Granulocytes (Polymorphonuclear Cells):

Neutrophils, Eosinophils and Basophils

Learning Objectives
1. Describe a neutrophil (kamikaze cell) origin, structure, location in
the body, circulation, etc.
2. Define integrin, selectin, diapedesis, and chemotaxis
3. List the steps required for a neutrophil to go from the blood stream to
a site of infection; include important molecules essential to the
process
4. Define phagocytosis, phagosome, and phagolysosome
5. Describe the general types of infections for which neutrophils are
most important
6. Describe situations, conditions and/or drugs that decrease neutrophil
function and explain a common sequela to decreased neutrophil
function
7. List the types of clinical conditions that stimulate an eosinophil
response and explain how eosinophil response can be enhanced by
acquired immunity

Abscess

Introduction to Granulocytes
Granulocytes a general term that refers to white blood cells that contain
granules. The granules contain a variety of substances that are important for the
cells effective function
Three types of granulocytes (also called polymorphonuclear (PMN) cells
because they have nuclei with condensed chromatin and in a lobular shape)
Neutrophils granules contain substances important for controlling/killing
bacteria
Eosinophils granules contain substances important for killing parasites;
these substances also play a role in allergies
Basophils granules contain a variety of vasoactive substances like
histamine; important for controlling parasites and play a role in allergies.
Basophils are very similar to tissue mast cells.

Neutrophils
Generalities
Very important first responders in bacterial infections
Will arrive in minutes to hours in high numbers at sites of inflammation and
attempt to kill the invader
They circulate 8-10 hours and then migrate to tissue; live 1-2 days in normal
tissue, much shorter in inflamed tissue
The neutrophil is an end stage cell; they do not repair or replace damaged
membranes but kill themselves in the process of killing bacteria
Neutrophil movement
Rolling
 Mediated by selectin molecules; selectins are surface protein molecules
important for cell adhesion
The rolling or margination of neutrophils occurs because of selectin binding
between neutrophils and the endothelium; selectins are adhesion molecules
that mediate loose binding
Margination is mostly in the post capillary venules because this is where the
venule widens out and the neutrophil can roll; not as much sheer force in
this part of the vasculature
Because of margination, the neutrophil count in a blood sample (circulating
pool) is lower than the number of neutrophils in the blood; those that are
marginated are called the marginal pool of neutrophils
Fear, epinephrine, glucocorticoids cause endothelium to down-regulate their
selectin molecules which results in demargination of the neutrophils; the
neutrophil count goes up; on a CBC this is referred to as a stress leukogram
Tight binding
Mediated by integrin molecules; integrins are surface protein molecules
important for cell adhesion
In response to inflammatory stimulus (e.g. C5a, bacterial products) the
endothelium and neutrophils change
Endothelium will up-regulates integrin adhesion surface molecules
Neutrophil has integrin receptors that will bind tightly to the integrin
adhesion molecules on the surface of the endothelial cells
Tight binding of neutrophils to the endothelium via the integrin molecules
allows them to stop rolling
Diapedesis
Once the neutrophil is tightly bound to the endothelium it can squeeze
between the endothelial cells and leave the blood stream to enter the
tissues (in response to inflammatory stimuli, the endothelium has increased
permeability which makes it easier for cells to exit (diapedese) out of the
vasculature into the tissues)
Chemotaxis
Chemotaxis is the movement of cells along a chemical concentration
gradient. The general term used to refer to the chemicals the induce
chemotaxis is chemotaxin. Examples of chemotaxins are C5a, chemokines
like IL8, leukotriene B4 (arachidonic acid metabolism product) and various
bacterial products
Neutrophils follow from the lowest concentration to highest concentration
of the chemotactic chemical gradient
The neutrophil flows its membrane forward releasing enzymes like
collagenase and elastase from its granules; these enzymes digest connective
tissue allowing the neutrophil to move along the chemotactic gradient to the
site of the infection
Neutrophil phagocytosis of bacteria
Phagocytosis is the cellular process of ingesting or engulfing a particle,
eating by cells
Neutrophils can phagocytose anything more hydrophobic than itself;
material more hydrophilic than the neutrophil won't bind. Many bacteria
have hydrophilic capsules and escape neutrophil phagocytosis.
 Opsonization (prepare to eat, coating of a bacteria)
Coating hydrophilic material with antibody or a complement component
(e.g. C3b) will allow the neutrophil to bind to the opsonized bacteria via its
receptors for antibody and C3b
Neutrophils have membrane receptors that are important in phagocytosis:
Fc receptors (FcR) - bind to the Fc portion of antibody that has bound the
antigen it recognizes
C3b receptors (CR1) - binds to C3b when it is bound to a bacteria, etc.
Steps in phagocytosis
Attachment binding to the surface of the bacteria using the antibody or
C3b receptors
Engulfment the neutrophil membrane wraps around the bacteria
Formation of a phagosome - the cell membrane fuses around the bacteria
and forms a membrane bound vesicle that contains the bacteria called a
phagosome;
The cell granules (lysosomes) then fuse with the phagosome and form the
phagolysosome now the lysosomal enzymes are in contact with the
bacteria and begin to degrade the it
Acute inflammation
Acute inflammation can be defined by the accumulation of neutrophils in the
tissues
Acute inflammation is initiated by tissue damage and/or bacteria leading to
complement activation and/or the release of other mediators. This response
results in increased vascular permeability, changes in endothelium adhesion
molecules and the accumulation of plasma components and neutrophils at
the site of the damage/bacteria. This happens in a few hours.
In the skin, the area is red because of vasodilation and more blood at the
surface; it is swollen because of the fluid accumulation; it will feel warm
because of the increased blood flow, and it will be painful because of the
swelling and mediators that act on nerve endings. These are the cardinal
signs of acute inflammation: redness, swelling, heat, and pain.
Modulation of neutrophil function decreased or increased
Neutrophil function can be suppressed by many things; decreased
neutrophil function makes the animal more susceptible to bacterial infection
Conditions/situations/drugs that decrease neutrophil function
Distress: increased cortisol levels decrease neutrophil movement into
tissues and killing function; endogenous cortisol can decrease adhesion
molecules and inhibit neutrophil movement to sites of infection and can also
decrease the various killing responses of neutrophils
Some viral infections (e.g. respiratory viral infections decrease neutrophil
function and can make an animal more susceptible to bacterial pneumonia)
Bacterial virulence factors leukotoxin of Mannheimia hemolytic kills
neutrophils
Neonates neutrophils function below adult levels but this gradually
improves over first weeks of life
Drugs administration of glucocorticoids decrease neutrophil function
Neutrophil function can be enhanced by:
 Native defense cytokines (e.g. IL1 and TNF) which are released within
hours of initial infection have been shown to upregulate the neutrophils
ability to kill bacteria
T helper cytokines (e.g. IFN gamma) activate neutrophils and macrophages
to make them better killers. Cytokines from T helper cells are part of
adaptive immunity and on initial exposure to an antigen will take 5-7 days to
begin to be made.

Eosinophils
Introduction
Part of innate immunity and similar to neutrophils in some ways including
being polymorphonuclear, granulocytes; their granules stain red with eosin
dye
Eosinophils are to parasites what neutrophils are to bacteria; eosinophil
numbers increase in a parasitized animal
Eosinophils also play a role in Type 1 (immediate type) allergic responses
and the eosinophil count can go up in animals with allergies
Mechanism leading to destruction of a parasite (similar to neutrophils
responding in a bacterial infection)
Move out of the blood stream and into the tissues by diapedesis
They follow chemotactic gradient to the parasite due to chemotaxins from
the parasite or mast cells that degranulate
If IgE is coating the parasite the eosinophil will bind to Fc portion of IgE
(neutrophils it is IgG)
Binding to the IgE and cytokines will activate the eosinophils killing
mechanisms which are:
Degranulation of lysosomal contents released onto surface of parasite
cuticle and destroy it
Production of oxygen radicals on parasite surface like superoxide anion
and hydrogen peroxide
Production of lipid mediators like leukotrienes and platelet activating
factor
Activation of eosinophils by cytokines
Cytokines from T cells (adaptive immunity) and macrophages (innate
immunity) can act on eosinophils and result in:
Increased adherence, degranulation, and cytotoxicity and enhanced killing
of parasites
IL5 from T memory cells is an especially important cytokine for eosinophil
production and function
 Complement: Activation and Function

Learning Objectives
1. List the three ways the complement cascade is initiated and molecules
responsible for initiation
2. Classify the complement system into innate or adaptive immune systems
3. Explain the source and location of complement proteins; where they are
made and when and where they are located in the body
4. Define membrane attack complex (MAC) and explain its role in defense
5. List the biological functions of complement that contribute to defense
against bacterial infections including membrane damage, vascular
changes, chemotaxis, anaphylatoxin activity, opsonization and which
components of complement contribute to these functions
Complement activation,
Complement (C) (note spelling it is not complement fixation, and
compliment) complement cascade are often
Part of innate defense mechanisms used synonymously to mean
A series of 20-30 proteins always present in the complement cascade was
blood plasma; these proteins circulate in an initiated and went to
inactive state completion.
Many of the complement proteins are
produced by the liver
Complement system is an enzyme cascade (similar to the coagulation cascade);
after initiation, one enzyme activates hundreds of the next enzyme which
activates hundreds of the next, and so on. There are three enzymes in the
complement cascade.
It is very rapidly induced; will be activated within seconds of the introduction of a
microbe
It is a potent system and is highly regulated; if it is induced and not regulated
(turned off) the result is death
Complement contributes to defense in a variety of ways, including bacterial
membrane damage, recruiting neutrophils to sites of infection, targeting bacteria
for destruction, inducing inflammatory processes that help fight infection

Three Pathways for Activation of Complement

Classical pathway
Initiated when a complement fixing antibody (IgM, IgG) binds antigen
Since antibody is required to initiate, this pathway does not occur early in a
primary response because antibody is not present
This pathway is an example of innate (complement) and adaptive immunity
(antibody) working together
Lectin (a protein that binds to a carbohydrate) pathway
Initiated when mannose-binding lectin (MBL), a serum protein, binds to
mannose on microbes
Mammalian cells do not have mannose on their surface
 Innate immunity no previous exposure to the antigen is required to initiate
this pathway and no memory develops
Alternative pathway activated by special property of microbial surface
and C3b
Initiated by C3b binding on the cell wall of bacteria, fungi, some viruses
C3 is the most important C component and is in highest concentration in
the blood, it spontaneously breaks down to C3a and C3b
C3b binds to mammalian cells also but is quickly inactivated (See
Regulation of Alternative Pathway below)
Like the lectin pathway, the alternative is innate immunity

Comparison of the Three Pathways

All three differ in how they are initiated
All three pathways are essentially the same from C3 through the terminal
pathway
The terminal pathway, C5b-C9, is the same for all three pathways and
results in formation of the membrane attack complex (MAC) which inserts
into membranes and results in the death of the cell
The lectin and alternative pathways are innate immunity and the classical
pathway is both innate and adaptive immunity since it is initiated by antibody

Cleavage of Complement Proteins by Enzymes Formed During Complement

Activation
During activation of C, the enzymes formed result in cleavage of C proteins into
two pieces
Inactive complement proteins are cleaved into two pieces, and are named a and
b of the complement protein
For example, C2 is cleaved into C2a and C2b
The a is the small soluble molecule that results from the cleavage. So, C2a, C3a,
C4a, C5a are small soluble proteins with important biological function (discussed
later)
The larger molecule that results from the cleavage of the protein is designated as
the b portion, i.e. C2b, C3b, C4b, C5b. These molecules bind covalently to
nearby membranes. The binding must happen rapidly or these molecules are
inactivated by water. The requirement for rapid binding or inactivation is
important for regulating the complement system and ensuring it is acting near
the site of activation/infection and not at some distant site where it would just
cause damage.

Functions of Complement
The membrane attack complex (MAC)
Assembly of MAC
Cleavage of C5 results in formation of the C5b which binds to the
membrane; this results in exposure of a site on C5b that can bind
complement components C6, C7, C8 and C9 (C5a is also formed and it has
other important functions, see below)
12 to 18 molecules of C9 then polymerize and form a transmembrane pore,
this is called the MAC
 The MAC has a hydrophobic exterior and hydrophilic interior; the MAC
basically pokes a hole in the bacterial cell membrane
Function of MAC
Lysis and cell death via formation of the pores in the membranes of the
bacteria
Most of the soil and environmental bacteria can be killed by MAC; their
membranes are not protected and they are killed by complement almost
instantly when they enter the body
Many of the pathogens (those that cause diseases) have mechanisms to
prevent MAC from getting to their membrane and so evade this killing
mechanism of complement
Vasodilation and increased vascular permeability
C2a, C3a, C5a act on endothelium resulting in vasodilation and increased
vascular permeability
This allows neutrophils, macrophages, and lymphocytes to more easily exit
the blood vessel and enter the tissues
It also allows fluid containing antibody and components of complement to
easily get to site of infection
Chemotaxis of phagocytic cells
Some components of complement (e.g. C5a) causes neutrophils to go to the
area where bacteria are located; C5a also activate neutrophils and
stimulates their killing mechanisms
Opsonization
Some components of complement (e.g. C3b) bind or coat the bacteria and
this makes it easier for phagocytic cells (neutrophils and macrophages) to
attach to and phagocytose the bacteria; neutrophils and macrophages have
receptors for C3b

In summary, complement is an important part of the innate immune system.

Immediately after infection, complement and neutrophils begin working together to
control the infection until the adaptive immune system (B cells and T cells) can respond.
It can take several days for the adaptive immune system to respond.

 Macrophages

Learning Objectives
1. Describe the origin and circulation of a macrophage
2. List the names, location, and function of the resident macrophages in the
body, include those found in the bone, liver, brain, kidney, lung (2), and
connective tissue
3. Compare and contrast the macrophage and the neutrophil, including life-
span, location, functions, and killing mechanisms
4. Define acute and chronic inflammation by the predominant cell type at
the site of infection
5. List three central roles the macrophage plays in the immune system
6. Define sentinel cell
7. Describe proinflammatory cytokines; include the names of major
proinflammatory cytokines, what induces their production, what cells
make them.
8. Describe the major effects of proinflammatory cytokines in an animal
when they are in low concentration, medium concentration, and high
concentration
9. Explain the immunologic basis for the most likely reason why a puppy
may develop a low grade fever and be lethargic 12-24 hours after
vaccination

Origin and Development of Macrophages

Develop in bone marrow as monocytes which are released into the blood stream;
monocytes make up about 1-3 % of white blood cells in the blood; they are an
inactive cell
Monocytes circulate in bloodstream a day or two, then migrate to tissue and
mature to macrophage which is the active form of the cell
Monocytes in tissues differentiate into different types of macrophages depending
on the tissue they are in; live in tissues for months

Tissue Macrophages: Names, Locations, Special Functions of Macrophages in

the Body
Osteoclast
Located in bone and important in the resorption of bone
Pulmonary Alveolar Macrophage (PAM)
Located in the alveolus of the lung
They patrol the alveoli looking for inhaled particles dust, virus, bacteria e.g.
garbage collectors of the lung
PAMs phagocytose and attempt to kill ingested particles; the activated PAM
then often leave the alveolus by one of two paths:
Up the mucociliary escalator and are coughed out or swallowed, or
Exit through the alveolar wall and go to draining lymph node to present
antigen to lymphocytes
Normal healthy lungs contain these resident macrophages distributed in the
alveolar area it is not normal to have neutrophils in lungs
 Kupffer Cell
Located in the sinusoids of the liver
Blood circulates from the gastrointestinal tract via the portal vein to the liver
and past the Kupffer cells before going to the rest of the body
When bacteria pass through the epithelium in intestine and enter the
bloodstream, e.g. during enteritis, they will pass through the liver and be
trapped and removed by the Kupffer cells
Histiocytes - connective tissue macrophages, found throughout the body
Microglia Cells
Located in the brain
Important for removing plaque, damaged neurons and infectious agents in the
brain
Primarily function in maintaining the normal physiologic state of brain
The blood-brain barrier should keep microbes out of the brain; however, if the
blood-brain barrier is compromised the microglia cells can function to fight the
infection

Central Roles of Macrophages in the Immune System

Initial defense
Sentinel cell
Sentinel cells are cells that detect early microbial invasion and send out
early warning signals (e.g. proinflammatory cytokines)
Macrophages secretes proinflammatory cytokines (messengers) in response
to danger signals (DAMPS and PAMPs) from bacteria and these cytokines
initiate the inflammatory process
Antigen presentation to T cells the macrophage is a professional antigen
presenting cell. The macrophage can process and present antigen on MHCII to
memory CD4+/T helper lymphocytes for the adaptive immune system
Effector functions
Macrophages phagocytose and kill bacteria and other types of cells, e.g. tumor
cells
Like neutrophils, macrophages phagocytose and kill bacteria
They kill with lysosomal enzymes and products of oxidative metabolism
(nitric oxide is their most potent killing product)
Macrophages are activated to become better killers by cytokines produced by
T cells; a T cell cytokine activated macrophage can kill bacteria that a
neutrophil or a resting macrophage cannot; this is a very important component
of cell-mediated immunity
Macrophages will begin to accumulate at the sites of infection if the infection
is not easily cleared; the accumulation of macrophages at a site of infection is
called chronic inflammation

Sentinel Cells and Proinflammatory Cytokines

Sentinel cells - those that stand guard in the body
Types of cells that serve as sentinels: Definitions:
Mast cells, PAMPs: external and internal
Macrophages and molecular structures widely
distributed among microbes
Dendritic cells DAMPs: endogenous
molecules produced or
 Location of sentinel cells:
In tissues
Under skin and the epithelium of mucosal surfaces
Along vessels
Function and response of sentinel cells
Sentinel cells detect pathogen associated molecular patterns (PAMPs)
and damage associated molecular patterns (DAMPs)
Different pathogens and tissues have different molecular patterns and the
sentinel cells have receptors for groups of these different molecular
patterns. Collectively the DAMPs and PAMPs are sometimes called danger
signals.
Sentinel cells respond to danger signals by producing proinflammatory
cytokines
Proinflammatory Cytokines
Proinflammatory cytokines include IL-1, TNF, IL-6 and HMGB-1 (high mobility
group box protein-1, also called alarmin, and is released from damaged
tissue cells).
These cytokines have many effects and the effects vary depending on the
concentration/amount of the proinflammatory cytokines produced
Systemic effects result when moderate amounts of proinflammatory
cytokines are secreted. When enough proinflammatory cytokines are produced
they:
Act on the hypothalamus, and induce :
Fever IL1 and IL6 can act on the hypothalamus and cause fever
High body temperature can enhance the activity of some of the
enzymes to destroy bacteria, also some viruses do not replicate as
well at high temperatures
Speeds up metabolism
Sickness behavior (you feel bad)
No appetite
Lethargic
Keep the animal away from the herd
Act on the liver induce the production of acute phase proteins
Acute phase proteins are a group of proteins that increase tremendously
in the first 24 hours of infections. These proteins help control the
infection while the adaptive immune response is preparing to fight the
infection in an antigen specific way
Act on the bone narrow to stimulate neutrophilia - increased production of
neutrophils
Cytokines are proteins and they are newly synthesized by the sentinel cells
upon stimulation; in addition, many of the responses they induce also require
synthesis of proteins. This means the amino acids are needed to fight the
infection and the animal is anorexic so is not eating. Amino acids are released
from the muscle for use by the immune system; if it continues for days this can
cause the animal to become weak and lose muscle mass and weight
All of these effects are beneficial to control infection in the individual and in
the population by helping to prevent spread of the infection. If the sick animal
 separates from the herd the infectious agent isnt spread to the rest of the
herd.
Another important function of proinflammatory cytokines is the initiation of the
adaptive response; they provide important signals to cells of the adaptive
immune response; they signal to the lymphocytes that there is danger and a
response is needed
Extensive tissue damage, like that which occurs after burns, severe trauma,
surgery, can cause these systemic effects also; HMBG1, a proinflammatory
cytokine, is released by damaged cells

Other Clinical Signs and Effects of Proinflammatory Cytokines

In low quantities, the proinflammatory cytokines induce local
inflammation, which includes:
Macrophage activation, endothelium activation, complement activation
In moderate amounts the proinflammatory cytokines have systemic
effects discussed in the section above
In high quantities the proinflammatory cytokines cause septic shock and
potentially death
The proinflammatory cytokines induce vasodilation, increased vascular
permeability and upregulation of endothelial adhesion molecules. In high
quantities this happens in a lot of blood vessels and leads to:
A drop in blood pressure and low cardiac output,
Vascular injury, thrombosis and disseminated intravascular coagulopathy
(DIC); often a terminal event
Pulmonary edema, air spaces fill with fluid, and leads to acute respiratory
distress syndrome (ARDS)
Septic shock a clinical example of this is the septic shock that can result in
cows with gram negative mastitis. Endotoxin from the gram negative
bacteria induces high quantities of proinflammatory cytokines
Summary: Proinflammatory cytokines are necessary in low quantities because
they initiate the bodys response to the invader, but they are lethal in high
quantities.

Vaccinations and Proinflammatory Cytokines

In order to be effective a vaccine must induce secretion of the proinflammatory
cytokines (IL1, IL6 and TNF)
Normally, following one vaccination, low quantities of proinflammatory cytokines
are produced; the response remains local and there isnt any clinically observable
reaction; however, there are times (e.g. following injection of several different
vaccines) when moderate to high levels of proinflammatory cytokines are
generated.
The response to vaccines is additive, so if multiple vaccinations are given at the
same time they each may induce low levels on their own but added together
result in moderate levels of pro-inflammatory cytokines and systemic effects.
Some examples of when vaccines can induce enough proinflammatory cytokines
that cause systemic, clinically observable signs:
 A MLV (modified live vaccines) may cause low grade fever, lethargy and
anorexia which is due to moderate levels of proinflammatory cytokines induced
by the vaccine
Young animals and lighter weight dogs are more apt to display these effects
post-vaccination
Multiple vaccines (injections) given during one office visit to a dog 10kg or
less;
Vaccination with multiple gram negative bacterins (sometimes called
endotoxin stacking); this can lead to moderate or high quantities of
proinflammatory cytokines; if only one bacterin was given at a time then likely
nothing would be seen clinically
Mishandling (e.g. freeze/thawing, excessive agitation) of a gram negative
bacterin
can cause the release of LPS; free LPS (endotoxin) can cause high levels of
proinflammatory cytokines to be produced

Chronic Inflammation
Defined by a mononuclear cell infiltrate; predominantly
macrophages which may fuse to form multinucleate cells Granuloma:
(Acute inflammation is defined by the accumulation of Inflammatory
neutrophils, as previously discussed) lesion
Occurs when foreign material (e.g. a persistent infectious characterized by
chronic
agent like mycobacteria or non-degrading foreign body) is
inflammation with
not destroyed by the initial response but instead persists mononuclear cell
Macrophages begin to accumulate in response to the infiltration (cells
continued signals from tissue damage and the presence of may fuse into
foreign material and dying macrophages multinucleate
Macrophages become M2 macrophages and begin to giant cells and
remove damaged tissue and wall off the infection
Because the irritant persists, the macrophages continue to secrete IL1, TNF
alpha, transforming growth factor, fibroblast growth factor
This environment stimulates fibroblasts to deposit fibrin (fibrosis)
Granuloma is formed and is characterized by macrophages and fibroblasts; this is
called a tubercle in tuberculosis