a

and MIP-1 b attract not only T cells, monocytes and macrophages, but also immature

DCs. Although T cells, natural killer cells and macrophages are the main producers of

cytokines, the ability of PMNs to synthesize and release various immuno-regulatory

cytokines can be crucial in the initial phase of an immune response. Cathelicidins and

defensins promote cell proliferation, vasculogenesis and wound repair. Cathelicidins

and defensins can act at the interface of innate and adaptive immunity modulating DC

function and antigen-specific immune responses (Brown and Hancock, 2006; Yang et

al., 2002).

Neutrophils have been lately "re-discovered" as very versatile cells, contrary

to their traditional description as terminally differentiated effectors of inflammation

(Nathan, 2006). In fact, recent observations of their capacity to respond to a wide

variety of cytokines and chemotactic molecules, to change phenotype under specific



circumstances, to participate in the resolution of inflammation, and to regulate

angiogenesis and tumor fate (Witko-Sarsat et al., 2000). Matrix metalloproteinase-7

and b-defensin- 1 gene knockout mice are more susceptible to, and fail to clear,

infections (Moser et al., 2002; Nizet et al., 2001). Cathelicidins and defensins secreted

at sites of infection and/or injury are chemotactic for effector cells, induce the

transcription and secretion of chemokines and induce histamine release from mast

cells (Befus et al., 1999). Neutrophils are exquisite targets of proinflammatory

cytokines, e.g. IL-l and TNF-a, of chemokines such as IL-8, and growth factors such

as granulocyte/ monocyte colony stimulating factor (G-CSF and GMCSF). Indeed,

these cytokines have been shown to amplify several functions of neutrophils,

including their capacity of adhering to endothelial cells and to produce ROS, as

described above; likewise, chemokines act as potent attractants and favour their

orientated migration toward the inflammatory site.

2.4.8) Apoptosis

Neutrophils are short-lived cells and comprise a fundamental component of the non

specific immune armours of free radicals and proteases. Neutrophils over-recruitment,

uncontrolled activation and defective removal contribute to initiation and propagation

of many chronic inflammatory conditions, so neutrophils are subsequently removed

by the process of apoptosis and are engulfed by macrophages to resolve the

inflammatory response (Savill, 1997; Savill et al., 2002). The daily turnover ofhuman

neutrophils is 0.8-1.6 x 109 cells/kg body weight. The disposal of apoptotic cells is

regulated by a highly redundant system of receptors, bridging molecules and 'eat me'

signals. Dying neutrophils are the most abundant and important targets for such

recognition and engulfment by phagocytic cell. Apoptotic neutrophils display

morphological and biochemical characteristics of an apoptotic cell, including cell

shrinkage, compaction of chromatin and loss of the multi-lobed shape of the nucleus

(Savill et al., 1993). Constitutive neutrophil death is an essential mechanism for

modulating neutrophil homeostasis. Accelerated neutrophil death leads to a decrease

of neutrophil counts (neutropenia), augments the chance of contracting bacterial or

fungal infections, and impairs the resolution of such infections. On the other hand,

delayed neutrophil death elevates neutrophil counts (neutrophilia), which is often

associated with myeloid leukemia, and acute myocardial infarction (Luo and Loison,

2008).



Neutrophils accumulate rapidly at sites of infection in response to

proinflammatory cytokines-TNF-a, GM-CSF, IL-l, IL-15, IL-6 and chemokines IL-8

as well as bacterial endo/exotoxins. There is concomitant potential to cause severe

tissue destruction. Therefore, it follows that timely and vigilant execution of

neutrophils after phagocytosis for preventing damage to healthy tissues by

inflammatory process as encountered in systemic inflammatory response syndrome

(SIRS) and multiple organ failure (MOF) (Ayala et al., 2003). Spontaneous apoptosis

of neutrophils (Savill eta/., 1989) reflects clustering of death recptors (CD95). Life

span of this granulocyte is modulated variously by the presence of growth factors and

cytokines that set up a fine tuning between the constitutive action of the

predominantly present pro apoptotic members Bid, Bak, Bim, Bax and their

counteraction by the transiently induced unstable anti apoptotic members Mcl-1, A-1

(Akgu1 et al., 2001). GM-CSF signaling and proteasome inhibition delay neutrophil

apoptosis by increasing the cellular levels and stability of Mcl-1 (Derouet et al.,

2004).

The apoptotic signals for neutrophils are; TNFa [Death receptor; Fas (CD95),

TNF receptorl (p55)] and Nerve growth factor (NGF). Seely et al. (2003) described

two distict and independent pathway for neurophils apoptosis; activation ofNFKB and

the caspase pathway. Inflammatory cytokines and growth factors including IL-l~' IL-

2, IL-6, IL-8, IL-15, G-CSF, GM-CSF, C5a, LPS, IFN-y, glucocorticoids can prolong

neutrophil survival (Akgul et al., 2001). In addition to constitutive apoptosis,

inducible apoptosis mediated by the Fas pathway is suppressed by a variety of

inflammatory mediators, including IL-8, G-CSF, GM-CSF, IFN-y and TNF- a

inflammatory mediators may alter intracellular factors within neutrophils in order to

delay apoptosis; these factors include mitochondrial stability and caspases activity

(Seely et al., 2003). But neutrophils from CD95 deficient mice (lacking Fas) undergo

constitutive or spontaneous apoptosis at the same rate as control mice, arguing against

a role for the Fas system in constitutive apoptosis (Fecho and Cohen, 1998).

Prolonged incubations (<12 h) ofhuman neutrophils with TNF-a can cause a decrease

in apoptosis, TNF- a can also induce apoptosis in a sub-population of cells at earlier

times of incubation (<8 h) (Murray et al., 1997). Engagement ofthe ~2 integrin Mac-1

through its adhesion to its ligands, intercellular adhesion molecule- 1 (ICAM-1) and

fibrinogen, signals survival cues in neutrophils. However, in the presence of pro

apoptotic signals, such as TNFa, Mac-1 engagement accelerates apoptosis.



Furthermore, Mac-1 dependent phagocytosis of complement-opsonized pathogens

triggers rapid neutrophil apoptosis (Mayadas and Cullere, 2005).

Neutrophils lose their functional properties during apoptosis as outcome of

down-regulation of surface receptors (e.g. CD15, CD16, CD32, CD35, CD88,

CD 120b) and immunoglobulin superfamily members (e.g. CD31, CD 50, CD66,

CD63, CD87) for efficiently binding and activation to extracellular ligands

(Dransfield et al., 1994; Hornburg et al., 1995). Aged neutrophils have impaired

respiratory burst and reactive nitrogen intermediates, rendering them less able to

destroy bacteria and susceptible to apoptosis. Phagocytosis remains unimpaired in the

elderly neutrophils while microbicidal capacity of PMN is significantly decreased

with advancing age (Niwa et al., 1989; Plackett et al., 2004). Compounds modulating

their survival modulate expression profile of the DC markers on neutrophils. Higher

MHC-II, CD80, CD86, CD83 and CD40 expression levels were detected on the

surface of the cultured neutrophils for 24 h and annexin V-positive cells showed a

higher expression level of the DC markers. These apoptotic neutrophils expressing

DC markers on their surfaces have no stimulatory activity on T cells (Park et al.,

2007). Moreover, several genes encoding proteins involved in antigen presentation are

up-regulated during the initial stages of neutrophil apoptosis (Kobayashi et al.,

2003a). It has been shown that MHC-II is synthesized by neutrophils after being

stimulated with anti-apoptotic cytokines, such as IFN-y or GM-CSF (Fanger et al.,

1997; Radsak et al., 2000).

Apoptotic neutrophils do not only remove by macrophage without

inflammation and tissue damage but also increase the antimicrobial activity of

macrophage. Apoptotic neutrophils and purified granules have shown to inhibit the

growth of extracellular mycobacteria (Tan et al., 2006). Uptake of apoptotic cells

actively inhibits the secretion of proinflarnmatory mediators such as TNF-a and

increases the anti-inflammatory and immunosuppressive cytokine TGF-P by activated

rnacrophages (Ren et al., 2008). Administration of apoptotic cells can protect mice

from LPS-induced death, even when apoptotic cells were administered 24 h after LPS

challenge. The beneficial effects of administration of apoptotic cells included reduced

circulating proinflarnrnatory cytokines, suppression of neutrophils infiltration in target

organs and decreased serum LPS levels (Ren et al., 2008). Furthermore presence of

apoptotic cells during rnonocytes activation with LPS increases their secretion of the

anti-inflammatory and imrnunoregulatory cytokine IL-10 and decreases secretion of

Role o_lnitric oxide in neutrophil maturation and fimction 31


the proinflammatory cytokines TNF-a, IL-l, and IL-12 (Oku et al., 2002; Voll et al.,

1997).

New findings indicate that the interaction of phosphatidyl serine (PS) on

apoptotic neutrophils with its receptor on macrophages is not as critical for the

specific clearance of neutrophil corpses it was previously believed. Clearance of

dying neutrophils in a highly proteolytic milieu containing both host and bacteria

derived proteinases dramatically modified. Pre-incubation of apoptotic neutrophils

with cathepsin G or thrombin have been shown to inhibit their uptake significantly by

macrophages (Guzik and Potempa, 2008). Myeloperoxidase independent of its

catalytic activity through signaling via the adhesion molecule CD 11 b/CD 18 rescued

human neutrophils from constitutive apoptosis and prolonged their life span (Carrigan

et al., 2005). MPO evoked a transient concurrent activation of ERK and Akt,

phosphorylation of Bad, prevention of mitochondrial dysfunction and subsequent

activation of caspase-3. Furthermore, acute increases in plasma MPO delayed murine

neutrophil apoptosis assayed ex vivo. Therefore, MPO contributes to prolongation of

inflammation (El Kebir et al., 2008).

Homeostatic regulation of neutrophil production and apoptosis is essential to

maintain constant number of neutrophils in blood. Normal neutrophils migrate to

tissues, where they become apoptotic and are phagocytosed by macrophages and

dendritic cells. This leads to phagocyte secretion of IL-23, a cytokine controlling IL

l 7 production by T cells. IL-l 7 released from subsets of T cells regulates

granulopoiesis through G-CSF. Antibody blockade of the p40 subunit of IL-23

reduces neutrophil numbers in wild-type mice and shows homeostatic mechanism for

the regulation of neutrophil production in vivo (Stark et al., 2005).

Role o.lnitric oxide in neutrophil maturation andfimction 32


II.) NITRIC OXIDE (NO)

Evolution has resorted to nitric oxide (NO), a tiny lipophilic reactive radical gas, to

mediate both regulatory and cytotoxic functions (Bredt and Snyder, 1994).

Recognition of the endothelium derived relaxing factor (EDRF) as nitric oxide (NO)

initially suggested that NO was synthesized only by the endothelial lining of vessel

wall. However, it has been found that NO was synthesized constitutively by the

enzyme nitric oxide synthase (NOS) in various cells, the best studied of which are

vascular endothelial cells (Moncada et al., 1989), macrophages (Gross et al., 1991),

and neurons (Bredt and Snyder, 1994). NO was designated as "molecule of the year"

by Science journal in 1992. Furthermore Furchgott, Ignaro and Murad were honoured

with Nobel Prize in 1998 for their contribution in NO biology. Blood cells such as

eosinophils, platelets, neutrophils, monocytes and macrophages also synthesize NO.

Among them, neutrophils constitute an important proportion and are also the major

participants in a number of pathological conditions with suggestive involvement of

NO.

NO is generated by a class of nicotinamide adenine dinucle?tide phosphate

(NADPH)-dependent NO synthases (NOS), which catalyze the conversion of L

arginine to L-citrulline and NO. NOS exists in three isoforms, neuronal NOS (nNOS),

endothelial NOS (eNOS) and inducible NOS (iNOS). Constitutive NOS (eNOS)

including eNOS and nNOS are calcium dependent and produce low level of NO;

however inducible iNOS is augmented by inflammatory cytokines and calcium

independent and produce high NO for prolong time (Alderton et al., 2001). TheN

terminal oxygenase domain of NOS has binding sites for biopterin, heme and L-Arg;

while, the C-terminal reductase domain contains binding sites for NADPH, FMN and

FAD and closely resembles the Cytochrome P450. Calmodulin (CaM) binding

triggers the transfer of electrons from the reductase to the oxygenase domain (Bredt

and Snyder, 1994). Heme and biopterin (BH4) are the most important requirements for

enzyme dimerization and for achieving a stable conformation for electron transport.

The independence of the iNOS for calcium is due to the tightly bound calmodulin

(Alderton et al., 2001). Domain structure of all the NOS isoform is represented in

Fig.4.

Primary function of NOS is to produce NO, but it also produces Oi-, ONOO

and N03- depending on the environment (Stuehr et al., 2004). In normal productive



cycle in the presence of all cofactors NOS synthesizes NO. While all the three NOS

isoforms (nNOS. eNOS & iNOS) can also generate 0 2- in the experimental conditions

such as absence of L-Arg or reduction in BH-t concentration, by mechanism known as

uncoupling. When, the NO concentration accumulates in the range of micromolar

range ONoo· and N0 3 - are generated from NOS known as futile cycle (Stuehr et al..

2004).

Dimer interface

Oxygenase domain

NADPH 1433

NADPH 1153

FAD NADPH 71& 1203

Reductase domain

Fig 4: Domain structure of human nNOS, eNOS and iN OS (Alderton et al. , 200 I).

2.5) Functions of NO

nNOS H

eNOS

iN OS

H

Unlike most small signaling molecules, the biological effects of nitric oxide are

determined by their chemical reactions, such as binding to the regulatory heme in

soluble guanylate cyclase (sGC), rather than traditional protein receptor- ligand

interactions. NO is a highly diffusible molecule in both aqueous and hydrophobic

environments whose biological concentration is determined partially by its distance

from the point of synthesis and partly by the cellular redox environment. The

diffusion coefficient for 0 2 is 2800 1.1m2s- 1 and for NO is 3300 1.1m2s- 1 (Thomas et al. ,

2008). Its target cell specificity depends on its concentration, compartment, exposure

time, chemical reactivity, vicinity and priming of target cells. In tissue, cell s are

usually within 50-300 1.1m or between 1-30 cell lengths, where NO is rapidly

consumed.

The chemical biology of NO divides these potential reactions into two

categories: direct and indirect (Thomas et al. , 2008; Wink et al. , 1996). The direct



effects ofNO are those chemical reactions that occur fast enough to allow NO to react

directly with a biological target molecule. In contrast. the indirect effects require that

NO reacts with oxygen or superoxide to generate RNS, which subsequently react with

the biological targets. Direct effects generally occur at low concentrations, whereas

indirect effects occur at much higher concentrations. Subsequently, the molecular

mechanisms that mediate the biological activities of NO can be divided into three

categories- reaction with transition metal, nitration and nitrosylation. NO reacts

readily with transition metals, such as iron, copper and zinc abundantly present in

prosthetic groups of enzymes and proteins [guanylate cyclase, cytochrome P450 and

NOS (IgnatTO, 1991). Secondly, NO is able to induce the formation of S-nitrosothiols

on cysteine residues by a reaction called S-nitrosylation. Nitrosylation has been

shown to modify the activity of several proteins involved in cellular regulatory

mechanisms. Thirdly, NO reacts very quickly with superoxide anion (02-). resulting in

the formation of peroxynitrite (0 oo-). Peroxynitrite is a nitrating agent and a

powerful oxidant that is able to modify proteins, lipids and nucleic acids. The first

mechanism represents direct effects of NO and the two latter mechanisms are refetTed

as indirect effects of NO. The concentration of NO will determine its chemistry

(direct vs indirect), the distance it diffuses and the type of signaling targets (Fig 5).

Direct Effects ([NO] < 200 tlM)

I DNA Radicals I

Indirect Effects ([NO) > 400 nM)

1

+0,

+NO +NO

+CO~

Metal oi1r0 yl Formation

GC, P450, UbJMb

1 Oxidali vc Stress

Fig 5: Diagrammatic representation of NO direct versus indirect effects (Thomas et al. , 2008).

NO is the best example of a reactive molecule demonstrating both cytotoxic

and cytoprotective properties (Wink et al. , 1996). After its discovery, in the 1980s as

Role o.fnitric oxide in neutrophil maturation andjimction 35


EDRF, further investigations led to an explosion of NO research, and revealed the

importance of this diatomic molecule in nearly every tissue in the body. Low

concentrations of NO such as those that occur in vascular and stromal cells (i.e., from

eNOS and nNOS) regulate normal physiological processes, and the high levels such

as those expected in activated macrophages (via iNOS) are thought to serve a

cytotoxic/cytostatic function (Knowles and Moncada, 1994; Thomas et al., 2008).

However, at these higher concentrations, it is not always clear that cell death is the

ultimate outcome. Nitrosative stress has a protective side, where nitrosation of

caspase-3 and -8 as well as poly (ADP-ribose) polymerase (PARP) leads to protection

against apoptosis (Thomas et al., 2008).

The importance of concentration when talking about NO signaling can be

appreciated when one considers the distinct concentration dependence of NO

regulated proteins. At sustained NO levels between 10 and 30 nM, phosphorylation of

ERK occurs through a cGMP-dependent mechanism in MCF7 and endothelial cells.

At 30-60 nM NO Akt is phosphorylated (Thomas et al., 2008). While at threshold

concentration of about 100 nM, HIF-la is stabilize. At 400 nM NO, p53 is

phosphorylated and acetylated (Thomas et al., 2004). It is above 1 ~M NO that

nitrosation of critical proteins such as P ARP, caspase and others occurs. Conclusively,

lower NO concentrations promote cell survival and proliferation, whereas higher

levels of NO favor pathways that induce cell-cycle arrest, senescence, or apoptosis

(Thomas et al., 2004; Thomas et al., 2008). In addition to concentration, temporal

aspects of NO exposure are equally important. Certain proteins respond immediately

to NO exposure, whereas others require hours or even days to be activated. HIF-la,

for example, responds immediately to NO (Thomas et al., 2004). In contrast,

phosphorylation of p53 by NO takes several hours and sustained long after NO

exposure.

The presence of other radical spectes 1s also a key regulator of NO

concentration and will partially dictate its influence on target molecules. By viewing

NO in this context we can understand why there can be a seemingly infinite number

of biochemical responses to a single signaling molecule. It regulates the functional

activity, growth and death of many immune and inflammatory cell types including

macrophages, T lymphocytes, antigen-presenting cells, mast cells, neutrophils and

Role a_{ nitric oxide in neutrophil maturation andfimction 36


natural killer cells. In the following section I will emphasize on PMN s function

modulated by NO.

2.6) Nitric oxide mediated modulation of PMNs functions

Coming to the functional modulation of neutrophils the overall picture as depicted in

recent reviews indicates a profound though biphasic influence of NO on neutrophil

immune responses such as chemotaxis, adhesion, phagocytosis, respiratory burst and

apoptosis (Akgul et al., 2001; Li and Wogan, 2005; Sethi and Dikshit, 2000; Taylor et

al., 2003). NO mediated modulation of neutrophil physiology or functionality is still

under scrutiny contradictory evidences come from time to time adding further to the

complexity of the dilemma.

(a) Chemotaxis

Chemotaxis is the directed movement of cells in response to concentration gradient of

a chemo-attractant. Chemotactic stimulation of PMNs induces a cascade of events

which include actin reorganization, shape changes, development of polarity and

reversible adhesion, culminating in directed migration in a gradient of stimulus. NO

from both exogenous and endogenous sources limit leukocyte recruitment into normal

and inflamed vessels (Fukatsu et al., 1998; Gaboury et al., 1993; Sato et al., 1996).

While Okayama et al have shown that NO enhance neutrophil adhesion to endothelial

cells (Okayama et al., 1999; Okayama et al., 1998). Moreover, exogenous NO has

shown to enhance random migration of rabbit peritoneal neutrophils in a

concentration dependent manner, which is associated with rapid and transient

increases in cGMP levels (VanUffelen et al., 1996). Role ofNO in migration has also

been shown by use of NOS inhibitors, which elicit leukocyte emigration, and

prevented by L-Arginine (Kurose et al., 1995; Sato et al., 1996).

Neutrophil chemotaxis is induced in response to invading pathogens and

chemokines, which subsequently upregulate iNOS. Intra-peritoneal inoculation by a

lethal dose of Staphylococcus aureus in sepsis models prevented neutrophil migration

to the site of infection, which was abolished on pretreatment with aminoguanidine

(Crosara-Alberto et al., 2002). In a similar study Benjamin et al., (2002) observed that

iNOS-1-)mice subjected to lethal sepsis induced by cecal ligation and sublethal sepsis

by cecal ligation and puncture suffered high mortality despite effective neutrophil

migration due to lack of microbicidal activity in neutrophils of iNOs-/- mice. Zymosan



injection into the peritoneal cavity in both wild type and iNOS knockout mice elicited

similar chemotactic response of neutrophils yet a subtle difference in the kinetics

points to possible fortifying effects of NO on neutrophil chemotaxis (Ajuebor et al.,

1998).

NO induced chemotaxis and its inhibition have been shown by use of NO

donors (Beauvais et al., 1995; Kaplan et al., 1989; Kosonen et al., 1999; Malawista

and de Boisfleury Chevance, 1997) and was suggested to be unrelated to rise in

cGMP. High concentration of NO donors and cGMP inhibit chemotaxis whereas

lower concentrations promote this response, suggesting a biphasic regulation of

chemotaxis by NO (VanUffelen et al., 1996; Wanikiat et al., 1997). The mechanism

responsible for this effect is not completely understood but several reports in literature

suggest that the effect at low concentration is cGMP independent whereas at high

concentration it is cGMP dependent.

(b) Rolling & adhesion

Extravasation of neutrophils is a complex and highly coordinated phenomenon that

involves initial low affinity rolling of neutrophils mediated by selectins followed by

high affinity interactions mediated by integrins to the vascular endothelium

facilitating the process of transmigration. Rolling of leukocytes is mediated by L, P

and E selectins (Granger and Kubes, 1994). It has been demonstrated that inhibition

of NO synthesis promotes P-selectin dependent leukocyte rolling (Terada, 1996)

suggesting that NO may be a homeostatic factor in down regulating the leukocyte

rolling under normal conditions. Exogenous NO decreases leukocyte rolling under

normal conditions (Gaboury et al., 1993; Johnston et al., 1996). In the iNOS deficient

mice increase in the leukocyte-endothelium interaction following LPS induced

endotoxemia has been reported (Hickey et al., 1997).

L-Arginine supplementation increases and prolongs fMLP triggered neutrophil

aggregation in NO dependent mechanism involving ADP ribosylation and

rearrangement of actin cytoskeleton (Forslund et al., 2000). NO prevents neutrophil

endothelium interaction by reducing CD11 b/CD18b expression and inhibits ~2

integrins by interfering with the cell surface transduction of signals linked to

particulate guanylate cyclase activity (Banick et al., 1997; Kubes et al., 1991). LPS

treatment induces NOS and upregulates expression of E-selectin and ICAM-1 thus



influencing intercellular adhesion (Gluckman et al., 2000; Kosonen et al., 1999) a

phenomenon opposed by NO donors.

NO donors generating NO in higher than physiological level inhibit LPS or TNF-a

induced neutrophil adhesion to endothelial cells. Furthermore, endogenous NO or

supplementation with L-arginine is effective in preventing reperfusion injury and

target organ infiltration and damage attributed to neutrophils as in sepsis (Nakanishi et

al., 1992; Pemow et al., 1994; Siegfried et al., 1992). NO is an important homeostatic

regulator of leukocyte adherence (Akimitsu et al., 1995; Kubes et al., 1991; Mitchell

et al., 1998). NO prevents the leukocyte-endothelial cell adhesion by reducing the

CD11/CD18 expression (Kubes et al., 1991; Mitchell et al., 1998). It inhibits the ~2

integrins in a concentration dependent fashion by inhibiting the cell surface

transduction of signals linked to the activity of membrane bound guanylate cyclase

(Banick et al., 1997). Cell permeable analogues of cGMP also inhibit leukocyte

endothelial cell adherence (Kurose et al., 1995), suggesting an involvement of

NO/cGMP in the leukocyte-endothelial cell adherence.

(c)Phagocytosis

Neutrophils remove microbes and segregate them intracellularly into the phagocytic

vacuole by phagocytosis. Endogenous enzymatic generation of NO has been

implicated in bacterial internalization and subsequent killing by neutrophils. Human

neutrophils required the cytokine trigger in form of IL-l, TNF -a and IFNy, to induce

iN OS and subsequently nitration or nitrosylation of the bacterial targets possibly due

to the formation of peroxynitrite (Evans et al., 1996).

Endogenous enzymatic generation ofNO has been implicated in bacterial endocytosis

and subsequent killing by neutrophils. These observations can be further categorized

as a response of peripheral and peritoneal neutrophils. Rat peritoneal neutrophils

constitutively generating NO showed pronounced fungal killing in vitro, in

comparison to the peripheral neutrophils, which produce less amount of NO. In the

presence ofNOS inhibitor, L-NG-mono-methyl Arginine (L-NMMA), decrease in the

phagocytosis and killing was observed in an anucleate granule poor neutrophil

preparation, which was neutralized by L-arginine (Malawista et al., 1992). Phagocytic

activity of human neutrophils is augmented by supplementation with L-arginine, but

was antagonized by NG nitro-L-arginine (L-NNA), L-canavenine (L-CAN) or



aminoguanidine (Moffat et al., 1996). NO donors at high concentrations however,

inhibited phagocytosis.

(d) Degranulation

Neutrophil activation involves its degranulation. Azurophilic granules and specific

granules show different degranulation dynamics. This is primarily due to different

Ca2+ requirements for exocytosis, specific granules being more sensitive to a rise in

[Ca2+]i and, consequently, released before the azurophils (Sengelov et al., 1993).

Cyclic GMP and its analogues or agents, which increase intracellular cyclic GMP,

enhance degranulation (Ignarro, 1974). Enzyme release is a complex multi-step

process, which is influenced by migration, membrane recognition, adherence of

particle and ingestion, as well as granule exocytosis. NO donors inhibit the release of

~glucuronidase from human PMNs. Moreover Moilanen et al. found that NO donors

inhibited degranulation in PMNs (Moilanen et al., 1993), supporting the idea that

PMNs derived NO could act as a negative feedback signal to restrict the inflammatory

processes. Exogenous NO also enhances fMLP induced exocytosis in rabbit

peritoneal neutrophils. Higher concentrations, however, strongly inhibited exocytosis

(VanUffelen et al., 1997).

(e) Respiratory burst/ Free radical generation

Respiratory burst was reported first by Baldridge and Gerald (1932) during the

process of phagocytosis in neutrophils, due to the activity of NADPH oxidase, a

multi-subunit enzymatic complex. Respiratory burst is responsible for more than 90%

of the total oxygen consumption by these leukocytes (Babior et al., 1973). This leads

to generation of 0 2- into the phagosome or to the exterior milieu. Superoxide anions

are relatively in noxious, but form additional toxic oxygen species, in particular H20 2,

by spontaneous dismutation which may then oxidize halides, in particular cr, to

hypohalous acid, e.g. HOCl, catalyzed by myeloperoxidase released from azurophil

granules during degranulation. After encounter the invading organisms, the

neutrophils sequester the invading organism into an enclosed vacuole, known as a

phagosome. Upon stimulation, cytoplasmic proteins, p47Phox, p67Phox, and a Rae

related GTP protein translocate to the plasma membrane, binding to sites located on a

unique b-type hemoprotein, Cytochrome bsss. This hemoprotein, a dimer consisting of

gp91 Phox and p22Phox, binds FAD and NADPH that results in a flow of electrons to the



terminal acceptor Cytochrome b558 (Babior et al., 2002; Segal, 2005). Transfer of an

electron from the Cytochrome to oxygen yields superoxide. The production of

superoxide initiates a series of oxidative events, which result in microbial killing.

Patients with chronic granulomatous disease face life threatening infections primarily

because their phagocytic cells are unable to generate superoxide (Babior et al., 2002),

highlighting the importance of phagocyte derived superoxide in host defence.

NO and oxidative burst in neutrophils have been extensively investigated in

our lab. The observations convincingly indicate towards NO mediated augmentation

of free radical generation from PMNs (Seth et al., 1994; Sethi et al., 2001; Sethi et al.,

1999). Intracellular and extracellular calcium levels also have a modulatory impact on

NOS activity and free radical generation (Dikshit and Sharma, 2002). Recently, effect

of NO donors on neutrophil respiratory burst is suggested with involvement of K+

channels and kinases in NO mediated augmentation of respiratory burst (Patel et al.,

2009). Clancy et al., (1992) showed direct interaction of NO with the membrane

subunit of the NADPH oxidase complex, while Fuji et al., (1997) demonstrated an

inhibitory association ofNO with both membranous and cytosolic subunits. Lee et al.,

(2000) also reported an inhibitory effect at a higher concentration of NO. ONOO

exhibited a biphasic effect like NO, being stimulatory at lower concentrations through

the MEK/ERK/MAPK pathway, but inhibitory at higher concentrations (Sethi et al.,

1999). Recently, NO donors have found to decrease PMA- and/or fMLP-induced

phosphorylation of p4 7 on tyrosine and serine/threonine residues and PKC on serine

residues and ROS production with MAPK phosphorylation (Klink et al., 2009).

The antioxidant defense mechanisms are on constant vigil to maintain the

redox balance of the neutrophils. Neutrophil is protected against self-destruction by

intracellular superoxide dismutase, ascorbate, GSH and catalase (Roos and

Winterboum, 2002). Factors instigating oxidative burst may simultaneously trigger

NOS in neutrophils. Lipopolysaccharide (LPS), membrane component of gram

positive bacteria, a potent inducer ofiNOS lead to a significant increase in L-arginine

uptake and free radical generation with arachidonic acid from peripheral and

peritoneal neutrophils (Sethi et al., 2001). NOS inhibitors, aminoguanidine and 7-

nitroindazole, inhibit arachidonic acid-induced free radical generation from LPS

treated neutrophils. Moreover, pre-incubation with nitrite also elevate the free radical

generation and MPO activity (Sethi et al., 2001). Moreover, hypoxic neutrophils

following oxygenation exhibited a significant increase in the respiratory burst in a NO



dependent manner (Sethi et al., 1999). This observation is of significance in

explaining the damaging effects of neutrophils at the hypoxic environment of the

inflammatory loci.

(j) Apoptosis

NO and apoptotic regulation of neutrophils is though indicated, but a decisive and

distinctive picture is still awaited. The role of NO in modulating gene expression and

cell survival has been extensively elaborated (Kim et al., 2002; Kim et al., 1998; Li

and Wogan, 2005; Luo and Loison, 2008; Taylor et al., 2003). Role of endogenous

NO is controversial showing both pro and anti-apoptotic outcome. Levels of nitrite

increase in spontaneously aging neutrophils (Misso et al., 2000), and the anti

apoptotic effect of GM-CSF in prolonging neutrophil survival is associated with

decrease of nitrite content in these cells. On the contrary apoptotic trigger from anti

Fas ligand or TNF-a relate to a decrease in the nitrite content also suggesting a

survival signal from NO (Misso et al., 2000). During inflammation neutrophil survival

is prolonged and it is interesting to see that both NO donors and NOS inhibitors

provide protection. It thus highlights the complexity in action of such a simple

molecule like NO on neutrophils. Upon induction of apoptosis in the thymus by x-ray,

iNos-/- knockout mice exhibited higher levels of neutrophil infiltration (Shibata et al.,

2007).

NO is thus a very important regulator of PMNs functions and is involved in

various physiological and pathological conditions. Although much importance has

been focussed on NO which is generated by endothelium, platelets or other cells

however, controversy exists regarding its presence in human PMNs. These

controversies arise mainly due to the variations in the experimental procedures used

for its detection in this cell type and also because under physiological conditions,

these cells generate very low amounts of NO. A complete and better understanding of

the NOS in PMNs and its mechanism of modulation would help in evolving a better

understanding ofthe role of NOS in PMNs function.

2.7) Neutrophils and NO/NOS

Wheeler et al. (1997) identified neutrophils as the pnmary source of iNOS in

leukocyte enriched pellets isolated from the urine of patients with urinary tract

infections and induction in iNOS after bacterial infection (Wheeler et al., 1997).

Role a_{ nitric oxide in neutrophil maturation andfimction 42


Plasma nitrate concentration has been reported significantly higher in patients with

septicemia who have normal or elevated number of neutrophils in peripheral blood

than to those with neutropenia (Neilly et al., 1995). It has been demonstrated that

neutrophil derived NO is responsible for the augmented free radical generation

following hypoxia-reoxygenation (Sethi et al., 1999). Increase in the neutrophil nitrite

content and its role in Parkinson's disease has also been suggested (Barthwal et al.,

1999).

An increase in the release ofNO from PMNs after thrombosis (Dikshit et al.,

1993) suggests that these cells play an important role in the regulation of homeostasis

by having an inhibitory effect on platelet activation. Circulating neutrophils from

hypertensive patients are oxidatively more active than their normotensive counterparts

(Pontremoli et al., 1989). Recently, circulating neutrophils have been shown to

maintain physiological blood pressure by suppressing bacteria and IFNy-dependent

iNOS expression in the vasculature of healthy mice (Morton et al., 2008) as

neutrophil depletion led to low blood pressure and suggested as requirement to

maintain the optimal vascular tone. Malawista et al., (1992) reported that NO

generation by neutrophils is also involved in their antimicrobial function.

2.7.1) Nitric oxide synthases (NOS) in Neutrophils

Neutrophils have been predicted to generate NO at a rate of 10-100 nmoles/5min/l 06

cells, comparable to the endothelial cells, contributing much to the amount of NO in

circulation (Salvemini et al., 1989; Wright et al., 1989). Thus, neutrophils, which

represent 50-60% of the total circulating leukocytes, could add substantial amount of

NO in circulation with potentially a widespread impact on vascular homeostasis.

Neutrophils nitric oxide synthase was first discovered by its ability to relax

aortic rings. Peritoneal (Rimele et al., 1988) and peripheral (Dikshit et al., 1993) rat

PMNs elicited a vasodilator response when added to endothelium denuded aortic

rings. These cells were also found to inhibit platelet aggregation (Faint et al., 1991 ).

The inhibitory activity of neutrophils was found to be prevented by the preincubation

of cells with NG-monomethyl L-Arginine (Dikshit et al., 1993; Faint et al., 1991), NG

nitro-L-Arginine methyl ester (Faint et al., 1991). Furthermore, incubation of platelets

with neutrophils led to an increase in platelet cGMP levels. Besides bioassays,

measurement of nitrite (N02) (Miles et al., 1995; Rodenas et al., 1995) and NOS

activity by conversion of radiolabeled L-Arginine to radiolabeled L-citrulline



(Cedergren et al., 2003; Chen and Mehta, 1996; Saini et al., 2006) have been used to

detect NO production or to demonstrate the presence of NOS activity in rat and

humanPMNs.

Miles et al.,(1995) have shown that circulating PMNs (rat or human)

contained no iNOS mRNA, protein, or enzymatic activity. Furthermore, when

cultured for 4-6 h in vitro iNOS mRNA levels, iNOS protein and iNOS enzymatic

activity increased from normally undetectable levels in circulating rat PMNs. Similar

results were reported by Evans et al.,(1996) through induction iNOS by cytokine

treatment in human neutrophils. Furthermore induction in neutrophils iNOS after

bacterial infection was reported (Wheeler et al., 1997). However Amin et al.,(1995)

reported that iNOS was under detectable level in human neutrophils by western

blotting analysis and NOS activity. However, sensitive methods such as RT-PCR and

Northern blot have shown "constitutively expressed" iNOS mRNA from neutrophils

and indicated that very low levels ofNOS protein expressed in neutrophils.

Wallerath et al., (1997) Identified NO synthase isoforms expressed in bone

marrow human neutrophil granulocytes, megakaryocytes and platelets. Immuno

cytochemistry demonstrated nNOS, whereas no eNOS was detected in neutrophils.

Similarly, in RT-PCR, transcripts for nNOS but not for eNOS were identified. Thus,

concluded the constitutive NOS isoform in neutrophils is nNOS. PMNs isolated from

the human oral cavity (Nakahara et al., 1998) and from patients with sepsis syndrome

(Tsukahara et al., 1998) possess iNOS mRNA, protein and exhibit enzyme activity.

Miles et al. (Miles et al., 1995) were unable to detect either iNOS mRNA, protein or

enzyme activity in the extravasated human cells. Possible reasons for the inability of

workers to demonstrate iNOS activity may be that iNOS has been shown not to

present in cytosol but in membrane fractions (Wheeler et al., 1997). Human and rat

neutrophils have been shown to express nNOS mRNA constitutively (Greenberg et

al., 1998; Greenberg et al., 1996). However Greenberg et al. (1998) failed to show the

presence of NOS protein, while presence of nNOS mRNA and 150 kD protein in

circulating human PMNs has been shown by Wallerath et al. (1997). Human PMNs

have been shown to express significantly lower amounts of mRNA for NOS than rat

circulating cells (Greenberg et al., 1998; Miles et al., 1995).

Increase in the neutrophil nitrite content and its role in Parkinson's disease has

been suggested (Barthwal et al., 1999). Moreover Gatto et al., (2000) reported over

expression of neutrophil neuronal nitric oxide synthase in Parkinson's disease by



using RT-PCR, western blotting and hybridization. Constitutive expression of iNOS

in human neutrophils was evaluated by flow cytometry, western blotting and NOS

activity (Cedergren et al., 2003). iNOS was revealed by Western blotting but its

detection was dependent on diisopropylfluorophosphate for proteinase inhibition.

Presence of eNOS in neutrophils is still controversial as only one report regarding

expression of endothelial nitric oxide synthase isoform in human neutrophils

published so far (Frutos et al., 2001) and has been shown to modulate by tumor

necrosis factor-alpha and during acute myocardial infarction.

A detailed study from our lab by using RT-PCR and Western blotting

demonstrated the presence of nNOS and iNOS in rat PMNs (Saini et al., 2006).

Furthermore localization of nNOS and iN OS was shown in cytoplasm and firstly in

nucleus by confocal and immunogold electron microscopy and confirmed by L-[3H]

citrulline formation and DAF fluorescence (Saini et al., 2006). Furthermore nNOS

and iNOS colocalized with caveolin-1, was shown by immunocytochemical and

immunoprecipitation studies. Neutrophil NOS in spontaneously hypertensive rats was

studied by Griess reagent, Flowcytometry and RT-PCR. NO generation was found to

be augmented from SHR neutrophils in comparison to normotensive wistar rats

(Chatterjee et al., 2007). Furthermore expression of iNOS was significantly more in

the SHR neutrophils, while that of nNOS remained unaffected. Recently, Ascorbate

has been shown to sustain neutrophil NOS expression, catalysis, and oxidative burst

from our lab (Chatterjee et al., 2008).

Reports depicting the characteristics of NOS present in neutrophils are limited

as compared to investigations in other cells/cell lines. However, presence of both

nNOS and iNOS has been accepted unequivocally (Cedergren et al., 2003; Chatterjee

et al., 2008; Gatto et al., 2000; Greenberg et al., 1996; Saini et al., 2006), while

occurrence of eNOS in neutrophils (Frutos et al., 2001), is being advocated,

warranting further investigations. As localization of nNOS and iN OS was shown in

cytoplasm and firstly in nucleus by Saini et al., (2006) but course of its localization in

nucleus is still unknown and requires further evaluation.

2. 7 .2) Nitric oxide in Bone Marrow

NO mediates the action of growth factors and control the balance between

proliferation and differentiation in neuronal, cardiomyocyte, adipocyte, osteoblast and

endothelial cell cultures (Enikolopov et al., 1999). Henceforth the potential impact of

Role o_{nitric oxide in neutrophil maturation and fimction 45


NO in differentiation of diverse cellular systems is well established but its implication

during the terminal differentiation of neutrophils needs to be explored.

Nitric oxide production by bone marrow cells was explored after IFNy and

LPS stimulation. Maximal effects were observed with GM-CSF and LPS

combinations and were dependent on the presence of L-arginine and inhibited by NG

monomethyl-L-arginine. Furthermore, flow cytometry revealed that the granulocyte

containing fraction was largely responsible for nitric oxide production. (Punjabi et al.,

1992) Maciejewski et al (1995) using PCR and immunoprecipitation, found iNOS

mRNA and protein in BM cells after stimulation with IFNy or TNF-a. iNOS mRNA

was also detected by PCR in highly purified CD34+ cells, NG-Monomethyl-L-arginine

(L-NMMA), an NOS inhibitor, partially reversed the effects and inhibited apoptosis

of BM cells induced by these cytokines and suggest that NO may be one mediator of

cytokine-induced hematopoietic suppression (Maciejewski et al., 1995).

Nitric oxide produced by mouse bone marrow-derived dendritic cells (DCs) in

response to GM-CSF plus IL-4 has been shown to regulate the allogeneic T cell

responses (Bonham et al., 1996). NO synthesis was induced in DC by IFN-y and LPS,

and was blocked by L-NMMA. Liu et al., (2007) investigated the developmental

expression of eNOS during stem cell differentiation into endothelial cells using mouse

adult multipotent progenitor cells (MAPCs). eNOS expression disappeared

immediately after induction of differentiation which reoccurred at day 7 during

differentiation and increased upto 14 and 21 days during differentiation at mRNA,

protein content, and activity level. Thus it was concluded that eNOS dynamically

expressed during the differentiation of MAPCs into endothelial cells. IL-17

upregulate the expression of mRNA for both iN OS and constitutive, eNOS isoforms

in murine bone marrow cells, as well as enhances the phosphorylation of p3 8 MAPK

(Jovcic et al., 2004)

Cell surface expression and mRNA of CXCR4 on CD34+ cells was reported to

be increased in a dose- and time-dependent manner in response to NO donors (Zhang

et al., 2007). SDFl and its receptor CXCR4, along with matrix metalloproteinases

(MMPs), regulate bone marrow stromal cell (BMSC) migration. Nitric oxide donor

mediated upregulation of SDFl)/receptor CXCR4 and MMP9 enhances bone marrow

stromal cell migration into ischemic brain after stroke (Cui et al., 2007). Bone marrow



hematopoietic cells were exhibited a very faint expression of eNOS. It is now known

that bone marrow stromal-cell-derived eNOS is a substantial component of the stem

cell niche and is essential for the mobilization of stem and progenitor cells in vivo

(Aicher et al., 2003). Stem cell mobilization is mediated by proteinases such as

elastase, cathepsin G and the matrix metalloproteinases (MMPs) (Lapidot and Petit,

2002). G-CSF, a cytokine that is typically used for the mobilization of CD34+ cells in

patients, releases the proteinases elastase and cathepsin G from neutrophils (Lapidot

and Petit, 2002). Rossner et al., (2005) have generated MSC as myeloid DC

precursors with GM-CSF having potent suppressive activity via cell contact and NO

production in vitro on allogeneic and OVA-specific CD4+ and CD8+ T cell responses

(Rossner et al., 2005).

Endothelial NO synthesis might also be reduced in the bone marrow of

patients with coronary artery disease or with heart failure, resulting in the reduced

mobilization of progenitor cells. Patients with coronary artery disease or diabetes

showed significantly lower levels of endothelial progenitor cells (EPCs) in the

circulation. Uncoupling of the eNOS has been shown to causes diabetic endothelial

dysfunction. eNOS regulates mobilization and function of EPCs, key regulators of

vascular repair. A role of eNOS uncoupling has been suggested for reduced number

and function ofEPC in diabetes (Thurn et al., 2007).

2.7.3) Nitric oxide and Hematopoiesis

Suppression of the NO synthases (NOS) activity directly or after irradiation and BM

transplantation, increase the number of stem and progenitor cells in the bone marrow

(BM). In the transplantation model, this increase is followed by a transient increase in

the number of neutrophils in the peripheral blood (Michurina et al., 2004) by yet

unknown mechanism. While NO donors in vitro have shown to markedly decrease the

generation of myeloid and erythroid colonies by CD34+ cells (Shami and Weinberg,

1996). Asthma exacerbates the number of circulating CD34+ progenitors expressing

high levels of iNOS with NO acting in a paracrine and autocrine manner to prevent

cell growth and colony formation but is not sufficient enough to prevent their

proliferation in circulation (Wang et al., 1999). Treatment with NOS inhibitor has

been found to increase the number of neutrophils in the circulation (Michurina et al.,

2004). In a study on monocytic cell line, NO has been demonstrated to block the cell

cycle in the early G2/M phase through ADP ribosylation of actin (Takagi et al., 1994).



In another study it has been shown that in the presence of NO in myocytes after

ischemia-reperfusion injury, there is nuclear accumulation of p27, an inhibitor of

CDK, which prevents apoptosis (Maejima et al., 2003).

Moreover, nNOS has been shown to regulate hematopoiesis in vitro and in

vivo. Strong correlation between expression of nNOS in a panel of stromal cell lines

established from bone marrow and fetal liver and the ability of these cell lines to

support hematopoietic stem cells; furthermore, NO donor can further increase this

ability (Krasnov et al., 2008). Moreover, Recently hematopoietic stem cell

development has shown to be dependent on blood flow, NO donors regulated HSC

number even when treatment occurred before the initiation of circulation and rescued

HSCs. Knockdown of nNOS/eNOS blocked HSC development (North et al., 2009).

Endogenous NO causes vasodilation in rat bone marrow, bone and spleen during

accelerated hematopoiesis (Iversen et al., 1994). Thus, NO seems to be a key

regulator of haematopoiesis in bone marrow by modulating HSC development.

Recently, Nakata et al (2008) have shown spontaneous myocardial infarction

m mice lacking all nitric oxide synthase isoforms associated with multiple

cardiovascular risk factors of metabolic origin. The triple n/i/eNOs<-l-) mice exhibited

markedly reduced survival with manifested phenotypes including visceral obesity,

hypertension, hypertriglyceridemia, and impaired glucose tolerance, demonstrating

the critical role of the endogenous NOS system in maintaining cardiovascular and

metabolic homeostasis (Nakata et al., 2008).

2.8) Neutrophils in Pathophysiology

Impaired neutrophil function can lead to deadly results due to the overwhelming

bacterial infections. Several congenital diseases with impaired neutrophil functions

are often fatal, which have been described in table-S.



Table 5: Disorders related to Neutrophils and their clinical manifestation

Disorder

Chronic Granulomatous Disease (CGD)

Neutrophil GlucosedPhosphate Dehydrogenase (G6PD) Deficiency

Myeloperoxidase (MPO) Deficiency

Leukocyte Adhesion Deficiency

Molecular/Genetic Defect

Inherited X-linked and autosomal recessive disorders. respiratory burst hampered 1:200000 Inactive hexose monophosphate shunt pathway, G6PD deficient

most common inherited disorder (1: 2000 persons)

Structurally abnormal or reduced amounts of the ~2 subunit. rare disorder of leukocyte adhesion and chemotaxis. inability of the leukocyte to bind C3bi opsonized microorganisms

Congenital Absence Specific Granules (SGD)

of Defective regulation of the synthesis of various lysosomal proteins confmed to the myeloid series

Chediak-Higashi Syndrome

C/EBPe transcription factor

rare autosomal recessive disease, Mutation in LYST, encoding a cytoplasmic protein involved in protein transport

2.9) NO & Cell Proliferation

Abnormalities/ manifestation

Clinical

recurrent infections, phagocytosis but not killing and digestion, severe chronic granulomas

CGD-like clinical complications, extremely rare

Not have senous bacterial infections, Candida infections are frequent. Severe bacterial infections. Phagocytosis, enzyme release and 02- production are severely affected.

Decreased chemotaxis, impaired 02- production and low bactericidal activity. deficiency in microbicidal molecules (lactoferrin, defensins recurrent bacterial and fungal infections presence of giant lysosomal granules by fusion of azurophilic and secondary granules, Low resistance to infection due to phagocyte abnormalities

NO has been demonstrated to have both pro- and anti-apoptotic role depending on a

variety of factors including the type of cells involved, redox state of the cell, and the

flux and dose ofNO (Gordge et al., 1998; Griscavage et al., 1995; Li and Wogan,

2005; Liu et al., 2003; Villalobo, 2006; Wang et al., 2007). Anti-apoptotic effects are

associated with low levels of exposure from the activation of endogenous NO

synthases and slow release rates from NO donors (Li and Wogan, 2005) Specific

molecular targets include inhibition of Bcl-2 cleavage (Kim et al., 1998), inactivation

of caspases by S-nitrosylation (Liu and Stamler, 1999; Mannick et al., 1999),

induction of p53 gene expression, upregulation of FLIP (Chanvorachote et al., 2005),

and over expression of Bcl-2 and Bcl-XL with subsequent inhibition of cytochrome c

release (Azad et al., 2006) and cGMP-mediated effects. However, exposure of cells to



high NO concentrations results in extensive inhibition of mitochondrial ATP synthesis

and cell death results (Brookes et al., 2000). Low concentrations ofNO can stimulate

cell growth and protect many cell types from apoptosis, whereas high concentrations

ofNO can inhibit cell growth and induce apoptosis depending on cell type and redox

state (Liu et al., 2003; Villalobo, 2006).

Takagi et al., (1994) have shown presence of IL-6 blocks the cell cycle

through nitric oxide of mouse macrophage-like cells in the early G2M phase (Takagi

et al., 1994). On the other hand, NO donors with a suitable concentration enhanced

proliferation in some cell lines such as U937 human leukemic cells (Jea et al., 1998),

pancreatic tumor cells (Hajri et al., 1998), and myoblasts (Ulibarri et al., 1999). A

earlier study using NO-donating agents, SNP showed that NO could suppress the

growth and induce the monocytic differentiation of a human leukemia cell line, HL-

60 (Magrinat et al., 1992). Recently, Wang et al reported that NO inhibits the

proliferation of HL-60 cells by inducing Go/G1 arrest and apoptosis in a dose- and

time-dependent manner through AKT pathway. NO induces Bid cleavage, Bad

phosphorylation and Bax expression but down-regulates the expression of Bcl-2 and

Bcl-xL. G0/G1 arrest was resulted from NO-induced up-regulation of p21(waf/cip1),

p27(kip1) and down-regulation of cyclin D1, cyclin E (Wang et al., 2007).

Since NO is an unstable molecule and it converts into more stable metabolites

nitrite and nitrates. The anion nitrite (N02 -) constitutes a biochemical reservoir for

NO. Nitrite biology has been revolutionized in last few years (Gladwin et al., 2005).

Nitrite reduction to NO may be catalyzed by hemoglobin, myoglobin or other metal

containing enzymes and occurs at increasing rates under conditions of hypoxia or

ischemia (Cosby et al., 2003; Dezfulian et al., 2007; Gladwin et al., 2005; Huang et

al., 2005). The most common use of nitrate salts as antidote against cyanide poisoning

and to cure foods from ancient time, which not only imparts a pleasing colour to

meats but also is a very effective agent against the bacterium that causes botulism

(Butler and Feelisch, 2008; Gladwin et al., 2005). With the discovery of mammalian

NO synthase enzymes in the late 1980s, nitrite was largely considered to be only an

end product ofNO metabolism (Knowles and Moncada, 1994). In the past few years,

evidence has been mounting that nitrite may have important physiological and patho

physiological functions (Butler and Feelisch, 2008; Gladwin et al., 2005; Lundberg et

al., 2008). Bryan et al. recently reported that nitrite is a signaling molecule in its own

right, even under physiological conditions (that is, in the absence of ischemia).



Specifically, they demonstrated that nitrite increases cGMP formation, inhibits

cytochrome P450 activity and affects the expression of two archetypical proteins, heat

shock protein 70 and heme oxygenase-1 (Bryan et al., 2005). Multiple groups hav

reported that low doses of nitrite prevent ischemia reperfusion cellular infarction in

the Langendorf heart preparation (Webb et al., 2004), in the mouse liver and heart

(Duranski et al., 2005). Nitrite therapy significantly increased ischemic limb vascular

density and stimulated endothelial cell proliferation. (Kumar et al., 2008). Thus nitrite

has therapeutics potential for a number of pathology.


09_review of literature

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