Bio 151 lec 12 13 cmer & lmi

CELL-MEDIATED EFFECTOR
RESPONSE
BIOLOGY 151 Lecture 12

Parungao-Balolong 2011

Sunday, February 20, 2011

RECALL: Humoral vs Cell-
Mediated Response
✤ assumes different roles in
protecting the host

✤ HUMORAL

✤ antibodies

✤ antigen-specific

✤ CELL-MEDIATED

✤ specific and non-specific
to antigens


ANTIBODY PROTECTION
LIES OUTSIDE THE CELL....
✤
SCENARIO: If antibodies were the
only agents of immunity, pathogens
that managed to evade them and
colonize the intracellular environment
would escape the immune system,
RIGHT?

✤
GOOD THING..This is not the
case!

✤
Cell-mediated immunity detect and
eliminate cells that harbor intracellular
pathogens

✤
EVEN...tumor cells that have
undergone genetic modifications
(they express antigens not typical
Parungao-Balolong 2011 of normal cells)


THE CELLS OF
CMI....
✤ ANTIGEN-SPECIFIC CELLS

✤ CD8+cytotoxic T lymphocytes (TC cells or CTLs)

✤ Cytokine-secreting CD4+ TH cells that mediate delayed-type hypersensitivity (DTH)

✤ NON-SPECIFIC CELLS

✤ NK cells and non-lymphoid cell types (macrophages, neutrophils, and eosinophils)

✤ The activity of both specific and nonspecific components usually depends on effective
local concentrations of various cytokines

✤ T cells, NK cells, and macrophages are the most important sources of the cytokines
that organize and support cell-mediated immunity


HUMORAL AND CELL-MEDIATED
IMMUNITY ARE INTERDEPENDENT
✤
Cells such as macrophages,
NK cells, neutrophils, and
eosinophils can use
antibodies as receptors to
recognize and target cells for
killing

✤
Chemotactic peptides
generated by the activation
of complement in response
to antigen-antibody
complexes can contribute to
assembling the cell types
required for a cell-mediated
response


TYPES OF
EFFECTOR
T-CELLS
✤ CD4+ (TH1 & TH2)
✤ CD8+ (CTLs)
✤ Unlike naive cells
✤ less stringent activation requirements
✤ increased expression of cell-adhesion molecules
✤ increased production of both membrane-bound and soluble
effector molecules


THE CTLs: CYTOTOXIC T-
CELLS
✤ generated by immune activation of T cytotoxic (TC) cells

✤ have lytic capability: critical in the recognition and elimination of altered self-cells
(e.g., virus-infected cells and tumor cells) and in graft-rejection reactions

✤ class I MHC restricted (since virtually all nucleated cells in the body express class I
MHC molecules, CTLs can recognize and eliminate almost any altered body cell)

✤ 2 phases of CTL-mediated immune response:

✤ first phase activates and differentiates naive TC cells into functional effector
CTLs

✤ second phase, effector CTLs recognize antigen–class I MHC complexes on
specific target cells, which leads them to destroy the target cells


3 SEQUENTIAL STEP
REQUIREMENTS...
✤
An antigen-specific signal 1
transmitted by the TCR complex
upon recognition of a peptide–class
I MHC molecule complex

✤
A co-stimulatory signal transmitted
by the CD28-B7 interaction of the
CTL-P and the antigen-presenting
cell

✤
A signal induced by the interaction
of IL-2 with the high-affinity IL-2
receptor, resulting in proliferation
and differentiation of the antigen-
activated CTL-P into effector CTLs


CTLs KILL CELLS
IN 2 WAYS...

✤
A. Directional delivery of cytotoxic proteins (perforin and granzymes) that
are released from CTLs and enter target cells

✤
B. Interaction of the membrane-bound Fas ligand on CTLs with the Fas
receptor on the surface of target cells

✤
NOTE:

✤
The end result of both the perforin/granzyme and Fas-mediated
pathways is the activation of dormant death pathways that are present in
the target cell

✤
“CTLs don’t so much kill target cells as persuade them to commit suicide”


CTLs KILL
CELLS IN 2
WAYS...
✤ PERFORIN-GRANZYME PATHWAY

✤ when introduced into the target
cell from the CTL mediate
proteolytic events that activate
an initiator caspase

✤ Fas/FasL PATHWAY

✤ engagement on a target cell by
Fas ligand on the CTL causes
the activation of an initiator
caspase in the target cell



NON-SPECIFIC
CYTOTOXIC CELLS

✤ non-MHC dependent

✤ can also kill target cells

✤ NK cells, neutrophils,
eosinophils, macrophages

✤ via antibody-dependent
cell-mediated cytotoxicity (ADCC)

✤ bind to the Fc region of antibody on target cells and
subsequently release lytic enzymes, perforin, or TNF,
which damage the target-cell membrane


COMPARING NOTES...
✤ CDC ✤ ADCC

✤ CDC activity ✤ ADCC activity

✤ antibody bound to its antigen ✤ Immune cells such as macrophages
activates reaction cascade of a and NK cells recognize the antibody
group of serum proteins called bound to the target cell and destroy
complement system the target cells such as tumor cells

✤ CDC is triggered by the binding of
complement to the antibody bound
to cell surface an antigen

✤ Activated complement system lyses
the target cell



COMPARING NOTES...
✤ NEUTRALIZING ACTIVITY

✤ Neutralizing activity

✤ When ligands such as growth factor
bind to their receptors, ligands
activate target cells and cause
damages

✤ When therapeutic antibody binds to
its target, ligand or receptor, antibody
interferes ligand-receptor interaction
and prevents undesirable activities


NK CELLS AND THE PERFORIN-
INDUCED PORE FORMATION
✤ NK cells
mediate lysis of
tumor cells and
virus-infected
cells by
perforin-
induced pore
formation
✤ mechanism
similar to one
of those
employed by
CTLs

NK CELLS ACTIVITY
REGULATION
• Activity regulated by
balance of activating and
killing receptors encoded
in the NKC gene cluster
• Kill Mechanism
– Similar CTL
•Granule release
•Perforin
– Constitutive cytotoxic


LEUKOCYTE MIGRATION
AND INFLAMMATION
BIOLOGY 151 Lecture 13



WHAT YOU NEED
TO KNOW

✤
Lymphocyte Recirculation

✤
Cell-Adhesion Molecules

✤
Extravasation (Neutrophils and
Lymphocytes)

✤
Chemokines and Inflammation

✤
Inflammatory Process

✤
Anti-Inflammatory Agents



LEUKOCYTE MIGRATION
✤
Many type of leukocytes move from one part
of the body to another

✤
LYMPHOCYTES: circulate continually in the
blood and lymph and, in common with other
types of leukocytes, migrate into the tissues
at sites of infection or tissue injury

✤
RECIRCULATION:

✤
increases the chance that lymphocytes
specific for a particular antigen will
encounter that antigen



LEUKOCYTE MIGRATION
& INFLAMMATION
✤
INFLAMMATION: a complex response to
local injury or other trauma

✤
characterized by redness, heat,
swelling, and pain

✤
involves various immune-system cells
and numerous mediators

✤
CONTROLLED MIGRATION of LEUKOCYTE
POPULATIONS:

✤
necessary for assembly and regulation
of inflammatory responses


WHY MIGRATE?

✤
The immune system relies upon the
continual circulation of leukocytes
through the body

– For the Innate IR
– variety of lymphocytes, granulocytes,
and monocytes can respond

– For the Adaptive IR
– lymphocytes must contact Ag in either
tissue, lymph, or blood



✤ Lymphocytes constantly re- LYMPHOCYTE
circulate from blood to spleen,
lymph nodes, and 3° lymphoid RECIRCULATION
tissues

✤ Continual circulation provides
systemic protection

✤ A complete circuit can be
performed 1-2X per day

✤ ~1 in 10 5 lymphocytes can
recognize a specific Ag

✤ therefore, constant circulation
increases chance of
lymphocytes contacting Ag



CELL-ADHESION
MOLECULES (CAMs)

✤ So how does lueukocytes transit
the bloodstream?

✤ They must bind to an
endothelial cell first!

✤ Endothelial cells exhibit ‘cell
adhesion molecules’ – CAM’s

✤ Lymphocytes, granulocytes, and
monocytes form receptors
which bind to CAM’s



1. SELECTINS (glycoproteins)

✤ has a distal lectin-like domain (bind to specific
carbohydrate groups)

✤ interact primarily with sialylated carbohydrate
moieties (often linked to mucin-like molecules)

✤ NOTE: 3 molecules (L, E and P) = Expression...

✤ Most circulating leukocytes (L-selectin)

✤ Vascular endothelial cells (E-selectin and P-
selectin)

✤ responsible for the initial stickiness of
leukocytes to vascular endothelium



2. MUCINS
(ser and thr-rich heavily glycosylated proteins)
✤
extended structure allows them to
present sialylated carbohydrate ligands
to selectins

✤
EXAMPLES:

✤
L-selectin on leukocytes recognizes
sialylated carbohydrates on two
mucin-like molecules (CD34 and
GlyCAM-1) expressed on certain
endothelial cells of lymph nodes

✤
(PSGL-1) found on neutrophils
interacts with E- and P-selectin
expressed on inflamed endothelium


3. INTEGRINS (heterodimeric proteins =
alpha and beta chains)
✤ expressed by leukocytes and facilitate both adherence to
the vascular endothelium and other cell-to-cell
interactions

✤ grouped into categories according to which beta subunit
they contain

✤ bind to different CAMs that belong to the
immunoglobulin superfamily expressed along the
vascular endothelium

✤ must be activated before they can bind with high affinity
to their ligands

✤ NOTE: important in leukocyte extravasation

✤ leukocyte-adhesion deficiency (LAD) = recurrent
bacterial infections and impaired healing of wounds


4. ICAMS/ Ig-superfamily CAMs

✤ contain a variable number of
immunoglobulin-like domains

✤ ICAM-1, ICAM-2, ICAM-3, and VCAM
(expressed on vascular endothelial cells
and bind to various integrin molecules)

✤ MAdCAM-1 (has both Ig-like domains and
mucin-like domains)

✤ expressed on mucosal endothelium and
directs lymphocyte entry into mucosa

✤ binds to integrins by its
immunoglobulin-like domain and to
selectins by its mucin-like domain


NEUTROPHIL
EXTRAVASATION
✤
As an inflammatory response develops, various cytokines and other
inflammatory mediators act upon the local blood vessels, inducing increased
expression of endothelial CAMs = ACTIVATION or INFLAMMATION!

✤
Neutrophils are generally the first cell type to bind to inflamed endothelium
and extravasate into the tissues

✤
HOW: neutrophils must recognize the inflamed endothelium and adhere
strongly enough so that they are not swept away by the flowing blood

✤
Bound neutrophils must then penetrate the endothelial layer and migrate
into the underlying tissue

✤
NOTE: Monocytes and eosinophils extravasate by a similar process


NEUTROPHIL
EXTRAVASATION
✤ Four sequential steps: ✤ (3) arrest and adhesion

✤ (1) rolling ✤ (4) transendothelial migration

✤ (2) activation by chemoattractant
stimulus

Parungao-Balolong 2011 recognize → adhere strongly → clinging to the vessel wall → penetrate


NEUTROPHIL ✤ Cell-adhesion molecules and
EXTRAVASATION chemokines involved in the first
three steps of neutrophil
extravasation

✤ Initial rolling is mediated by
binding of E-selectin molecules on
the vascular endothelium to
sialylated carbohydrate moieties on
mucin- like CAMs

✤ A chemokine such as IL-8 then
binds to a G-protein–linked
receptor on the neutrophil,
triggering an activating signal.

✤ This signal induces a
conformational change in the
integrin molecules, enabling them
to adhere firmly to Ig-superfamily
molecules on the endothelium


NEUTROPHIL
EXTRAVASATION

✤ THE 4TH STEP:
✤ The steps in transendothelial migration and
how it is directed are still largely unknown
✤ May be mediated by further activation by
chemoattractants and subsequent integrin–
Ig-superfamily interactions or by a separate
migration stimulus


LYMPHOCYTE
EXTRAVASATION
✤
Various subsets of lymphocytes exhibit directed extravasation at
inflammatory sites and secondary lymphoid organs

✤
The recirculation of lymphocytes thus is carefully controlled to ensure that
appropriate populations of B and T cells are recruited into different tissues

✤
SIMILAR WITH NEUTROPHILS

✤
extravasation of lymphocytes involves interactions among a number of
cell-adhesion molecules

✤
overall process is similar to what happens during neutrophil extravasation
and comprises the same four stages (contact and rolling, activation,
arrest and adhesion, transendothelial migration)


LYMPHOCYTE EXTRAVASATION



CHEMOKINES: Key Mediators
of Inflammation
✤ a superfamily of small polypeptides, most
of which contain 90–130 amino acid
residues

✤ selectively, and often specifically, control
the adhesion, chemotaxis, and activation
of many types of leukocyte populations
and sub- populations

✤ they are major regulators of leukocyte
traffic

✤ primarily involved in inflammatory
processes, others are constitutively
expressed and play important homeostatic
or developmental roles


of Inflammation
✤
“Housekeeping” chemokines

✤
are produced in lymphoid organs and tissues or in non-lymphoid sites
such as skin, where they direct normal trafficking of lymphocytes,
such as determining the correct positioning of leukocytes newly
generated by hematopoiesis and arriving from bone marrow

✤
inflammatory chemokines

✤
are typically induced in response to infection

✤
contact with pathogens or the action of proinflammatory cytokines,
such as TNF-alpha, up-regulate the expression of inflammatory
cytokines at sites of developing inflammation


Chemokine-Receptor Profiles Mediate Leukocyte Activity



of Inflammation
✤ Chemokines cause leukocytes to move into various tissue sites
by inducing the adherence of these cells to the vascular
endothelium
✤ After migrating into tissues, leukocytes are attracted toward
high localized concentrations of chemokines resulting in the
targeted recruitment of phagocytes and effector lymphocyte
populations to inflammatory sites
✤ The assembly of leukocytes at sites of infection,
orchestrated by chemokines, is an essential part of
mounting an appropriately focused response to
infection


OTHER MEDIATORS OF
INFLAMMATION

✤ other mediators released by cells of the innate and acquired immune systems
trigger or enhance specific aspects of the inflammatory response

✤ released by tissue mast cells, blood platelets, and a variety of leukocytes,
including neutrophils, monocytes/macrophages, eosinophils, basophils, and
lymphocytes

✤ four interconnected mediator-producing systems: the kinin system, the
clotting system, the fibrinolytic system, and the complement system

✤ Hageman factor: common intermediate of KS, CS and FS

✤ When tissue damage occurs, these four systems are activated to form a web of
interacting systems that generate a number of mediators of inflammation


✤ kinin system: activated by tissue injury

KININ SYSTEM ✤ an enzymatic cascade that begins when a
plasma clotting factor (Hageman factor)

✤ activated Hageman factor then activates
prekallikrein to form kallikrein

✤ then cleaves kininogen to produce
bradykinin

✤ BRADYKININ: a potent basic peptide that
increases vascular permeability, causes
vasodilation, induces pain, and induces
contraction of smooth muscle

✤ KALLIKREIN: also acts directly on the
complement system by cleaving C5 into C5a
and C5b

✤ RECALL: C5a complement
component is an anaphylatoxin that
induces mast-cell degranulation,
Parungao-Balolong 2011 resulting in the release of a number
of inflammatory mediators

CLOTTING SYSTEM

✤ clotting system: triggered by
damage to blood vessels

✤ yields large quantities of
thrombin

✤ THROMBIN: acts on soluble
fibrinogen in tissue fluid or
plasma to produce insoluble
strands of fibrin and
fibrinopeptides

✤ INSOLUBLE FIBRIN: crisscross
one another to form a clot
Parungao-Balolong 2011 (serves as a barrier to the spread
of infection)

CLOTTING SYSTEM

✤ The clotting system is
triggered very rapidly after
tissue injury to prevent
bleeding and limit the
spread of invading
pathogens into the
bloodstream
✤ FIBRINOPEPTIDES: act as
inflammatory mediators,
inducing increased vascular
permeability and neutrophil
Parungao-Balolong 2011 chemotaxis

FIBRINOLYTIC SYSTEM

✤ fibrinolytic system: removal of
the fibrin clot from the injured
tissue

✤ PLASMIN: end product formed by
the conversion of plasminogen

✤ a potent proteolytic enzyme,
breaks down fibrin clots into
degradation products that are
chemotactic for neutrophils

✤ contributes to the
inflammatory response by
Parungao-Balolong 2011 activating the classical
complement pathway

RECALL: COMPLEMENT SYSTEM

✤ complement system: complement split-products serve as important mediators
of inflammation

✤ Binding of the anaphylatoxins (C3a, C4a, and C5a) to receptors on the
membrane of tissue mast cells induces degranulation with release of histamine

✤ HISTAMINE: induce smooth-muscle contraction and increase vascular
permeability

✤ C3a, C5a, and C5b67 act together to induce monocytes and neutrophils to
adhere to vascular endothelial cells, extravasate through the endothelial lining of
the capillary, and migrate toward the site of complement activation in the tissues

✤ Activation of the complement system thus results in influxes of fluid that carry
antibody and phagocytic cells to the site of antigen entry


PHOSPHOLIPIDS AS
INFLAMMATORY MEDIATORS



CYTOKINES AS INFLAMMATORY
MEDIATORS



RECALL: INFLAMMATION



INTERFERON GAMMA AND CHRONIC
INFLAMMATION

Chronic
Inflammation
Develops
When
Antigen
Persists



RECALL: INFLAMMATION
(within minutes)

✤ Swelling:
(Tumor)

vasodilation phagocytosis ✤ Redness:
(Rubor)
edema
✤ Pain: (Dolore)
✤ Heat: (Calore)
(within a few hours)

✤ Loss of
function:
Parungao-Balolong 2011 (Fasor)

ANTI-INFLAMMATORY
AGENTS
✤ Although development of an effective inflammatory
response can play an important role in the body’s
defense, the response can sometimes be detrimental
✤ Allergies, auto-immune diseases, microbial infections,
transplants, and burns may initiate a chronic
inflammatory response
✤ Various therapeutic approaches are available for
reducing long-term inflammatory responses and thus
the complications associated with them


ANTI-INFLAMMATORY
AGENTS

✤ ANTIBODY THERAPIES: reduce leukocyte
extravasation
✤ block the activity of various adhesion molecules
with antibodies
✤ CORTICOSTEROIDS: cholesterol derivatives
✤ NSAIDS: non-steroidal anti-inflammatory drugs



ANTI-INFLAMMATORY
AGENTS
✤ CORTICOSTEROIDS: cholesterol derivatives (prednisone, prednisolone, and
methylprednisolone)

✤ causes a decrease in the number of circulating lymphocytes as the result either of steroid-
induced lysis of lymphocytes (lympholysis) or of alterations in lymphocyte-circulation
patterns

✤ lipophilic (can cross the plasma membrane and bind to receptors in the cytosol) =
transported to the nucleus, where they bind to specific regulatory DNA sequences,
regulating transcription up or down

✤ EXAMPLE: induce increased transcription of the NF-kappaB inhibitor (I-kappaB)

✤ Binding of this inhibitor to NF-kappaB in the cytosol prevents the translocation of
NF-kappaB into the nucleus and consequently prevents NF-kappaB activation of a
number of genes, including genes involved in T-cell activation and cytokine
production


ANTI-INFLAMMATORY
AGENTS
✤
CORTICOSTEROIDS: cholesterol derivatives
(prednisone, prednisolone, and
methylprednisolone)

✤
reduce both the phagocytic and killing
ability of macrophages and neutrophils,
and this effect may contribute to their
anti-inflammatory action

✤
also stabilize the lysosomal membranes
of participating leukocytes, so that
decreased levels of lysosomal enzymes
are released at the site of inflammation



ANTI-INFLAMMATORY
AGENTS
✤ NSAIDs:
non-steroidal
anti-
inflammatory
drugs



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