Cells and Cellular Activities

of the Immune System

(Part 1):
Granulocytes and Mononuclear Cells
Bryan Michael N. Balunes, RMT

Granulocytes
Neutrophil
Eosinophil
Basophil

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of Immunity

Granulocytes
1. NEUTROPHIL (Polymorphonuclear Neutrophil)
Comprises 50% to 70% of total peripheral
leukocytes

Principal WBC associated with phagocytosis

and a localized inflammatory response
Provides host defense against bacterial and
fungal infections

Mature neutrophil pools:

Circulating pool blood
Marginating pool vascular endothelium

Movement from the circulating pool to the

peripheral tissues occurs by a process called
diapedesis (movement through blood vessel
walls)

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Granulocytes
1. NEUTROPHIL (Polymorphonuclear Neutrophil)
Primary
granules

Myeloperoxidase, elastase,
proteinase 3, lysozyme,
cathepsin G, defensins

Secondary Collagenase, lactoferrin,

granules
lysozyme, NADPH oxidase
Tertiary
Gelatinase, plasminogen activator
granules

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Granulocytes
2. EOSINOPHIL
Comprises 1% to 3% of total peripheral
leukocytes

Homeostatic regulator of inflammation

Possesses less phagocytic activity
Increased number:
Allergic reaction
Parasitic infection
Roles:
Neutralizes basophil and mast cell
products
Kills certain parasites

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Granulocytes
2. EOSINOPHIL
Primary
granules

Acid phosphatase, arylsulfatase

Secondary Major basic protein,

granules
eosinophil cationic protein,
eosinophil peroxidase,
eosinophil-derived neurotoxin

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of Immunity

Granulocytes
3. BASOPHIL
Comprises <1% of total peripheral leukocytes
Exists for only a few hours in the bloodstream

Same with eosinophil, it possesses less

phagocytic activity
Plays an important role in acute, systemic,
hypersensitivity reactions
Granules (induces & maintains immediate
hypersensitivity)
Histamine contracts smooth muscle
Heparin anticoagulant
Eosinophil chemotactic factor-A

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Phagocytosis
The process in which phagocytic cells
engulf and destroy foreign particles such
as microorganisms or damaged cells

Most important phagocytic cells

Macrophages
Segmented neutrophils (PMNs)

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of Immunity

Phagocytosis
STAGES OF PHAGOCYTOSIS
1. Initiation
2. Chemotaxis
3. Adherence
4. Engulfment

5. Phagosome Formation
6. Fusion and Phagolysosome
Formation
7. Digestion (Degranulation)
and Destruction

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Phagocytosis
STAGES OF PHAGOCYTOSIS
1. INITIATION
Phagocytosis is initiated as the
result of tissue damage or
microbial multiplication.

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Phagocytosis
STAGES OF PHAGOCYTOSIS
2. CHEMOTAXIS
Process by which cells tend to move in a
certain direction under the stimulation of
chemical substances (chemotaxin)
Types of Chemotaxis
Positive chemotaxis cells move
toward the stimulating substance;
leukocytes always show this type of
chemotaxis
Negative chemotaxis cells move
away from the stimulating substance
Chemotaxins soluble bacterial factors,
complement components, CRP, antigenantibody complexes, dead tissue
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Phagocytosis
STAGES OF PHAGOCYTOSIS
2. CHEMOTAXIS
Opsonization coating of the organisms
by molecules that speed up phagocytosis
Opsonins serum proteins that attach to
a foreign substance and helps prepare it
for phagocytosis
Fc portion of antibody
C3

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and Mononuclear Cells
2: Overview
of Immunity

Phagocytosis
STAGES OF PHAGOCYTOSIS
2. CHEMOTAXIS

Steps in Opsonization
Antibody attached to the surface of a
bacterium minimally binds the Fc receptor
of the phagocyte
Complement C3b is attached to the
bacterial surface and binds loosely to the
C3b receptor of the phagocyte
Both antibody and C3b are attached to the
surface of the bacterium and bound tightly
to the phagocyte, allowing greater
opportunity for the phagocyte to engulf the
bacterium.
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Phagocytosis
STAGES OF PHAGOCYTOSIS
3. ADHERENCE
A physical contact between phagocytic
and microorganisms, aided by opsonins
Steps for effective leukocyte recruitment to
the site of injury:
a. Capture (tethering) first contact of a
leukocyte with the activated endothelium
Occurs after margination, which allows
phagocytes to move in a position close to
the endothelium
P-selectin found on endothelial cells;
primary adhesion molecule for capture and
initiation of rolling
L-selectin also has an important role in
capture

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Phagocytosis
STAGES OF PHAGOCYTOSIS
3. ADHERENCE

Steps for effective leukocyte recruitment to

the site of injury (contd):
b. Rolling
c. Slow rolling
d. Firm adhesion
e. Transmigration

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Phagocytosis
STAGES OF PHAGOCYTOSIS
3. ADHERENCE

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Phagocytosis
STAGES OF PHAGOCYTOSIS
4. ENGULFMENT
Once the phagocyte has recognized that a
particle is foreign, engulfment occurs by
ameboid motion.
The phagocyte extends its cytoplasmic
membrane around the invading organism
(active
membrane
invagination;
outflowing of cytoplasm), which is
eventually surrounded and completely
enclosed.
The bacteria must be more hydrophobic
than
the
phagocyte.
(Bacteria
with
hydrophilic capsule are not normally
phagocytosed.)
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Phagocytosis
STAGES OF PHAGOCYTOSIS
5. PHAGOSOME FORMATION
The phagocytic cell membrane invagination
leads to the formation of an isolated vacuole
(phagosome) within the cell.
Microorganism is completely surrounded by a
part of the cell membrane

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Phagocytosis
STAGES OF PHAGOCYTOSIS
6. FUSION AND PHAGOLYSOSOME FORMATION
Cytoplasmic granules fuse with membrane of
phagosome to form the phagolysosome.

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Phagocytosis
STAGES OF PHAGOCYTOSIS
7. DIGESTION (DEGRANULATION) AND
DESTRUCTION
Degranulation of the phagocytic cell
(neutrophil) releases antibacterial
substances (lytic enzymes) from the
granules.

Release of lytic enzymes results in

destruction of neutrophils and their
subsequent
phagocytosis
by
macrophages.
Phagocytosis presents no
risk to the macrophage
unless the ingested material
is toxic or unless it damages
the lysosomal membrane.
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Phagocytosis
STAGES OF PHAGOCYTOSIS

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Phagocytosis
SUBSEQUENT PHAGOCYTIC ACTIVITY
If invading bacteria are not phagocytized, they may establish themselves
in secondary tissue sites (lymph nodes or various body organs).

The undigested bacteria produce a secondary inflammation, where

neutrophils and macrophages again congregate.
If bacteria escape from secondary tissue sites, a bacteremia will develop,
which can prove fatal in patients who are unresponsive to antibiotic
intervention.

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Phagocytosis
OTHER PURPOSES OF PHAGOCYTOSIS
Disposal of damaged or dying cells and removal of aging erythrocytes
from the spleen
Removal of tissue debris from repairing wounds
Removal of debris as embryonic tissues replace one another
Removal of cancer cells (by mononuclear phagocytes)
Suppression of growth of spontaneously arising tumors

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TOPICS FOR NEXT MEETING (Summarization starts here)

Continuation of Granulocytes and Mononuclear Cells
Lymphocytes and Plasma Cells
Antigens and Antibodies
Soluble Mediators of Immune System (complements, cytokines,
interferons, etc.)

QUIZ
Intro to Immunology
Overview of Immunity
Granulocytes and Mononuclear Cells (up to Phagocytosis)
PRE- AND POST-LEC QUIZZES
Topics for next meeting

DRAWING ASSIGNMENTS (sketch pad)

Turgeon
Figure 2-3 basic immunoglobulin configuration
Figure 2-4 basic structure of IgG
Figure 2-5 enzymatic cleavage of human IgG1
Figure 2-11 variants of antibodies
Figure 2-15 production of monoclonal antibody
Figure 3-2 process of phagocytosis
Figure 4-8 lymphocyte membrane marker development
Figure 4-10 function of Th1 and Th2

DRAWING ASSIGNMENTS (sketch pad)

Stevens
Figure 1-9 events in the inflammatory response
Figure 2-6 B-cell development in the bone marrow
Figure 2-7 B-cell activation in peripheral lymphoid organs
Figure 2-11 activation of T cells
Figure 2-12 mechanism of NK cell cytotoxicity
Figure 3-2 characteristics of hapten
Figure 3-4 major histocompatibility complex
Figure 3-6 antigen processing pathways for endogenous and
exogenous antigens
Figure 3-7 CD8+ cell
Figure 3-8 CD4+ cell
Figure 4-3 antibody variations
Figure 4-9 action of IgE on mast cells
Figure 5-4 individual T cell classes
Figure 6-1 classical complement cascade
Figure 6-6 convergence of classical, alternative and lectin pathways

Neutrophil Abnormalities
1. CHDIAK-HIGASHI SYNDROME
Autosomal recessive disorder
Abnormal granulation giant granules
Impaired chemotaxis and delayed killing of ingested bacteria

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Neutrophil Abnormalities
2. CHRONIC GRANULOMATOUS DISEASE (CGD)
Most common and best characterized of the neutrophil abnormalities
Inability to produce reactive forms of oxygen necessary for normal
bacterial killing
X-linked form: failure to exhibit increased anaerobic metabolism
during phagocytosis
Cells from patients with CGD can phagocytize non-H2O2-producing
bacteria (catalase-positive), gram-negative rods, and fungi
(Aspergillus) but they cannot destroy them.
The onset of CGD is during infancy, with one-third of patients dying
before age 7 years because of infections.

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Neutrophil Abnormalities
2. CHRONIC GRANULOMATOUS DISEASE (CGD)
Diagnosis:
Nitro-blue tetrazolium (NBT) dye reduction
Reduction converts the nearly colorless NBT dye into a blue precipitate
that can be assessed visually on a microscope slide.

Flow cytometric assay more objective and quantitative than NBT

reduction
Neutrophils are labeled with dihydrorhodamine (DHR)
Phorbol myristate acetate (PMA) used to activate DHR-labeled
neutrophils
The resultant oxidative burst will reduce the DHR, resulting in
fluorescence that may be quantitated on a flow cytometer.
CGD: less fluorescence (failure to undergo oxidative burst)

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of Immunity

Neutrophil Abnormalities
2. CHRONIC GRANULOMATOUS DISEASE (CGD)

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of Immunity

Neutrophil Abnormalities
3. COMPLEMENT RECEPTOR 3 (CR3) DEFICIENCY
Autosomal recessive
Presents as leukocyte adhesion deficiency
Associated with marked abnormalities of adherence-related functions:
Decreased aggregation after activation
Decreased adherence to endothelial cells
Poor
adherence
and
phagocytosis
of
opsonized
microorganisms
Defective spreading
Decreased diapedesis and chemotaxis
Patients may also lack an intravascular marginating pool of
neutrophils

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of Immunity

Neutrophil Abnormalities
4. MYELOPEROXIDASE DEFICIENCY
Autosomal recessive (chromosome 17)
Deficiency of myeloperoxidase (iron-containing heme protein,
responsible for the peroxidase activity; accounts for the greenish
color of pus)
Primary (azurophilic) granules are present, but myeloperoxidase is
decreased or absent.

Mild to moderate defect in killing bacteria in vitro

Marked defect in killing fungi (Candida spp.) in vitro

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Neutrophil Abnormalities
5. SPECIFIC GRANULE DEFICIENCY
Autosomal recessive
Caused by a failure to synthesize specific granules during
differentiation of neutrophils in the bone marrow
Patients have recurrent, severe bacterial infections of the skin and
deep tissues, with a depressed inflammatory response

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of Immunity

Neutrophil Abnormalities
6. LEUKOCYTE ADHESION DEFICIENCY TYPE 1 (LAD-1)
Caused by a deficiency of CD18
CD18 component of adhesion receptors on neutrophils,
monocytes, and T cells
Leads to abnormal adhesion, motility, aggregation, chemotaxis and
endocytosis by the affected leukocytes
Affects people of all racial groups

7. LEUKOCYTE ADHESION DEFICIENCY TYPE 2 (LAD-2)

Caused by absence or deficiency of CD15s
CD15 (sialylLewis X) carbohydrate molecule involved in
adhesive interactions
Failure to convert guanosine diphosphate (GDP) mannose to fructose
Reported only in people from the Middle East and Brazil
Patients have a characteristic facial appearance, short stature, limb
malformations, and severe developmental delay
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Neutrophil Abnormalities
8. LAZY LEUKOCYTE SYNDROME
Defective leukocyte mobility

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Monocytes-Macrophages
MONONUCLEAR PHAGOCYTE SYSTEM
Originally called reticuloendothelial system
Includes the following cells
Promonocytes and their precursors (bone marrow)
Monocytes (blood)
Macrophage (tissues)

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Monocytes-Macrophages
1. MONOCYTE
Largest cell in the peripheral blood
Makes up 4% to 10% of leukocytes

Primary
Peroxidase, acid phosphatase,
granules
arylsulfatase
Secondary Beta-glucuronidase, lysozyme, lipase
granules

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and Mononuclear Cells
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of Immunity

Monocytes-Macrophages
2. MACROPHAGE
Arise from monocytes (differentiation and all
division of macrophage takes place in the
tissues
Do not contain peroxidase, as compared to
monocyte
Exists as fixed or wandering cells

Fixed
macrophages
line
the
endothelium of capillaries and the
sinuses of organs (bone marrow, spleen,
and lymph nodes)

Specialized macrophages

Microglial cell (brain)

Alveolar macrophage (lung)
Kupffer cell (liver)
Histiocyte (connective tissue)
Glomerular mesanglial cell (kidney)
Osteoclast (bone)

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Monocytes-Macrophages
2. MACROPHAGE
Functionally, the most important step in
the maturation of macrophages is the
cytokine-driven conversion of the normal
resting macrophage to the activated
macrophage.

During infection, cytokines such as

interferon-gamma
(IFN-)
and
granulocyte
colony-stimulating
factor (G-CSF) are released from
T
lymphocytes
sensitized to
antigens from the infectious agent in
order for macrophages to be
activated.

When exposed to an endotoxin,

macrophages release tumor necrosis
factor alpha (TNF-, cachectin), which
can activate macrophages itself under
certain in vitro conditions

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Monocytes-Macrophages
MULTINUCLEATED GIANT CELL
Terminal stage of development in the mononuclear phagocyte cell line
Characterizes granulomatous inflammatory diseases such as tuberculosis

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Monocytes-Macrophages
HOST DEFENSE FUNCTIONS OF MONOCYTE-MACROPHAGE
1. Phagocytosis

2. Antigen Presentation and Induction of Immune Response

3. Secretion of Biologically Active Molecules

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Monocytes-Macrophages
HOST DEFENSE FUNCTIONS OF MONOCYTE-MACROPHAGE
1. PHAGOCYTOSIS
Macrophages ingest and kill invading microorganisms such as
intracellular parasites, M. tuberculosis, extracellular pathogens
(pneumococci), and some fungi.
Macrophages can phagocytize particulate and aggregated soluble
materials. This is enhanced by the presence of receptors on the
surface of the Fc portion of IgG and C3
Another important phagocytic function of macrophages is to dispose
of damaged or dying cells and tissue debris

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Monocytes-Macrophages
HOST DEFENSE FUNCTIONS OF MONOCYTE-MACROPHAGE
2. ANTIGEN PRESENTATION AND INDUCTION OF IMMUNE RESPONSE
Macrophages are believed to process antigens and physically present
this biochemically modified and most reactive form of antigen to
lymphocytes (particularly T-helper cells) as an initial step in the
immune response.
With proper recognition of antigen on the macrophage surface by
T lymphocytes, the macrophage secretes interleukin-1 (IL-1),
lymphocyte proliferation ensues, and the immune response is
facilitated.

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Monocytes-Macrophages
HOST DEFENSE FUNCTIONS OF MONOCYTE-MACROPHAGE
3. SECRETION OF BIOLOGICALLY ACTIVE MOLECULES
Monocytes and macrophages are able to synthesize a number of
biologically important compounds (transferrin, complement, interferon,
pyrogens, growth factors).
Monocytes and macrophages are primary sources of interleukin-1
(IL-1), which supports proliferation of B lymphocyte, production of
antibody, and production of lymphokine by T lymphocyte

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of Immunity

Monocyte-Macrophage Abnormalities
1. GAUCHERS DISEASE inherited abnormality of cellular lipid metabolism
Deficiency of beta-glucocerebrosidase, the enzyme that normally
splits glucose from its parent sphingolipid, glucosylceramide

Cerebroside accumulates in histiocytes (macrophages)

Most frequently affects children
Gauchers cells
Rarely found in the circulating blood
Found in the bone marrow, spleen, and other organs of the
mononuclear phagocyte system
Large cell with 1-3 eccentric nuclei
Wrinkled cytoplasm

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Monocyte-Macrophage Abnormalities
2. NIEMANN-PICK DISEASE similar to Gauchers disease
Deficiency of sphingomyelinase
Massive accumulation of sphingomyelin in mononuclear phagocytes

Affects infants and children, with an average life expectancy of 5

years
Picks cell
Similar in appearance to Gauchers cell
Foamy cytoplasm

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Inflammation
Series of biochemical and cellular changes that facilitate the phagocytosis
of invading microorganisms or damaged cells
Overall reaction of body to injury or invasion by an infectious agent
Characterized by the vascular response, the cellular responses, and by
cellular proliferation and repair

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Inflammation
CARDINAL SIGNS OF INFLAMMATION
Redness (rubor)
Swelling (tumor)
Heat (calor)
Pain (dolor)
Loss of function (functio laesa)

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Inflammation
PRIMARY OBJECTIVES OF INFLAMMATION
Localize and eradicate the irritant
Repair the surrounding tissues

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Inflammation
THE VASCULAR RESPONSE

Localized dilation of the capillaries and venules in order to allow more blood
to pass to the site of injury (termed hyperemia)

Plasma leaks from the vessels, making the blood more viscoid.

The lubrication action of the plasmatic zone is impaired slowing the blood
flow (and sometimes stopping it in severe injury termed stasis).

The endothelial cells become swollen and the spaces between adjacent cells
become widened, allowing plasma and cells to pass between them, forming
an exudate.

The exudate is formed as a result of increased vascular permeability, which

allows the plasma proteins to leak through the vessel wall, causing the
osmotic pressure effect of the plasma proteins to be lost

The ground substance becomes more fluid, allowing the exudate to diffuse
into surrounding tissues more readily, preventing an immediate rise in tissue
tension. This causes pain.

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Inflammation
THE CELLULAR RESPONSE
The cellular response in inflammation begins when the leukocytes move into
the plasmatic zone and stick to the altered vessel wall.
In the initial stages of exudate development, polymorphonuclear
neutrophils predominate, but in the later stages, these cells are replaced by
monocytes.
Neutrophils major cell type in acute inflammation
Macrophages major cell type in chronic inflammation
The emigration of significant numbers of neutrophils into the inflamed area is
dependent upon chemotactic factors.
The amount of chemotactic factor present in the inflamed area
determines the intensity and duration of neutrophil emigration, which
may last 24-48 hours
The emigration of mononuclear cells begins about 4 hours after the initial
stimulus and may reach a peak (after a single injury) at 16-24 hours.
The few monocytes that are found in the early stages of inflammation
are stimulated either directly by phagocytosis of debris or indirectly
by products of PMN phagocytosis and degranulation to produce
monokines (e.g., IL-1)
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Inflammation
RESOLUTION AND REPAIR
Final stages of the inflammatory response
Fibroblasts begin to proliferate within 18 hours (and with a peak of
48-72 hours).
The fibroblasts produce acidic mucopolysaccharides during
proliferation, which may neutralize the effects of some of the chemical
mediators that are still being released by damaged mast cells and
basophils
Possible outcomes of inflammatory process:

The affected area may be completely repaired with total restoration

of function.
The injury may lead to the formation of an abscess with at least
some loss of function
A granuloma (a tightly packed pocket of inflammatory cells that die
and degenerate from the center out) may be formed.

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Inflammation

IS Lec 3:
Granulocytes
and Mononuclear Cells
2: Overview
of Immunity

Inflammation

IS Lec 3:
Granulocytes
and Mononuclear Cells
2: Overview
of Immunity

Inflammation

IS Lec 3:
Granulocytes
and Mononuclear Cells
2: Overview
of Immunity

Inflammation

IS Lec 3:
Granulocytes
and Mononuclear Cells
2: Overview
of Immunity

Inflammation

IS Lec 3:
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and Mononuclear Cells
2: Overview
of Immunity

End of Lecture