1. NEUTROPHILSNEUTROPHILS
Dr. P. ConcepcionDr. P. Concepcion
Fellow-in-Training, Allergy & ImmunologyFellow-in-Training, Allergy & Immunology
Philippine General HospitalPhilippine General Hospital
April 25, 2014April 25, 2014
2. Sources:Sources:
Adkinson et’al; Middleton’s Allergy Principle andAdkinson et’al; Middleton’s Allergy Principle and
Practice; 8Practice; 8thth
ed; Vol 1; Elsevier Saunders; 2014ed; Vol 1; Elsevier Saunders; 2014
Abbas et’al; Cellular and Molecular Immunology; 7Abbas et’al; Cellular and Molecular Immunology; 7thth
ed;ed;
Elsevier Saunders; 2012Elsevier Saunders; 2012
Journals:Journals:
--Bruce K. et al:Bruce K. et al: Immunomodulatory Activity and Effectiveness ofImmunomodulatory Activity and Effectiveness of
Macrolides in Chronic Airway DiseaseMacrolides in Chronic Airway Disease
-- Soichiro et’al:Soichiro et’al: Mechanisms of Action and Clinical Application ofMechanisms of Action and Clinical Application of
Macrolides as Immunomodulatory Medications,Macrolides as Immunomodulatory Medications, Clin. Microbiol. Rev.Clin. Microbiol. Rev. 2010,2010,
Journal ASM. OrgJournal ASM. Org
-- Evangelos, J.,Evangelos, J., Macrolides beyond the conventional antimicrobials:Macrolides beyond the conventional antimicrobials:
a class of potent immunomodulators,a class of potent immunomodulators, International Journal of AntimicrobialInternational Journal of Antimicrobial
Agents 31 (2008) 12–20Agents 31 (2008) 12–20
-- Tauber, S.,Tauber, S., Immunomodulatory Properties of Antibiotics,Immunomodulatory Properties of Antibiotics, CurrentCurrent
Molecular Pharmacology, 2008, 1, Bentham Science Publishers Ltd.Molecular Pharmacology, 2008, 1, Bentham Science Publishers Ltd.
3. Presentation Outline:Presentation Outline:
Introduction of Neutrophils’ BiologyIntroduction of Neutrophils’ Biology
The Birth & the MorphologyThe Birth & the Morphology
Discuss each step of its KeyDiscuss each step of its Key
Role in innate immuneRole in innate immune defensesdefenses
Neutrophil Clearance and DeathNeutrophil Clearance and Death
Neutrophil-Associated DiseasesNeutrophil-Associated Diseases
Discuss some Immunomodulators in contextDiscuss some Immunomodulators in context
5. Important role in inflammatoryImportant role in inflammatory
responsesresponses
Comprise 50–75% of circulatingComprise 50–75% of circulating
leukocytes in humansleukocytes in humans
First circulating cells to migrate to theFirst circulating cells to migrate to the
site of infectionsite of infection
Uncontrolled activation causes tissueUncontrolled activation causes tissue
damage--- contribute to the pathogenesis ofdamage--- contribute to the pathogenesis of
chronic inflammation (eg sinuses andchronic inflammation (eg sinuses and
respiratory tract)respiratory tract)
Introduction:Introduction:
6. • TheThe neutrophilsneutrophils are specializedare specialized
for the phagocytosis andfor the phagocytosis and
destruction of micro-destruction of micro-
organisms and damaged ororganisms and damaged or
necrotic tissues.necrotic tissues.
Introduction:Introduction:
7. 6 Subtypes:6 Subtypes:
1. myeloblast1. myeloblast
2. promyelocyte----- primary (azurophilic) granules2. promyelocyte----- primary (azurophilic) granules
3. myelocytes---- secondary (specific) granules3. myelocytes---- secondary (specific) granules
4. metamyelocytes--- tertiary (gelatinase) granules4. metamyelocytes--- tertiary (gelatinase) granules
5. bands cells5. bands cells
6. mature neutrophils6. mature neutrophils
• Continuously generated from the BM (1-2 x
1011
cells/day --- amplified in times of stress
(eg infection)
• Neutrophil maturation in the bone marrow
takes approximately 10–15 days,
8.
9. Neutrophil – electron micrographNeutrophil – electron micrograph
Peter Newburger, MD
University of Massachusetts Medical School
10. A.A. An abundance ofAn abundance of
granulesgranules
B.B. Multi-lobed nucleusMulti-lobed nucleus
C.C. ProminentProminent
cytoskeleton forcytoskeleton for
locomotion andlocomotion and
chemotactic functionschemotactic functions
1. microfilaments1. microfilaments
2. microtubules2. microtubules
3. intermediate3. intermediate
filamentsfilaments
Morphology:Morphology:
11.
12.
13. CirculationCirculation
Most abundant WBC in the bloodMost abundant WBC in the blood
The half life = 4-10 hoursThe half life = 4-10 hours11 (ave: 6hours(ave: 6hours2)2)
Can migrate to the site of infectionCan migrate to the site of infection
If not--- it undergoes Apoptosis andIf not--- it undergoes Apoptosis and
phagocytosed by resident macrophagesphagocytosed by resident macrophages
1. Adkinson et’al; Middleton’s Allergy Principle and Practice; 8th
ed; Vol 1; 2014
2. Abbas et’al; Cellular and Molecular Immunology; 7th
ed;; 2012
14. CirculationCirculation
Peripheral blood neutrophils arePeripheral blood neutrophils are
divided between;divided between;
A Circulating pool-A Circulating pool- present in large andpresent in large and
small blood vesselssmall blood vessels
A Marginating pool-A Marginating pool- that is arrested inthat is arrested in
capillaries.capillaries.
Margination - regulated by selectin-Margination - regulated by selectin-
mediated capture from themediated capture from the
bloodstream.bloodstream.
15. RollingRolling
Rolling adhesion of neutrophils to theRolling adhesion of neutrophils to the
endothelium is mediated byendothelium is mediated by L-selectinL-selectin
on the neutrophil andon the neutrophil and P- and E-selectinP- and E-selectin
on the endothelium.on the endothelium.
16. RollingRolling
Rolling allows interaction between CXCRolling allows interaction between CXC
chemokines such as:chemokines such as:
IL-8
(Endothelial Cell’s surface)
β2 integrin expressions
17. AdhesionAdhesion
P, L-selectins &
CD44
(Neutrophils)
P, E-selectins
(Endothelial Cells)
Firm adhesion to
the endothelium
LFA-1: Leukocyte function-ass’d Ag; Mac-1: Macrophage 1-Ag
The βThe β22 integrin’s 4 differentintegrin’s 4 different
heterodimers:heterodimers:
1. CD11a/CD18 or LFA-11. CD11a/CD18 or LFA-1
2. CD11b/CD18 or Mac-1;2. CD11b/CD18 or Mac-1;
3. CD11c/CD18 or p150,953. CD11c/CD18 or p150,95
4. CD11d/CD18.4. CD11d/CD18.
18. DiapedesisDiapedesis
Transmigration of Neutrophils from theTransmigration of Neutrophils from the
intravascular compartment to the site ofintravascular compartment to the site of
infection by deformation and elongation.infection by deformation and elongation.
20. • Transendothelial migration of neutrophils
Diapedesis:Diapedesis: Endothelial cell interactions
Transcellular
(directly through endothelial
cells)
Transmigratory cups high
ICAM-1
VCAM-1
21. Diapedesis:Diapedesis: Epithelial cell interactions
This process involves three stages:This process involves three stages:
Epithelial adhesionEpithelial adhesion
MigrationMigration
Post-MigrationPost-Migration
22. Cross Section of Blood Vessels
MARGINATION
Epithelial adhesionEpithelial adhesion
25. ChemotaxisChemotaxis
Once through the endothelial basementOnce through the endothelial basement
membrane, neutrophils migrate along amembrane, neutrophils migrate along a
chemotactic gradient.chemotactic gradient.
Neutrophil chemotactic proteins includeNeutrophil chemotactic proteins include
Chemokines (e.g., IL-8)Chemokines (e.g., IL-8)
Complement split products (e.g., C5a)Complement split products (e.g., C5a)
Bacterial products (e.g., N-formyl methionylBacterial products (e.g., N-formyl methionyl
peptides),peptides),
Lipid mediators (e.g., LTBLipid mediators (e.g., LTB44))
26. • IL-8-IL-8- produced by macrophages, epithelialproduced by macrophages, epithelial
cells and neutrophils.cells and neutrophils.
• IL-8 is a very strong chemoattractant forIL-8 is a very strong chemoattractant for
neutrophils and T-lymphocytes.neutrophils and T-lymphocytes.
Chemotaxis: Chemokine (Endogenous
Factor)
28. • C5aC5a (C3a, C4a)(C3a, C4a) act on specific receptors toact on specific receptors to
produce similar local inflammatory responsesproduce similar local inflammatory responses
(anaphylatoxins).(anaphylatoxins).
• All three induce smooth muscle contractionAll three induce smooth muscle contraction
and increase vascular permeability.and increase vascular permeability.
Chemotaxis: Chemokine (Endogenous
Factor)
29. • C5aC5a also acts directly on neutrophilsalso acts directly on neutrophils
to increase their adherence to vesselto increase their adherence to vessel
walls, their migration toward sites ofwalls, their migration toward sites of
antigen deposition, and their ability toantigen deposition, and their ability to
ingest particles.ingest particles.
Chemotaxis: Chemokine (Endogenous
Factor)
34. Degranulation:Degranulation:
Neutrophil enzymesNeutrophil enzymes
• Azurophilic or PrimaryAzurophilic or Primary
• These are the first granules formed in theThese are the first granules formed in the
developing neutrophil (Promyelocyte)developing neutrophil (Promyelocyte)
• Specific or SecondarySpecific or Secondary
• These granules are formed later in theThese granules are formed later in the
development of the neutrophil (myelocyte)development of the neutrophil (myelocyte)
39. Neutrophil enzymesNeutrophil enzymes
• Myeloperoxidase (MPO)Myeloperoxidase (MPO):: is an abundantis an abundant
granular enzyme (accounts for 5% of drygranular enzyme (accounts for 5% of dry
weight of the neutrophil).weight of the neutrophil).
• This enzyme combines hydrogenThis enzyme combines hydrogen
peroxide with chloride ions to formperoxide with chloride ions to form
hypochlorous acid (HOCl = bleach).hypochlorous acid (HOCl = bleach).
41. Neutrophil enzymesNeutrophil enzymes
• LysozymeLysozyme:: like MPO, is a microbicidallike MPO, is a microbicidal
enzyme.enzyme.
• LysozymeLysozyme digests debris from cell wallsdigests debris from cell walls
of bacteria that have already beenof bacteria that have already been
processed by other enzymes.processed by other enzymes.
• Another function ofAnother function of lysozyme is tois to
modulate inflammation bymodulate inflammation by suppressingsuppressing
neutrophil chemotaxis and oxidativeneutrophil chemotaxis and oxidative
metabolism.metabolism.
42. Within 30 seconds after a
neutrophil ingests a
particle, it begins to
secrete specific granule
components into the
phagosome via
phagolysosomal fusion.
Within 3 minutes,
azurophil granule
components are
discharged into the
phagolysosome.
45. NETosisNETosis
Neutrophil Extracellular Traps (NET)Neutrophil Extracellular Traps (NET)
Composed of DNA and histonesComposed of DNA and histones
Antimicrobial proteins from its granulesAntimicrobial proteins from its granules
- MyeloperoxidesMyeloperoxides
- ElastaseElastase
- DefensinsDefensins
- Protienase 3Protienase 3
- Cathepsin GCathepsin G
- CalprotectinCalprotectin
46. NETosisNETosis
Distinctive form of cell deathDistinctive form of cell death
- Disintegration of the nuclear envelopeDisintegration of the nuclear envelope
- Mixing of the NET components and granulesMixing of the NET components and granules
contentscontents
- Occurs when cell membrane ruptures andOccurs when cell membrane ruptures and
cell dies.cell dies.
- Upto 4 hours after activationUpto 4 hours after activation
51. PULMONARY TRACTPULMONARY TRACT
Marginating pool
(20-60x higher)
Diapedesis
Travel tru the
Pulmonary capillary
Increase transit TIME
Increase concentration
of Neutrophils
Exposure to:
Inhalants
Cigarette
infections
Decrease transit TIME
(Bone Morrow)
Release of Immature
Neutrophils to the Blood
Vast network of
Capillary beds
Smaller vessel
Diameter
52.
53. NeutrophilicNeutrophilic
AsthmaAsthma
• Mucus hypersecretion
• Impaired efferocytosis
• Bacterial persistence
- Symptomatic asthma andSymptomatic asthma and
airway hyper-airway hyper-
responsiveness in theresponsiveness in the
presence of a neutrophilicpresence of a neutrophilic
bronchitis, with sputumbronchitis, with sputum
neutrophil counts > 61%.neutrophil counts > 61%.
- 10-30% of cases of stable10-30% of cases of stable
asthma in adultsasthma in adults
62. Summary:Summary:
Discussed the neutrophils’ biologyDiscussed the neutrophils’ biology
The BirthThe Birth
The MorphologyThe Morphology
Discussed each step of its Key Role in innateDiscussed each step of its Key Role in innate
immune defenses (Phagocytosis, ROI,immune defenses (Phagocytosis, ROI,
NETosis)NETosis)
Neutrophil-Associated Diseases (N. Asthma)Neutrophil-Associated Diseases (N. Asthma)
Discuss some Immunomodulators ofDiscuss some Immunomodulators of
neutrophils functions (macrolide)neutrophils functions (macrolide)
Editor's Notes
Much of the information was taken from Middleton & others from Abbas and few Journals
The outline of this presentation starts from introduction
Followed by the production and maturation… their unique structures for us to differentiate them from other leukocytes..
Series of sequential key steps for innate immune defenses
Other names used are 1. Granulocytes; 2. PMNs; 3. PMNLs; 4. Poly’s; 5. Segmenters
Neutrophils play an important role in inflammatory responses that are critical for host defense against infection.
They are most abundant population of WBC and
The first ones that mediate to the site of infection
Responsible to the earliest phases of inflammatory reaction. (show Table)
> However if the activation in uncontrolled, it would contribute to the pathogenesis of chronic inflammatory conditions that involve the sinuses and respiratory tract, and
In short, neutrophils are specialized phagocytic cells that destruct and kill micro-organisms and
The birth of Neutrophils are generated continuously from hematopoietic stem cells of the bone marrow, and this process is called myelopoiesis.
Developing neutrophils can be divided into six subtypes, including the myeloblast, the promyelocyte where primary (azurophilic) granules appear, the myelocyte where cell division ceases and secondary (specific) granules appear, the metamyelocyte where tertiary (gelatinase) granules appear, followed by band cells and finally mature neutrophils, characterized by their multilobed nucleus and cytoplasm containing granules.[1]
Multi lobed nucleus, and granule-abundant cytoplasm…
Its parts can be identified using this diagram…. Like….. (mention few of the parts)
Electron micrograph; smear; scanning EM
> Difference between the resting and the IL-8 stimulated neutrophil
Paracellular (between endothelial cells)
- Requires the tight junction between endothelial cells to loosen (mediated by PECAM & JAMs- express at intercellular tight juction of endothelial and epithelial cells, along with LFA-1 and Mac-1 on the neutrophils)
Transcellular (directly through endothelial cells)
Mediated by transmigratory cups high in ICAM-1 and VCAM-1--- bind crawling neutrophils and allow to make way thru the cells
Paracellular (between endothelial cells)
- Requires the tight junction between endothelial cells to loosen (mediated by PECAM & JAMs- express at intercellular tight juction of endothelial and epithelial cells, along with LFA-1 and Mac-1 on the neutrophils)
Transcellular (directly through endothelial cells)
Mediated by transmigratory cups high in ICAM-1 and VCAM-1--- bind crawling neutrophils and allow to make way thru the cells
The process of neutrophil migration can be divided into 3 stages
C5a activation
But also remember the role C3b, C4b in the opsonization and phagocytosis later in this presentation
Elastase: is a serine protease which specifically hydrolyzes elastin.
Elastin is the major component of elastic fibers which stretch in the walls of blood vessels, lungs, and ligaments.
The activity of elastase is controlled by an inhibitor termed 1-anti-trypsin.
Collagenase: cleaves collagen into two distinct and specific peptide fragments
Collagenase is released by intact neutrophils during phagocytosis as a collagenase precursor (procollagenase) and is activated by trypsin, hypochlorous acid or rheumatoid synovial fluid.
Azurophil granules: peak degranulation is 90 minutes.
Specific granules: These enzymes are released within 15 seconds after contact with the pathogen.
Aqui, o NADPH transfere, via FAD e grupo heme, os 2 elétrons para o oxigênio molecular, iniciando a produção dos reativos intermediários do oxigênio.
HOCl- hypochlorous acid- toxic oxygen radicals
The pulmonary capillary bed is the main site containing marginating neutrophils and measuring 20–60 times that of the concentration of large systemic blood vessels.
Most neutrophils have to deform and elongate to travel through the pulmonary capillaries due to the vast network of the capillary bed, and the vessels being of a smaller diameter in comparison to spheric neutrophils.
The requirement of neutrophils to deform to travel through the pulmonary capillaries increases their transit time, resulting in a higher concentration of neutrophils in this space. Immature neutrophils can be released prematurely into the circulation in times of infection or inflammation, and these cells preferentially sequester into the lung microvessels.[3] Exposure to inhalants, such as cigarette smoke, can decrease the transit time of neutrophils through the bone marrow, and cause the release of immature neutrophils into the bloodstream.[4] Contact with cytokines (e.g., G-CSF, GM-CSF, IL-1) and chemokines (e.g., IL-8) can influence this process through the release of proteases (e.g., MMP-9) and the shedding of L-selectin.[5] Once released into the bloodstream, neutrophils have a half-life of 4–10 h, and can migrate into the tissues.
Mucus hypersecretion; impaired efferocytosis; bacterial persistence…
Neutrophils are commonly found in the airway lumen in healthy people and are in increased numbers of subgroups with stable asthma (neutrophilic asthma), during exacerbations and also in cases of fatal asthma. The precise role of neutrophils in the pathogenesis of asthma remains unclear.
Recruitment of neutrophils to the airways is orchestrated, in part, by the potent chemokine IL-8 (CXCL8). Epithelial cells release IL-8 on exposure, to a variety of stimuli, to promote their movement to the airways. Neutrophils themselves are a source of IL-8 and, in this way, may influence their own activation and sequestration from the circulation. Once activated, neutrophils release a variety of proinflammatory proteins, which induce further inflammatory processes and have the potential to induce remodeling of the airways. Proteolytic enzymes also influence the chemotaxis and activation of neutrophils. NE can induce IL-8 production and MMP-9 can enhance IL-8 potency by augmenting amino terminal processing of IL-8.[38]
The role of neutrophils in asthma is unclear, but increases in neutrophils have been reported in severe asthma requiring intubation,[39] sudden-onset fatal asthma, and life-threatening asthma, [40] [41] suggesting a role for these cells in the most severe forms of disease. Neutrophils are increased in a number of airway compartments, including the lumen[42] and submucosa.[43] Neutrophil numbers increase with increasing severity of asthma.[44] Neutrophil numbers and products are increased during exacerbations in both adults[45] and children with asthma [46] [47] and have been associated with damaged epithelium.[48] Neutrophilic inflammation is a feature of respiratory viral infections in both healthy subjects and those with asthma.[49] While generally overlooked in less severe forms of asthma, increased neutrophils are also present in milder forms of asthma. More recently there have been reports of subgroups of patients with stable asthma who have a persistent neutrophilic bronchitis. [50] [51] [52] Subjects with neutrophilic asthma have a number of unique inflammatory abnormalities to distinguish them from typical eosinophilic asthma (where sputum eosinophils are outside the normal range) and
Matrix metalloproteases-9;
These small, arginine-rich peptides play an important role in host defense to infections.
Four defensins that are present in neutrophils are the human neutrophil peptides (HNP-1 to HNP-4).
These peptides kill pathogens by causing permeabilization of the bacterial membrane.
Mature defensins are present in high concentration in the azurophilic granules (5–7% of the neutrophil's total protein).
>Defensins also modulate the inflammatory response, as they can bind to protease inhibitors such as α1-antitrypsin.
Schematic representation of the anti-inflammatory mode of action of macrolides. –, inhibition; +, stimulation; AP-1, activator protein-1; NF-B, nuclear
factor-B; TNF, tumour necrosis factor-alpha; IL-8, interleukin-8.
Eventually leads to decrease, if not inhibit, the neutrophil activation
Intracellular signal transduction pathways that have been proposed to be involved in macrolide immunomodulation. There are three major pathways that are influenced by macrolides.
1. Receptor tyrosine kinases (RTKs) are receptors for many polypeptide growth factors and cytokines.
2. TLRs recognize bacterial molecules. For example, in response to LPS stimulation, TLR4 and adaptor molecules (not shown) activate the IRAK family and TAK1. TAK1 then stimulates two distinct pathways, the IKK complex and the MAPK pathway. The latter leads to the induction of AP-1, while the former activates NF-B through the degradation of IB proteins and the subsequent translocation of NF-B (194).
3. G-protein-coupled receptor (GPCR)- or RTK-mediated activation of phospholipase C (PLC) produces inositol triphosphate (IP3). IP3 is a ligand for the intracellular IP3R channel of the endoplasmic reticulum’s internal Ca2 stores. Activation of PLC also leads to the production of diacylglycerol (DAG), which in turn activates protein kinase C (PKC). PKC and Ca2/calmodulin signaling are then activated (44). Macrolides inhibit intracellular Ca2 increase. Abbreviations:
FIG. 2. Beneficial effects of macrolides in the inflamed airway. In a chronically inflamed airway, there is epithelial cell damage, infiltration of
inflammatory cells, goblet cell hyperplasia, hypersecretion, mucociliary dysfunction, and recurrent airway infection. Macrolides have been reported to
attenuate inflammation and cellular damage in a variety of ways, as represented in this diagram. Downward-facing arrows, inhibition; upward-facing
arrows, enhancement.
