Elements of innate immunit

.
Outline
[Link] and contrast the external and internal innate defense s
ystems.
b. Define the functions of : neutrophils, eosinophils, basophils, ma
st cells, monocytes, macrophages,dendritic cells
c. List the different names for macrophages as they reside in differ
ent tissues.
d. Describe phagocytosis and list cells that perform it.
e. Describe receptors involved in the innate immune response
f. Describe NK cells
 Innate immunity

present from birth, provide the first line of def

ense against pathogens. Most m.o encounter
ed daily in the life of a healthy individual are d
etected and destroyed within min. to hrs by in
nate defenses
Development pathways
of various cell types
from pluripotent b.m
stem cells.

Stem cell
 Physical and chemical barriers
Before entry of m.o skin:
a. physical barrier: most m.o cannot penetrate intact skin. exception: S a
ureus can enter through sebaceous gland and hair follicles.

b. chemical barrier: acidic pH of sweat (pH of 3 to 5) , sebaceous s

ecretions, fatty acids, hydrolytic enzymes (e.g Lysozyme) .

C. Biological barrier: bacteria of which there are around 1000 species upon
human skin from 19 phyla. The total number of bacteria on an average hum
an has been estimated at 1012. The normal flora protect us from disease by:

1. Competing with invaders for space and nutrients

2. Producing compounds (bacteriocins) which kill other bacteria
3. Lowering the pH so that other bacteria can't grow
 Mucous membrane

• Mucus traps microorganisms that enter the digestive and

respiratory systems
• Mucus-coated hairs in the nose trap inhaled particles
• Mucosa of the upper respiratory tract is ciliated
• Cilia sweep dust- and bacteria-laden mucus away from lower respir
atory system. Alcohol, smoking, narcotics suppress this defense sys
tem
• Stomach mucosa secrete concentrated HCl and protein-digesting e
nzymes
• Hydrolytic enzymes in saliva and lacrimal fluid e.g lysozyme
• Low pH of vagina
After entry of m.o into blood
Humoral innate mechanisms:
Interferons (IFN):
• are proteins made and released by host cells in response to the pre
sence pathogens such viruses.

• IFNs belong to cytokines. IFNs are named after their ability to "interf
ere" with viral replication within host cells. Uninfected host cells resi
st new infection by virus.

• IFNs activate immune cells, such as natural killer cells and

macrophages;

• Certain symptoms, such as aching muscles and fever, are related t

o the production of IFNs during infection.
• Complement system
The complement system helps or “complements” the ability
of antibodies and phagocytic cells to clear pathogens fro
m an organism.

The complement system consists of a number of small prot

eins found in the blood, generally synthesized by the
liver, and normally circulating as inactive precursors (
pro-proteins). The end-result of the activation cascade is
massive amplification of the response and activation of t
he cell-killing membrane attack complex.
•
• Antimicrobial Peptides
defensins: are cationic peptides,
.They kill a wide variety of bacteria.
.Neutrophils are rich sources of these peptides,
.Defensins kill microbes rapidly,typically within minutes.

•Enzymes such as Lysozymes, are glycoside hydrolases,

• damage bacterial cell walls by catalyzing hydrolysis of peptidogly
can. abundant in a number of secretions, such as tears, saliva,
human milk, and mucus.
•present in cytoplasmic granules of the
polymorphonuclear neutrophils (PMN).
Cells involved in the Innate Immune Syst
em (Neutrophils, Macrophages, dendritic cells, NK )
Neutrophils
• Neutrophils, eosinophils and basophils, are known as granulocytes
due to the presence of granules in their cytoplasm, or as polymorph
onuclear cells (PMNs) due to their distinctive lobed nuclei.

• A neutrophil count of 2.5–7.5 x 109/L is a standard normal range. Th

e average lifespan of (non-activated human) neutrophils in the circul
ation is less than 8 hs.
Upon activation, they migrate into tissues,
where they survive less than 24 hs.
In mice, half-lives of 8 to 10 hours.
• Neutrophils are the most abundant type of phagocyte, normally repr
esenting 50 to 60% of the total circulating leukocytes,
• are usually the first cells to arrive at the site of an infection.
• The bone marrow of a normal healthy adult produces more than 10
0 billion neutrophils per day.

• Phagocytic cells contain the enzyme-rich lysosomes.

• Neutrophil granules contain a variety of toxic substances that kill or i
nhibit growth of bacteria and fungi (respiratory burst).

• The main products of the neutrophil respiratory burst are strong

oxidizing agents including hydrogen peroxide, free oxygen radicals
and hypochlorite and nitric oxide.
Monocytes and Macrophages

• In tissues, organ-specific macrophages are differentiated from phagoc

ytic cells present in the blood called monocytes.
• In humans, these monocytes are identifiable by high cell surface expre
ssion of CD14, lack of expression of CD16, and expression of the che
mokine receptor CCR2.

• Functions:
a. phagocytosis of Ags
engulf and destroy the bacteria through the generation of a “
respiratory burst”, causing the release of reactive oxygen species.

b. Ag presentation to specific T cells.

C. macrophages produce chemokines, such as TNF-alpha, IL-1, IL-8,

and IL-12 to promote an inflammatory response.
• Histological forms of macrophages include t
he following:
a. Kupfer cells in the liver
b. Alveolar macrophages: in the lung
c. peritoneal macrophages: free floating
d. Microglia cells in the CNS
The network of macrophages population in the body is calle
d Reticuloendothelial system (RES).
The Process of Phagocytosis
• Activation of the Phagocyte
•Resting phagocytes are activated by inflammatory mediators: bacterial
products, complement proteins , inflammatory cytokines , and prostaglandins .
b. Chemotaxis of Phagocytes
Chemotaxis is the movement of phagocytes toward an increasing c
oncentration of some products as LPS.
c. Attachment of the Phagocyte to the Microbe or Cell
•is the attachment of microbes to phagocytes by way of an Opsonins that includ
e :-antibody molecule called IgG,
-the complement proteins C3b and C4b, and
-acute phase proteins such as mannose-binding lectin (MBL)
and C-reactive protein (CRP).
d. Ingestion of the Microbe or Cell by the Phagocyte
• polymerization and then depolymerization of actin filaments
a lysosome fuses with the phagosome: the pH is correct (3.5 - 4.0) for the aci
d hydrolases to break down cellular proteins
 e. Destruction of the Microbe
There are 2 killing systems in neutrophils and macrophages: the oxyg
en-dependent system and the oxygen-independent system.
1. the oxygen-dependent system: production of reactive oxygen s
pecies (ROS) microbicidal because they are powerful oxidizing ag
ents

• The enzyme NADPH oxidase converts oxygen into superoxide an

ion (O2-). This can combine with water by way of the enzyme dismut
ase to form hydrogen peroxide (H2O2) and hydroxyl (OH) radicals.

• In the case of neutrophils, but not macrophages, the hydrogen peroxi

de can then combine with chloride (Cl2-) ions by the action of the enz
yme myeloperoxidase (MPO) to form hypochlorous acid (HOCL), s
inglet oxygen and nitric oxide (NO) .
2. oxygen-independent system
• Some lysosomes contain defensins , cationic peptides that alter cy
toplasmic membranes;
• lysozyme , an enzyme that breaks down peptidoglycan
• lactoferrin , a protein that deprives bacteria of needed iron
• cathepsin G , a protease that causes damage to microbial membra
nes
• elastase , a protease that kills many types of bacteria
• collagenase ; and various other digestive enzymes that exhibit an
timicrobial activity by breaking down proteins, RNA, phosphate com
pounds, lipids, and carbohydrates.
Dendritic cells (DCs)
•are immune cells forming part of the mammalian immune system.
a. function as antigen-presenting cells.
b. act as messengers between the innate and adaptive immunity.
•2 subsets of DCs have been identified:
plasmacytoid synthesize the IFN α and β in the early phase of i.r. (derive
d from the common lymphoid progenitor).
Conventional or Myeloid DCs have a major role in the induction of T cell
i.r. (derived from the common myeloid progenitor)

•Lymphoid and myeloid DCs evolve from lymphoid and myeloid precursors, r
espectively, and thus are of hematopoietic origin. By contrast, follicular DCs
are of mesenchymal origin and do not express MHC class II, but are so named
because they are located in lymphoid follicles and have long "dendritic" proce
sses.
• DCs are found in tissue that has contact with the outside environ
ment, such as lung mucosa, epithelial cells of the skin (Langerhan
s cells), and the linings of the nose and the gastrointestinal tract. Th
ese regions form the interface between the body and the environme
nt and are constantly exposed to foreign proteins and pathogens.
• They can also be found in an immature and mature state in the bl
ood. Once activated, they migrate to the lymph nodes, where th
ey interact with T cells and B cells to initiate and shape the adaptive
immune response

• DCs that circulate in blood do not have all the typical features o
f their counterparts in tissue, i.e. they are less mature and have
no dendrites
• The type and function of the DCs determine whether the initiated im
mune response is cytotoxic T cells, T-cell tolerance, memory B-c
ells, or T-helper (Th) cells
T cells

DC
Natural Killer cells
•Are large granular lymphocytes, present in an unimmunized individual
s. They make 5-10% of the lymphocytes circulating in the blood are NK
cells. They have a short lifespan of about two weeks.
•NK cells provide rapid responses to virally infected cells and respond to t
umor formation, acting at around 3 days after infection.
•NK cells are unique, as they have the ability to recognize stressed cells in t
he absence of antibodies and MHC, allowing for a much faster immune re
action.

•Have intracellular granules that contain preformed biologically potent

molecules that are released when NK cell make contact with target cells.
a. These molecules cause the formation of pores in the membrane o
f target cells leading to its lysis.
b. enter the target cell and cause apoptosis through enhancement o
f nuclear DNA fragmentation.
How NK cells discriminate normal from target cell?
•MHC class I is expressed on all nucleated cells. NK cells express rece
ptors called Killer cell-inhibitory R (KIR), which bind to MHC class I
molecules expressed on normal cells. Upon MHC-KIR binding, intra
celluar inhibitory signals are generated which cause inhibition of specifi
c transcription factors. This result in inhibition of NK cells.

•Virus-infected cells or tumor cells have significantly reduced num

bers of MHC class I molecules on their surfaces. When such cells e
ncounter NK cells, they fail to engage KIRs and become susceptible to
cytotoxicity.
NK cells
 Innate lymphoid cells (ILCs)
• ILCs are BM–derived cells with lymphocyte morphology that were discovered
as cells that produced CKs similar to those made by T cells but lacked TCRs.
• We call them “lymphoid cells,” not “lymphocytes,” because they do not express
clonally distributed diverse Ag receptors like the T lymphocytes.
• ILC do not express PRRs and consequently, they are not directly activat
ed in response to pathogen-associated molecular patterns.
• In contrast, they respond to CKs, alarmins and inflammatory mediator
s derived from myeloid and epithelial cells, and produce immunoregu
latory CKs.
• ILC do not express markers of T and B lineage (they are CD3- , CD19-
), myeloid cells, or granulocytes, and all express CD132 (γ chain of the
IL-2 R), CD127 (IL-7Rα), CD25 (α chain of the IL-2 R),
• There are different subsets of ILCs that arise from the same common lympho
id precursor that gives rise to B and T cells
• ILCs participate in our immune response to pathogens in all organs, in partic
ular at mucosal surfaces.: They are key in the innate immune response du
e to their ability to rapidly secrete immunoregulatory CKs.

• The feature of ILCs that makes them potentially important for early host defen
se is that they are always resident in epithelial barrier tissues, poised to react aga
inst microbes that breach those barriers.
ILC1

• Both NK and ILC1 are activated in response to proinflammatory CKs

such as IL-12, IL-15 and IL-18, produce interferon (IFN)-γ and tumor n
ecrosis factor (TNF) and express the activation receptors NKG2D.
• Unlike NK cells, ILC1 lack most of the activation and inhibitory recept
ors of the Ly49 and KIR family , making the “missing self” recognition s
ystem exclusive of NK cells .
• In addition, the cytotoxic machinery of NK cells (expression of gran
zymes and perforins) is absent in ILC1, so they are not cytotoxic. Ther
efore, NK cells could be considered the innate counterpart of CD8 T ly
mphocytes and ILC1, the innate counterpart of CD4 Th1 lymphocytes.
• In addition, while ILC1 are tissue-resident cells, NK cells circulate
mostly in blood . Therefore, ILC have properties similar to tissue-resid
ent memory T cells
 Three subsets of innate lymphoid cells, called I
LC1, ILC2, and ILC3, produce different CKs a
nd express different transcription factors, analo
gous to the Th1, Th2, and Th17 subsets of CD4
+ T lymphocytes

IL-15
IL-4, 6
TNF
ILC2
• ILC2 are preferentially located in intestine, lung and skin dermi
s epithelia, and are activated in response to CKs such as IL-33, IL-
25.

• ILC2 are non-cytotoxic cells that secrete CKs characteristic of t

he Th2 profile (IL-4, IL-5, IL-13).

• They play an important role during the immune response to intesti

nal helminths, and in adipose tissue they regulate metabolic ho
meostasis and obesity, exerting regulatory effects on eosinophils
and the development of anti-inflammatory macrophages that regulat
e insulin sensitivity.
 ILC3
• ILC3 are located in mucosa-associated lymphoid tissues and in lam
ina propria. ILC3 cells produce IL-22 and IL-17.

• ILC3 cells seem to play an important role in the maintenance of the int
estinal microbiota and the prevention of colonization by pathogeni
c bacteria, because IL-22 acts on cells of the intestinal epithelium, stim
ulating the turnover of enterocytes, and the production of antimicrobial p
eptides and mucus.

• IL-17 participate in immunity against Candida albicans in oral muco

sa and may also exert pathogenic effects in colitis.
In addition, IL-17 promotes the production of antimicrobial peptides and th
e recruitment of neutrophils.
 ILC-reg

• Another ILC subpopulation was described, which harbors a regulato

ry phenotype, and hence named regulatory ILC (ILCreg).

• Initially described in mouse and human intestine secrete high amou

nts of IL-10 and TGF-β and are devoid of CD4 and Foxp3 expressio
n.

• They show a distinct gene expression profile compared with other IL

C and play an important role in the resolution of innate intestinal i
nflammation through the suppression of ILC1 and ILC3 via IL-1
0 secretion, in a mouse model of colitis.
 R involved in the innate immune system
Pattern Recognition R
1)Toll-like receptors (TLRs)
• They are large family of receptors, each of which recognizes specific microb
ial molecular pattern. They play a crucial role in innate cell recognition.
• Cell membrane TLR:
TLR1, TLR2 recognize Gram positive bacterial lipoteichoic acid
TLR 4, 5 recognize Gram negative bacterial LPS
TLR7,8 recognizes ssRNA, TLR3 recognizes dsRNA
TLR9 recognize dsDNA
2) Nucleotide-binding oligomerization domain (NOD-like R) or NL
R:
.recognize intracellular pathogens (Cytosolic).
.activated by peptidoglycan, RNA, toxins and flagelin.
.Mutation in NLR genes is associated with several autoimmune disease
s including cold urticaria, Crohn’s disease and asthma
2) Mannan-binding Lectin (MBL)
-allows phagocytes to recognize microbial polysaccharides
-This activates the MB lectin pathway of complement system.
-C3b complement component coat the m.o. causing opsonization.
3) Scavenger R:
.Recognize specific anion polymers and acetylated LDL expressed by certai
n pathogens.
.recognize dying RBCs leading to their removal. `
.is carried out in the spleen which contains large numbers of macrophages
expressing scavenger R.
4) F-Met-Leu-Phe R
Neutrophils express such R that recognizes N-formylated peptides expressed
by certain bacteria.
5) RIG-like R
• RIG-like receptors (RLRs) are cytosolic sensors of viral RNA that respond to viral ds
RNA by inducing the production of the antiviral IFN α and β and inhibit viral replica
tion.
 Inflammation
• An important function of phagocytic cells is their participa
tion in inflammatory reactions.

• A physiological process and a major component of the b

ody’s defense mechanisms is initiated by :
Endogenous factors: such as tissue necrosis or bon
e fracture
Exogenous factors: include mechanical injury (cut),
physical injury (burns), chemical injury ( corrosive chemic
als), immunologic injury (hypersensitivity) and biologic inj
ury (infections)
Signs of inflammation: Pain, redness, edema and heat
These can be explained by :
•increased vascular permeability that results from the separation of t
he tightly joined endothelial cells lining the blood vessels. increased blo
od flow, vasodilation, extravasation of fluids and cellular influx (edema).
•Increased expression of adhesion molecules on the endolthelial cel
ls to promote the binding of circulating WBCs to the vessel.

•Redness is caused by increased vascular diameter which leads to

increase blood flow, thereby causing heat and redness.
•Pain due to the swelling and buildup of tissue starts pressing against n
erve endings.
•The PMN leukocytes accumulate within 30-60 min., if the cause of i
nfl. Response persists, within 4-6 h the area will be infiltrated by mac
rophages and lymphocytes.
Localized inflammatory responses are generated as a result
of the activation of the kinins (potent stimulators responsible f
or itching and pain) and the coagulation systems (plasma enzy
mes that are activated in a cascading manner following damage to blood ve
ssels).

Systemic inflammatory responses include:

-the induction of fever, increased in WBC count, increase synthesis of
ACTH and cortisone hormones,
-In crease in acute phase proteins such as C-reactive protein (CRP) that
bind to m.o. and activating the complement system.
-The cytokines that play a key role in the inflammatory response include: I
L-1, IL-6, TNF-α.
Chronic inflammation:
•Sometimes it is difficult to remove the causes of inflammation such as
tuberculosis, rheumatiod arthritis and glomerulonephritis), chronic infla
mmation occurs.
•Administration of anti-inflammatory drugs such as aspirin, ibuprofi
n and cortisone decrease the symptoms but do not affect the root caus
e of the inflammation, when they are withdrawn, the symptoms may ret
urn.
Fever:
•is caused by many bacterial products namely the endotoxins of Gra
m-negative bac.
Endotoxins→ monocytes and mac release endogenous pyrogen
s cytokines (IL-1, IL-6, TNF- α and IFN) →few hrs fever with accompa
nying chills and malaise.
•When skin is overexposed to the UV light (sunburn), keratinocytes are
damaged, causing the release of IL-1.