Hematopoiesis – the production of all blood cells (white and red) in the bone marrow

Hematopoietic
stem cells

Myeloid lineage Lymphoid lineage

1
Hematopoiesis – a stressful but necessary event for our survival

The hematopoietic process is probably one of the most-demanding tasks for our body. It needs
to:
1) be able to produce a steady number of red and white blood cells every day to supply
oxygen and nutrients to every tissue, and to efficiently patrol the body;
2) be able to boost the production of red blood cells in case of low oxygen tensions and of
leukocytes in case of infection;
3) be able to perform those actions for the whole life of an organism, by saving and keeping
constant a certain amount of hematopoietic stem cells.

It has been calculated that:

• Approx. 1011-1012 cells are produced daily by the hematopoietic process
• Hematopoietic stem cells replicate once each 48-50 weeks, meaning that they replicate
100 times in 80 years.

2
Immunity – innate/adaptive or humoral/cell-mediated?
We already said that immunity is defined as resistance to infectious disease. There are two
different ways to “approach” the study of immunological mechanisms, either by considering
their specificity/ontogenetic derivation or their effector mechanisms.

Specificity and ontogeny: Effector mechanisms:

innate and adaptive humoral and cell-mediated

Innate immunity is: Humoral immunity is:

• Aspecific • Mediated by macromolecules
• Faster (antibodies, complement system, anti-
• Parentally acquired microbial peptides)
• Equal for all human beings • Occurs in blood and mucosae
• Effective against bacteria and toxins

Adaptive immunity is: Cell-mediated immunity is:

• Very specific • Mediated by leukocytes
• Slower • Occurs in almost every body district
• Somatically variable • Effective against bacteria, worms and
• Particular for each human being viruses
3
Innate and adaptive immunity

Abbas et al.

4
Innate immunity
The innate immunity is our first line of defense, and relies on cellular and humoral mechanisms
to actively match the invading pathogens as fast as possible. Its main targets are microbes and
cells infected by microbes. The outcome of innate immunity is INFLAMMATION and
activation of the ANTIVIRAL STATE.

Recognition of microbes and infected cells occurs via:

SIGNALS RECEPTORS

TLRs, RIGs, NOD,

Pathogen-Associated
Mannose receptor,
Molecular Patterns Microbes Phagocytes
Dectins, Scavenger
(PAMP)
receptors

Damage-Associated Antibodies,
Damaged/ complement system, Mucosae and
Molecular Patterns Infected
(DAMP) antimicrobial circulating
cells
peptides

5
Innate immunity – PAMPs, DAMPs and PRRs
Pathogen- Associated Molecular Patterns (PAMPs) are approx. 103 different molecules which
are normal components of viruses and bacteria (while our cells do not normally express them).
They are mostly structural components of bacterial walls, or viral genomic elements. Their
recognition by innate immunity ensures non-self discrimination and major damage of
microbes. When cells get infected by microbes, they will eventually die by necrosis, releasing
intracellular proteins. Those self nuclear/cytoplasmic proteins recognized by innate immunity
are called Damage-Associated Molecular Patterns (DAMPs). Receptors for PAMPs and DAMPs
are called Pattern Recognition Receptors (PRRs).

6
Innate immunity – PAMPs and DAMPs

PAMPs Microbes
ssRNA Viruses
Nucleic Acids dsRNA Viruses All of these molecules are
Non-meth. CpG Viruses, Bacteria necessary for the normal
functions and survival of
Pilin Bacteria
Proteins microbes. Their recognition
Flagellin Bacteria ensures wide coverage and a
LPS Gram- bacteria strong interference on
Wall lipids microbial life cycle.
LTA Gram+ bacteria
Mannans Fungi
Carbohydrates
Glucans Fungi, Bacteria
DAMPs
Stress proteins HSP
Crystals Monosodium urate
Nuclear proteins HMGB1

7
Innate immunity – PRRs
Toll-like receptors (TLRs) are a family of
receptors evolved from Drosophila’s Toll
receptors. They exist as membrane-bound and
cytosolic forms, and show the widest ability to
recognize PAMPs. They also recognize DAMPs.
In humans 9 TLRs are known (TLR 1-9).

Their activation by intracellular or extracellular

ligands determines transcriptional activation
of:
• antiviral genes (Type-I interferons)
• cytokines genes
• chemokines genes
• adhesion molecules genes
• co-stimulatory molecules
• oxidative burst genes

Abbas et al. 8
Innate immunity – PRRs
NOD-like receptors (NLRs) are cytosolic receptors for PAMPs and DAMPs. More than 20 NLRs
are known, but the best characterized are NOD1 and NOD2. NOD1 recognizes preferentially
bacterial peptidoglycans, while NOD2 recognizes muramyl dipeptide from Gram+ and Gram-
bacteria. NLRs activation leads to gene activation in a similar way to TLRs. NOD1 and NOD2 are
especially important against Helicobacter pylori and defects in NOD2 might be involved in
Chron’s disease.

RIG-like receptors (RLRs) are cytosolic receptors for viral RNA (both ssRNA and dsRNA). Best
characterized RLRs are RIG-I and MDA5, which recognize different viral nucleic acids. Once
activated, they will lead to transcription of Type-I interferons and antiviral defenses activation.
These include suppression of transcription factors needed for viral genes (like EIF2a), the
synthesis of RNAse which degrade viral RNA and suppression of viral proteins assembly
complexes.

9
Innate immunity – other PRRs
Microbial carbohydrates are recognized by calcium-dependent Lectins (C-type Lectins). These
include:
• Mannose receptor (CD206), which recognizes D-mannose, L-fucose and N-acetyl-D-
glucosamine.
• Dectins, which recognize fungal b-glucan (dectin-1) and mannose-rich oligosaccharides
(dectin-2).
• Langherin (CD207), DC-SIGN, and others

Other microbial PAMPs are recognized by:

• Scavenger receptors (like SR-A and CD36). Initially identified as receptors for oxidized LDL
(involved in atherosclerosis), they bind to bacterial polyanions (LPS, LTA, nucleic acids etc.).
• N-FMLP receptors (like FPR and FPRL1). Those receptors bind to n-formylmethionine
leucyl-phenylalanine, a sequence which characterizes all bacterial proteins but not human
ones (apart from mitochondrial proteins).

10
The innate immunity in action – a 3 step process

STEP 1 STEP 2 STEP 3

Circulating defenses: Cellular defenses:

Epithelia: - Phagocytes
- Natural antibodies
- Physical barrier - NK cells
- The Complement system
- Defensive molecules

Abbas et al.
11
The innate immunity in action – anatomical barriers

Epithelia play a big role in the very first match

with invading pathogens. As most of the
microbes enter the body via the skin, the
respiratory and the gastrointestinal tract, these
epithelia evolved specific strategies to
counteract pathogens, like:
• Expression of sialomucins on the external
side of the plasma membrane, which
electrostatically repel bacterial walls
• Production of antimicrobial peptides (like
defensins and cathelicidins), which both
directly attack the microbes and help their
recognition by innate immunity cells
• Hosting of intraepithelial lymphocytes.
While some of them are “classical” ab T-
cells, many are gd T-cells, which are
particularly good at recognizing bacterial
phospholipids. They can directly attack
microbes and stimulate phagocyte
responses.
Abbas et al.
12
The innate immunity in action – circulating defenses
Once microbes enter the blood flow or the extracellular fluids, they get in contact with the
humoral effector mechanisms of the innate immunity: natural antibodies, the complement
system and the pentraxins/collectins/ficolins family members.

Natural antibodies belong to the pentameric IgM class. They

are mainly produced by B1 lymphocytes in a spontaneous
way which does not require any presence of the pathogen.
These natural antibodies recognize PAMPs and DAMPs,
especially carbohydrates, lipids and lipoproteins. The AB0
blood group system depends on the presence of natural
antibodies.

Once bound to microbes, they activate the complement and

stimulate phagocyte response to eradicate the threat.

13
The innate immunity in action – circulating defenses
The complement system is a network of many (> 20) soluble and membrane-bound proteins
which are normally present in the blood flow in the form of inactive precursors (pro-enzymes).

The term complement (C) was used to refer to a heat-instable serum

component which was able to lyse bacteria when incubated at 37oC, but lost
when incubated at 56oC for 30 minutes (antibodies are thermo stable!).

• The complement system is activated by microbial macromolecules or by

antibodies bound to them. There are 3 complement activation pathways.
• Complement activation proceeds through sequential cleavage and
activation of circulating inactive forms of C proteins. Once cleaved, those
precursors become active and acquire proteolytic activity.
Jules Bordet
• The final stages of complement activation lead to formation of MAC
(Membrane Attack Complex), which is a canal (similar to acquaporins)
structure allowing water to enter into the microbes and kill them by
osmotic lysis.
• Microbes bound to complement fragments can be more-easily recognized
and killed by phagocytes
• Self cells, but not microbes, express proteins which inhibit complement
activation.
14
The innate immunity in action – complement activation

C3 has a spontaneous
cleavage turnover in
body fluids, so it can
directly recognize
microbes.

The classical and

lectins pathway
provide specificity to
complement activation

Abbas et al. 15
The innate immunity in action – complement activation

Abbas et al.

16
The innate immunity in action – other functions of the complement

The complement system provides a fast and reliable way to directly attack microbes, but it is
also connected to the cells of the innate immunity thanks to COMPLEMENT RECEPTORS (CR):
• CR1 (or CD35) promotes the phagocytosis of microbes bound to C3b and C4b. It is
expressed by almost all immune cells, in which determines activation, but also by red blood
cells. This provides a mean for the elimination of microbes by hemocatheresis.
• CR2 (or CD21) promotes the activation of B-cells and antibody production. It’s also
expressed by specific epithelial cells and is involved in Epstein-Barr virus pathogenesis.
• CR3 (or CD11b/Mac-1) binds a fragment deriving from C3b (iC3b) and is a strong promoter
of phagocytes’ activation.
• CR4 (or CD11c) belongs to the same family of CR3 and has similar functions, even though is
more restricted to specific cell subtypes (like dendritic cells)
• CR5 (or CRIg) binds to C3b and iC3b and is expressed only by liver’s Kupffer cells. It is
mostly involved in the direct elimination of circulating microbes by hemocatheresis.

17
The innate immunity in action – cellular immunity

Cellular defenses belonging to the innate immunity involve all myeloid cells and some specific
lymphoid cell types.

Myeloid Lymphoid

Phagocytes
NK cells and B1 cells
Phagocytosis and Mast cells gd T-cells
RESPIRATORY BURST NATURAL
BIOGENIC AMINES CYTOLYTIC ANTIBODIES
CYTOKINES and
MECHANISMS
CHEMOKINES
BIOGENIC AMINES

18
The innate immunity in action – phagocytosis and the respiratory burst

Elsevier 2005

19
The innate immunity in action – phagocytosis and the respiratory burst

Once the microbe is engulfed, the phagosome (phagocytic vesicle) fuses with the lysosome. Its
acidification activates the pro-enzymes that it contains and allows microbial degradation.
• Elastase and Cathepsin-G. Wide-spectrum proteases able to degrade many bacterial
proteins. Elastase is a serin-protease while Cathepsin-G is chymotripsin-like protease.
Those enzymes are activated by the low pH and by radicals.

In the phagolysosome, the respiratory burst occurs. This is the most powerful antimicrobial
mechanism of the phagocytes. It involves both the consumption of oxygen and specific
aminoacids to produce toxic compounds.
• Reactive oxygen species (ROS) include H2O2, O2.-, O. and are generated by the NADPH
oxidase. Those compounds are able to destroy/destabilize all major classes of
biomolecules, and thus destroy bacterial walls, membranes and genomes. Deficit of
NADPH oxidase is responsible for the chronic granulomatous disease (CGD), a very severe
deficiency of the innate immunity.
• The inducible nitric oxide synthase (iNOS) catalyzes the conversion of arginine to citrulline.
During this process, nitric oxide is formed. In the phagolysosome, NO interacts with H2O2
and O2.- to form peroxynitrite (ONOO-) which is extremely toxic as the ROS.

20
The innate immunity in action – biogenic amines and prostanoids

Phagocytes and mast cells secrete immunomodulatory compounds upon their activation,
including amines and prostanoids.

Histamine is produced via decarboxylation of

histidine. Once released, it will determine:
• Vasoconstriction of the major arteries
• Vasodilatation of arterioles
• Contraction of endothelial cells with
increased permeability of the capillaries and
venulae. This facilitates the immune cells to
penetrate in the site of infection (leukocyte
extravasation). Histamine is responsible for
itching at inflamed sites (think of mosquitos
bites)

21
The innate immunity in action – biogenic amines and prostanoids

Phagocytes and mast cells secrete immunomodulatory compounds upon their activation,
including amines and prostanoids.

Prostanoids are produced from

arachidonic acid by the cyclooxygenase
(COX) enzymes (target of NSAIDS).
Prostaglandins (PGs), leukotrienes (LTs)
and thromboxanes (TXs) have different
systemic and local effects, but they
generally either:
• Stimulate inflammation and
leukocyte extravasation (LTs, and
TXs and some PGs)
• Suppress local inflammation (PGs
like PGE2)

22
The innate immunity in action – lymphoid cells

Microbes can be recognized and killed by lymphoid cells too. Involved in this process are
mainly NK cells, NKT cells and gd T-cells. Recognition can be either direct or mediated by
antibodies.

DIRECT MEDIATED

F1000 2011 Nature 2003

23
The innate immunity in action – ADCC

NK cells can also directly recognize microbes if they are covered by antibodies (opsonized).

Abbas et al.

When antibodies bound to a microbe (or a damaged cell) are recognized by the NK cell
through their antibody receptor (FcgRIII), activation will occur even if the cells express
suppressive stimuli.
Note that opsonization works the same way to aid phagocytes with microbes recognition
and engulfment.

24
The innate immunity in action – Inflammation

The 5 symptoms of
inflammation:
• Tumor (swelling)
• Rubor (redness)
• Calor (heat)
• Dolor (pain)
• Functio laesa (functional
damage)

Consequences of immune
system activation!

25
The innate immunity in action – Inflammation

Inflammation is the normal, second line of defense of our body against pathogens. It is defined
as acute inflammation when it proceeds normally and resolves spontaneously. It will
eventually turn to chronic inflammation, a pathological process leading to tissue destruction,
if the stimulus (microbial invasion) cannot be resolved by the immune cells. The clinical signs
of inflammation depend on the release of soluble immune mediators, including amines,
prostanoids, cytokines and chemokines.

direct regulation of immune processes

cytokines coordinated regulation of surrounding, non-immune cells

chemokines chemotactic factors for immune cells

26
The innate immunity in action – cytokines

Cytokines can:
• Stimulate the functions of
phagocytes and lymphocytes,
empowering their antimicrobial
activity.
• Stimulate the proliferation of
leukocytes, to empower body
defenses.
• Stimulate lymphocytes
differentiation, ensuring specific
immunity to be effective.
• Stimulate multiple organs
functions, to cooperate with
leukocytes activity.
• Suppress immune functions, to
end the reaction and restore the
normal functions of the body.

27
Abbas et al.
Part Definition and general properties of
cytokines

. Definition
• A group of low molecular weight polypeptides or
proteins which are secreted by activated
immunocytes or some matrix cells and possess high
activity and various functions.
• Their major functions are to mediate and regulate
immune response and inflammatory reactions.
 . General properties of cytokines
1.Most cytokines are low molecular weight
polypeptides or glycoprotein(8~80 KD), and
most of them are monomer.
l Monomer, dimer, trimer

l Soluble, membrane-bounded
2. Natural cytokines are secreted by activated
cells
• Such as activated immune cells,matrix cells
and some tumor cells.
• Ag, SAg, mitogen
3. One kind of cytokines can be produced by different
cells. One kind of cells can secrete different cytokines.

IL-2 IL-4, 6

Th1 IL-3,GM-CSF,TNF-a Th2

IFN-γ, TNF-β IL-5

4. Cytokines act on target cells by the way of
paracrine, autocrine or endocrine.
• Cytokines can act on the cells that produce them
(autocrine), on other cells in the immediate vicinity
(paracrine), or on cells at a distance (endocrine) after
being carried in blood or tissue fluids.
5.Cytokines initiate their actions by binding to
specific membrane receptors on target cells.

• Receptors for cytokines

often bind their ligands
with high affinities.
• Kd 10-10-10-12
Antibody and antigen: Kd 10-7-10-11
MHC-peptide and TCR: Kd 10-5-10-7
• Low concentration (pmol/L)

• Most cells express low levels of cytokine receptors,

and this is adequate for inducing response.

• So only small quantities of cytokines are needed to

occupy receptors and elicit biologic effects.
6. The effects of cytokines are often pleiotropism,
redundant, synergy, antagonism, and form a
cytokine network.

• Pleiotropism refers to the ability of one cytokine having

multiple effects on diverse cell types.
• Redundancy refers to the property of multiple
cytokines having the same or overlapping
functional effects.
• Synergy refers to the property of two or more
cytokines having greater than additive effects.
• Antagonism refers to the ability of one
cytokine inhibiting the action of another.
 IL-1 IL-
IL-1 IL-6
6 TNF- IL-1 IL-6 IL-7 SCF
a IL-11
M- TNF-a
CS
FG GM-CSF
IL- M- IL- 4
1 TN CS G-CMF
F-a F
M-CSF
4
M-CSF
GM-C IL-
SF
IL-1 TN NF-a
FGF
F-a TG
F-b PD IL-1 T
a
GF
Mj L-8 TN F -
IL-1 I

4
IL-1
2 SF
M-C

IL-
G
-g

0
IFN 0 IL-

1
IL-
MF IL-1 -4
G-C IL
-g

13 b
IFN

-
TGF
IL-2

NK IL-2
IL-

IL-6
2I

IL- 4
L-1

IFN-g
2

IL-10 IL-13 IL-4 IL-4

TH2
IL-2 TH1 IL- 4

IL-4 IL-5 IL-6 IL-13

IL-2 IFN-g
IL-4 IL-4 IL-10 TGF-b
IL-4
B IL- 6
Tc

NK1+T
 Part Classification of cytokines

ØInterleukin, IL
ØInterferon , IFN
ØTumor necrosis factor, TNF
ØColony stimulating factor, CSF
ØChemokine
ØTransforming growth factor
The innate immunity in action – cytokines

43
Abbas et al.
The innate immunity in action – chemokines

Chemokines are generally divided

into:
• CC chemokines (b-chemokines), if
they have two adjacent cysteines
at the C-terminus
• CXC chemokines (a-chemokines),
if the two N-terminal cysteines
are separated by another residue
(X)

44
The innate immunity in action – activation of anti-viral state

The anti-viral state is a coordinated set of events occurring mostly in a paracrine way to
prevent viruses dissemination and further infection. It is considered as an ancillary mechanism,
as the major defense against viruses requires lymphocytes (adaptive immunity). It is mediated
by Type-I interferons, which are produced when viral PAMPs activate PRRs.

Type-I interferons
IFN-a (9 different types)
IFN-b (1 type)

Segregation of lymphocytes
in the lymph nodes, where
they will “learn” what type
ACTIVATION OF THE
of virus is infecting Potentiation of NK cells ANTIVIRAL STATE
Th1 differentiation of T-cells
Increased MHC-I expression

Innate (passive)
immunity
Adaptive immunity
45
The innate immunity in action – activation of anti-viral state

The so-called anti-

viral state is a passive
defense, which
prevents viruses to
replicate in cells
which have been
targeted by Type-I
IFN. But, it does not
kill infected cells.

The need for

Adaptive immunity

46
Abbas et al.
Inflammation switch-off – feedback mechanisms

Involving powerful cells (phagocytes, NK cells, etc.) armed with aggressive enzymes and toxic
products (lytic enzymes, vasoactive molecules, ROS, NO, etc.), inflammation always damage the
tissues. It is therefore necessary that it ends as soon as the microbial invasion is resolved.

Interleukin-10 (IL-10) is the prototypical anti-inflammatory cytokine. Its production is triggered

by inflammatory stimuli, but starts after the synthesis of inflammatory cytokines like TNF, IL-1,
IL-12 etc. Once secreted, it switches off the transcription of inflammatory cytokines genes.

IL-1 functions are suppressed by a soluble antagonist which binds to the same receptor as IL-1
but which has no biological functions, called IL-1 receptor antagonist (IL-1ra), produced by
phagocytes. There is also a Type-II receptor for IL-1 which is a decoy receptor with no activity
(scavenger of IL-1).

Inflammatory stimuli induce the expression of autophagy genes (Atg). Autophagy is the
process of a cell which degrades (eat) its own organelles. By “eating” organelles where PRRs or
cytokines are stored, a cell diminishes its ability to activate during inflammation.

Several suppressor of cytokines transduction pathways (SOCS, Suppressors Of Cytokine

Signaling) are induced by inflammatory stimuli and shut down pro-inflammatory cytokine
signals.
47