CHAPTER 12: Leukocyte Development o Proliferation (mitotic) pool: consists of cells

that are dividing: Common myeloid

Kinetics, and Functions progenitors (CMP): CFU-granulocytes,
erythrocytes, monocytes and
Leukocytes
megakaryocytes or CFU-GEMMs ;
 Aka WBC, so named because of being colorless granulocyte- macrophage progenitors
compared to RBC (GMPs); myeloblasts, promyelocytes and
 Different types vary depending on: myelocytes
o Being viewed after staining with a  Listed in order of maturation
Romanowksy stain (5-6 types) o Maturation pool : cells undergoing nuclear
o Identified according to their surface antigens maturation for marrow reserve available for
using flow cytometry (atleast 10 different release
types)  Metamyelocytes, band neutrophils
and segmented neutrophils
 Granulocytes : cytoplasm filled with granules whose
nuclei are segmented or lobulated  HSCs, CMPs and GMPs are not distinguishable with
o Eosinophils: basic protein that stain with Romanowsky stain
o May resemble type 1 myeloblast or
acid stain such as eosin
o Basophils: acidic and stain with basic stain lymphoid cells
o Identified thourgh surface antigen detection
such as methylene blue
o Neutrophils: granules react with both acid by flow cytometry
and basic stains  Myeloblasts: 0% -3% of nucleated cells in BM and
 Gives them a pink to lavender color subdivided into 3 types ; 2 & 3: “granular blasts”
 Called PMN’s due to prominent
nuclear segmentation
 Mononuclear cells: monocytes and lymphocytes
o Not segmented, round, oval, indented or
folded
 Leukocytes develop from hematopoietic stem cells
(HSCs) in the bone marrow
o Most undergo differentiation and maturation
and then released into circulation o Type 1: high nucleus to cytoplasm ratio of
o Varies with sex, age, activity, time of day, 8:1 – 4:1
ethnicity, stress, bone marrow production  slightly basophilic cytoplasm with
 4.5 x 10^9 / L to 11.5 x 10^9 / L for adults fine nuclear chromatin and 2-4
visible nucleoli
 Overall function: Immunity
 No visible granules with
o Innate(nonspecific) : phagocytosis by
Romanowsky stain
neutrophils
o Type 2: presence of azurophilic granules in
o Specific(adaptive) : antibody prod’n by
cytoplasm ; doesnt exceed 20 granules / cell
lymphocytes and plasma cells
o Type 3: darker chromatin and a more purple
 Kinetics: movement of cells through developmental
cytoplasm
stages, into the circulation to the tissues and incudes
 more than 20 granules / cell that
time spent in each phase of cell’s life.
doesn’t obscure the nucleus
Granulocytes  rare in normal BM but seen in
types of acute myeloid leukemia
Neutrophils  Promyelocyte : 1% - 5% of nucleated cells in BM
 Segmented (vast majority) or band shape
 Vast majority of leukocytes
Neutrophil Development

 Share a common progenitor with monocytes known

as the granulocyte-monocyte progenitor (GMP)
 Granulocyte colony-stimulating factor (G-CSF)
o Major cytokine responsible for the o Larger than myeloblast
stimulation of neutrophil production o Nucleus is round to oval and eccentric
 3 pools/ stages of developing neutrophil in BM o Paranuclear halo or “hof” is seen in normal
o Stem cell pool: consists of HSC capable of but not in malignant promyelocytes of acute
self renewal and differentiation promyelocytic leukemia
 o Basophilic and azurophilic granules in o Nuclear indentation than began in
cytoplasm metamyelocyte stage exceeds one half the
 1st in the series of granules to be diameter of the nucleus but without segment
produced during neutrophil o Elevated band count = infection
maturation  Still in question
o Nucleus is similar to myeloblasts except for  Segmented neutrophils : 7% - 30%
chromatin clumping (heterechromatin) o Secretory granules continue to form
o 1-3 nucleoli but maybe obscured by granules o Presence of between 205 nuclear lobes
 Neutrophil myelocytes: 6% - 17% connected by threadlike filaments
 Morphologic diff on bands
o Highest number in PBS (50%-70%)
 2.3 to 8.1 x 10^9 / L (abs terms)
 4-7 yrs and above
Neutrophil Kinetics

 Neutrophil kinetics: movement of neutrophils and

precursors between different pools in BM, peripheral
o Final stage of mitosis blood and tissues
o Production of primary granules ceases, o 0.9 - 1.0 X 10^9 cells / kg per day
begins to manufacture secondary neutrophil o Proliferative pool: contains 2.1 x 10^9
granules cells/kg
o Subdivided into early and late myelocytes: o Maturation pool: contains 5.6 x 10^9
 Early myelocytes: similar to cells/kg or a 5 day supply
promylocytes but with grainy pale o Transmit time of myeloblast-myelocyte
pink cytoplasm in Golgi apparatus  6 days
= dawn of neutrophilia o Transmit time to maturation pool
 Spreads slowly on cell  4-6 days
until it appears lavender o G-CSF: Granulocyte release form BM
pink than blue  In peripheral blood, neutrophils are divided into
 Late myelocytes : smaller than circulating and marginated neutrophil pool
promyelocytes ; nucleus is more o MNP: localized in walls of capillaries
heterochromatin o Ratio of two pools are roughly equal
 Neutrophil metamyelocytes : 3%-20% however MNP in capillaries of lungs has a
larger proportion of peripheral neutrophils
o Halflife of neutrophil: 7 hours
 Integrins and selectins: allows neutrophil to
marginate and exit the blood and enter tissues
o Diapedesis
o Neutrophils that doesn’t migrate into tissues
will undergo apoptosis and be removed by
macrophages in the spleen
o Cells are no longer capable of division  Inside the tissue, If there is absence of infection,
o Morphologic change : shape of nucleus neutrophil’s life span is measured in hours
 Kidney bean or peanut shaped o Infection prolong the neutrophil’s lifespan
 With clumped chromatin through anti-apoptopic signals
o Absent nucleoli o MAC-1 : triggers apoptosis in neutrophils
o Synthesis of tertiary or gelatinase granules
may begin Neutrophil function
o Slighthly smaller than myelocyte
 Major function of neutrophil : phagocytosis and
o Cytoplasm contains very little RNA and destruction of foreign materials or microorganisms
therefore little or no basiphilia o Process involves seeking (chemotaxis,
 Neutrophil bands : 9%- 32% motility and diapdesis) and destruction
o 0-5% on peripheral blood smear (phagocytosis and digestion)
o Evidence of RNA(cytoplasmic basophilia) is  Begins when chemotactic agents bind to
absent ; tertiary granules are formed neutrophil receptors (maybe produced by
o Secretory granules or vesicles may begin to microorganisms, damaged cells, etc)
form  1st neutrophil response: roll along
o Highly clumped nucleus endothelial cells of blood vessels using
 stronger adhesive molecules than those of  Has enzymes from neutrophil granules and
nonstimulated marginated neutrophils is able to trap and kill gram + and – bacteria
 Consists of transient contacts and fungi.
between neutrophil selectins and  NETs are generated at the time neutrophils
adhesive molecules on surface of die as a result in antibacterial activity.
endothelial cells  NETosis
 Secretory granules containing  Third function of neutrophils is their secretory
additional adhesive are fused to the function : source of transcobalamin I or R binder
neutrophil’s membrane protein, necessary for proper absorption of Vit B12
 CD11b/CD18 from the granules  Also a source of cytokines
contribute to stationary binding
between neutrophils and Eosinophils
endothelial cells
 It is then followed by diapdesis or  1% - 3% of nucleated cells in BM
transmigration of neutrophils between or o Slightly more than a third are mature
thorugh endothelial cells o Quarter are eosinophilic metamyelocytes
 Process mediated by integrins and o Remainder are eosinophilic promyelocytes
integrin assoc. proteins or myelocytes
 Tertiary granules containing gelatinase and  0.4 x 10^9 / L in peripheral blood (1-3%)
collagenase are released by tranmigrating
Eosinophil development
neutrophils
 Gelatinase degrades denatured  Similar to neutrophils, arised from CMP
collagen and aciavtes chemokines  Established through interaction between cytokines
and interleukin-8 IL-3, IL-5 and GM-CSF and three transcription
 Neutrophils migrate in a directional manner factors (GATA-1, PU.1, and C/EBP)
toward greatest concentration of chemotactic o IL-5: critical for eosinophilic growth and
agents survival
 Once at the site of infection, neutrophils  Eosinophilic promyelocyte: identified cytochemically
begin phagocytosis by utilizing enormous o Due to presence of Charcot-Leyden crystals
inventory of surface receptors in the primary granules
 With recognition, attachment and  Early myelocyte: 1st maturation phase identified as
engulfment of pseudopodia on the particle eosinophilic under Romanowsky staining
forms a phagosome within the neutrophil
 Eosinophil myelocytes
cytoplasm.
 Allows reduced NADH oxidase
complex to assemble which leads
to reactive oxygen species such as
hydrogen peroxide
 Hydrogen peroxide is converted to
hypochlorite by myeloperoxidase
 After a series of metabolic changes is the
release of bactericidal molecules into the
phagosome o Presence of large, pale, reddish-orange
 Combination of reactive oxygen secondary granules along with azure
species and non oxygen dependent granules in blue cytoplasm
mechanisms is generally able to o Nucleus is similar to neutrophil myelocytes
destroy most pathogens o Electron dense crystalline core: detected on
 Following emptying their contents into transmission electron micrographs on
phagosomes, secondary and primary eosinophilic granules
granules may fuse to the plasma membrane  Eosinophilic metamyelocytes
which results to the release of contents into
the extracellular matrix
 Acts as chemotactic agents to
phagocytize dead neutrophils as
well as inflammatory agents
 Second function of the generation of neutrophil
extracellular traps or NETs
 Represent chains of nucleosomes form
unfolded nuclear chromatin material (DNA)
o Resemble neutrophil counterpart
 o Secondary granules increase in number, and o Differentiated by cytokines including IL-3
3rd type called secretory granule is generated  Due to small numbers, they are difficult to observe
o Lipid bodies and small granules: detecred on and characterize
electron microscope o Described simply as immature and mature
 Mature eosinophils  Immature: round to lobulated nuclei with slightly
o Bilobed nucleus condensed chromatin
o Refractile orange-red secondary granules o Blue cytoplasm with blue-black granules
o Extensive secretory vesicles/granules o Granules are water solube therefore may be
 Number is increased when dissolved If bloodfilm is washed too much
eosinophil is stimulated  Mature: lobulated nucleus often obscured by granules
o Chromatin pattern: if visible ; clumped
Eosinophilic Kinetics o Rarely occurring nuclear segmentation
 Last myelocyte mitotic divison to mature eosinophils o Colorless cytoplasm and contains large
is about 3.5 days number of large blue-black granules
 Mean turnover 2.2 x 10^8 cells/kg/day o If dissolved, they often leave a reddish
 Large storage pool on BM: 9-14 x 10^8 cells/kg/day purple rim surrounding what appears to be a
 Circulating half life is 18 hours vacuole
o Prolonged when eosinophilia occurs Basophilic Kinetics
o Tissue destination is columnar epithelial
surfaces in the respiratory, gastrointestinal  poorly understood due to small number
and genitourinary tracts.  according to recent study, life span of mature
 Survival time: 2-5 days basophil is 60 hours
o relatively longer than of the other
Eosinophilic functions granulocytes
 Classical exocytosis: granules move to the plasma o due to activation by cytokine IL-3
membrane, fuse with the plasma membrane, and  anti-apoptopic pathways are
empty their contents into the extracellular space. initiated that causes prolonged
 Compound exocytosis: second mechanism in which lifespan
granules fuse together within the eosinophil prior to Basophilic functions
fusing with the plasma membrane
 Piecemeal degranulation: secretory vesicles remove  in the past, they are regarded as poor relatives by
specific proteins from the secondary granules. These mast cells and minor players in allergic inflammation
vesicles then migrate to the plasma membrane and o due to IgE receptors found in their surface
fuse to empty the specific proteins into the membrane, when cross-linked by antigen,
extracellular space results in granules release
 Deletion of double positive thymocytes  capable of releasing large quantities of subtype 2
o Migration to thymus in newborns helper T cell cytokines such as IL-4 and IL-3 that
 Promotion of proliferation of effector T cells regulate subtype 2 helper T cell
 Initiator of either type 1 or 2 response due to ability  also induce B cells to synthesize IgE
to secrete performed cytokines in stimulus-specific  Basophilic activation is not restricted to antigen-
manner specific IgE cross linking, but can also be triggered in
 Regulate mast cell function through major basic nonsensitized growing list of parasitic antigens,
protein that causes mast cell degranulation and lectins, viral superantigens binding to nonspecific IgE
cytokine production antibodies
 Increased in parasitic infections o Mast cells: effectors of IgE mediated
o Prevention of retroinfection chronic allergic inflammation
 Capable of destroying helminths through major basic  Mature basophils are capable of synthesizing granule
protein and production of reactive oxygen species protein based on activation signals
 Anti IL-5 monoclonal antibody: given to asthmatic o Basophils can be induced to produce a
patients that manifests airway inflammation due to mediator of allergic inflammation known as
eosinophilia granzyme B
o Mas cells: induce basophils to produce
Basophils retinoic acid
 A reulator of immune and resident
 0-2% and less than 1% of nucleated cells in BM
cells in allergic diseases
 True leukocytes because they mature in the BM
 Play a role in angiogenesis through expression of
Basophilic development vascular endothelial growth factor and its receptors
 Promotes eosinophilia on helminth infection and
 Derived from the progenitors of the BM promotes efficient worm expulsion
Mast cells o Based on flow cytometry
immunophenotyping
 not considered as leukocytes  Promonocyte pool : 6 x 10^8 cells/kg, and procudes 7
 tissue effector cells of allergic response and x 106 monocytes/kg per hour.
inflammatory reactions o Under normal circumstance, promonocytes
 precursors circulate in peripheral blood for a brief undergo two mitotic division in 60 hours to
period of time on their way to tissue destination produce a total of 4 monocytes
 mast cells have several phenotypic and functional o Under increased demand, it undergoes 4
similarities with both basophils and eosinophils divisions to yield a total of 16 monocytes in
 its progenitors MCPs originate from BM and spleen 60 hours
o are then released to blood before reaching  No storage pool for monocytes in BM
tissue and mediate action o Released immediately into the circulation
 KIT ligand (stem cell factor) : major cytokine for upon maturation
mast cell maturation and differentiation o Therefore, when BM recovers from failure,
 Functions as effector cells in allergic reactions monocytes are seen in peripheral blood
through release of lipid mediators, proteases, before neutrophils
proteoglycans and cytokines  Large reservoir of immature monocytes resides in the
o Result of cross-linking of IgE on mast cell red pulp of the spleen
surface by specific allergens o The respond to tissue injury such as
 Can also be activated independently of IgE myocardial infarction
o Leads to inflammatatory reactions  Monocytes in peripheral blood can be found in
 Act in both innate and adaptive immunity marginal or circulating pool
o Marginal pool : 3.5 times the circulating
Monocytes
pool
 Circulating: 2%-11% ; Absolute # : 1.3 X10^9 / L o Remain in circulation for 3 days
 Similar to neutrophil, both are derived from  Different pattern of chemokine receptors = different
granulocyte-monocyte progenitor tissue targets and functions
 Monocyte-CSF: major cytokine for growth and
differentiation
 Morphologic stages: Monoblast- promonocytes-
monocytes
 Promonocytes

o Slightly indented or folded nucleus

o Delicate chromatin pattern
o Blue and scattered azure granules
 Fewer and smaller than those in  Once in tissue, it differentiates into macrophage,
promyelocytes osteoclast or dendritic cells
 Monocytes  Macrophage usually has an oval nucleus and a netlike
o Larger than neutrophils because they tend to chromatin pattern
stick and spread out on glass or plastic o Pale vacuolated often filled with debris
o Slightly immature cytoplasm
o Goal: enter tissues and mature into  Resident macrophage such as Kupffer cells survive
macrophages, osteoclasts or dendritic cells longer than tissue neutrophils and Inflammatory
o Round, kidney shaped horseshoe indented macrophage
nucleus o Kupffer cells: 21 days
o Lacelike and looser chromatin pattern o Inflammatory : measured in hours
o Blue-gray cytoplasm with fine azure
granules referred as azure dust or a ground Monocyte/Macrophage functions
glass appearance
Innate immunity
 3 subsets of human monocytes: classical,
intermediate and nonclassical
 recognizes a wide range of bacterial pathogens by to produce a wide variety of antibodies and
means of pattern recognition receptors (toll-like surface receptors.
receptors) 4. Although early lymphocyte progenitors such
o Stimulates cytokine production and as the common lymphoid progenitor
phagocytosis originate in the bone marrow, T and NK
 Macrophage: synthesize nitric oxide lymphocytes develop and mature outside of
o Cytotoxic against viruses, bacteria, fungi, the bone marrow.
protozoa, helminths and tumor cells
Lymphocyte development
 Contains Fc receptors and complement receptors
o Phagocytosis of foreign organisms /  Subdivided into antigen independent and antigen
materials that have been coated with Ab dependent phases
o Antigen independent: development in BM
Adaptive immunity
and thymus ; referred as central or primary
 Macrophage and dendritic cells degrade antigen and lymphatic organs
present antigen fragments on their surfaces (antigen- o Antigen dependent: occurs in spleen, lymph
presenting cells) nodes, tonsils and mucosa-associated
o Activates both T and B lymphocytes to lymphoid tissue such as Peyer’s patches in
initiate adaptive immunity response the intestinal wall ; referred as peripheral
 Dendritic cells: most efficient orsecondary lymphatic organs
 3 stages of B lymphocytes: pro-B, pre-B and
Housekeeping functions immature B cells
o During these stages, immunoglobulin gene
 Removal of debris and dead cells rearrangement occurs so that each B cells
 Destruction of senescent RBC and maintenance of produces unique immunoglobulin antigen
stoarage pool of iron for erythropoiesis receptor
 Synthesis of proteins, coagulation factors, o Immature B cells: not yet been exposed to
interleukins, growth factors and enzymes antigen (antigen-naïve B cells)
Lymphocytes  Leaves the BM to migrate to
secondary lymphatic organs, and
 3 groups: T cells, B cells, and NK cells take residence in specific zones
1. T and B cells: adaptive immunity  AKA hematogones: have a
2. NK cells: innate immunity homogenous nuclear chromatin
 3 characteristics of Adaptive immunity pattern and extremely scanty
1. Relies on enormous number of lymphocytes, cytoplasm
each having surface receptors for different  Normally seen in newborn
specific molecular structure on a foreign peripheral blood and BM and with
antigen acute lymphoblastic leukemia
2. Memory cells will then be produced that  Secondary lymphatic organs or in the blood is where
will react faster to that same antigen upon B cells may come in contact with antigen
reexposure o Results in cell division and production of
3. Self antigens will then be ignored under memory cells such as effector cells
normal circumstance ( tolerance )  Effector B cells are antbody
 2 cateogories producing cells known as plasma
1. Humoral immunity : production of antibody cells and plasmacytoid
 B lymphocytes, called B cells lymphocytes
because they are produced in BM  3%-21% are B cells
2. Cellular immunity: attacking foreign  Resting lymphocytes: 5:1 – 2:1 N:C ratio
organisms or cells directly o Chromatin is arranged in blocks
 T cells: produced by thymus  T lymphocytes: developed by an epithelial organ
 NK cells: produced by BM and located in the upper mediastinum called thymus
thymus o Lymphoid progenitor cells migrate from BM
 Lymphocytes differ in other leukocytes in many ways to thymic cortex, and progress to pro-T, pre-
1. Lymphocytes are not end cells. They are T and immature T cells by regulation of
resting cells, and when stimulated, they cytokines produced by thymic epithelial
undergo mitosis to produce both memory cells
and effector cells. o During those stages, antigen receptor gene
2. Unlike other leukocytes, lymphocytes rearrangement to produce T cell receptors
recirculate from the blood to the tissues and that are unique to each T cell
back to the blood.  T cell with receptor that act with
3. B and T lymphocytes are capable of self antigen undergo apoptosis
rearranging antigen receptor gene segments
  T cells are subdivided into 2 categories, whether or o Modulate function of other cells including
not they possess CD4 or CD8 antigen on their surface macrophage and T cells
o Immature T cells whose receptor act with
self antigen, proceeds to the thymic medulla
where apoptosis of slef reactive T cells
occur
o The remaining T cells migrate to the
secondary lymphatic organs, where they
take residence in specific zones.
o They eventually come in contact with
antigen
o Results in cell activation and production of
either memory cells or effector T cells or
both
o Transformation of resting lymphocytes into
activated form is the source of medium and
large lymphocytes that have increased
cytoplasm and eventually make up only
about 10% of circulating lymphocytes
o Effector T cells: reactive/variant lymphocyte
 T cells: 51%-88% of circulating lymphocytes
 NK cells: heterogenous group of cells with respect to
surface antigen ; 4%-29% of circulating lymphocyte
o Majority are CD56+, CD16+, CD3- CD7+
large granular lymphocytes
o Mature NK cell is larger compared to resting
lymphocytes because of increased cytoplasm
o Cytoplasm: peroxidase negative with
azurophilic granules
Lymphocyte functions

 B lymphocyte: antibody production, antigen

presentation to T cells and necessary for CD4
activation
 T lymphocytes: divided into CD4+ and CD8+ T cells
o CD4+ : further subdivided into TH1, TH2,
TH17, and Treg (CD41CD251 regulatory T)
cells.
 TH1: immune response against
intracellular pathogens
 TH2: host defense against
extracellular parasites including
helminths, induction of asthma and
allergic disease
 TH17: immune response against
extracellular bacteria and fungi
 TH reg cells: self tolerance by
regulating immune response
o CD8 : killing target cells by secreting
granules containg granzyme
 Activating apoptopic pathways in
target cells
 Referred as cytotoxic T
lymphocytes
 NK lymphocytes: innate immunity
o Capable of killing tumor cells and virus-
infected cells without prior sensitization