ASSAY OF CELLULAR

INNATE IMMUNITY
PHAGOCYTE ACTIVITY
EXERCISE IMMUNOLOGY
NO.2
SOTIRIJA DUVLIS
CELLS OF INNATE IMMUNITY

GRANULOCYTE CELLS Leukocytes that act as independent, single organisms and

are the second step of the immune system after the humoral barrier (inflammation and
the complement system). They are grouped into several groups
 Neutrophilic (40-75% of circulating leukocytes), eosinophilic (1-6%), basophilic (<1%)
 Mononuclear phagocytes: macrophages and monocytes
 Dendritic cells,
 Mast cells (fat cells)
 All of these cells identify and eliminate pathogens, either by attacking large
pathogens by making contact or by phagocytizing and killing them.
 These cells are also important mediators in the activation of acquired immunity
MACROPHAGES
 Macrophages are activated by numerous stimuli during the immune response.
 Phagocytosis of certain antigens or contact with receptors that detect molecules present in bacterial antigens
often serve as the initial activation stimulus.
 Macrophage activity can be further increased by both cytokines secreted by activated T-cells and mediators of
the inflammatory response.
 Activated macrophages are more effective than quiescent macrophages in their pathogen removal potential
for several reasons.
1. They exert greater phagocytic activity and increased ability to kill ingested microbes
2. and to activate T cells.
 activated macrophages, but not resting macrophages, secrete a variety of cytotoxic proteins that help eliminate
a wide range of targets, including virus-infected cells, tumor cells, and intracellular bacteria.
PHAGOCYTOSIS IS FOLLOWED BY DIGESTION AND ANTIGEN
PRESENTATION

 Macrophages are capable of ingesting and digesting external antigens, such as whole organisms and
insoluble particles, endogenous substances, such as injured or dead host cells, cellular debris, and
activated clotting factors.
 binding of the antigen to the macrophage cell membrane. Complex antigens, such as whole bacterial
walls or viral particles, tend to adhere well and are rapidly phagocytosed,
 isolated proteins and encapsulated bacteria tend to adhere poorly and are more difficult to phagocytose.
 Ingestion begins with the formation of an outgrowth (pseudopodia) that encloses the material with a
membrane-bound structure called a phagosome, fusion
 the phagosome moves to the interior of the cell and fuses with the lysosome to form a phagolysosome.
 Lysosomes contain numerous hydrolytic enzymes that digest the ingested material. The digested
contents of the phagolysosome are then removed by a process called exocytosis
 GRANULOCYTE CELLS
 Granulocytes are classified as neutrophils, eosinophils, or
basophils based on cell morphology and cytoplasmic staining
characteristics. A neutrophil has a multilobular (multilobular)
nucleus and granular cytoplasm that stains with both acid and
base stains, it is often called a polymorphonuclear (PMN)
leukocyte because of its large nucleus.
 The eosinophil has a two-part (bi-lobular) nucleus and a
granular cytoplasm that stains with acid red eosin dye (hence
its name).
 Basophilic leukocytes have a rounded nucleus and quite
granular cytoplasm that stains with basic methylene blue. -
They are non-phagocytic cells whose role is the release of
active substances that play a role in allergic reactions Both neutrophils and eosinophils are
phagocytic, while basophils are not.
 Neutrophil leukocytes - They are mostly present in the blood, they are created in the bone marrow and there they
GRANULOCYTE
mature, so they enter the blood CELLS
fully mature and no longer have the ability to divide. They live in the blood for
less than a day, and in the tissues for 4-5 days. Their main function is phagocytosis, they also have receptors for the
Fc part of immunoglobulins and complement, so they can also participate in facilitated phagocytosis
 During an infection, the bone marrow produces more neutrophils than usual and they are the first cells to arrive at
the site of inflammation. This results in a transient increase in the number of neutrophils in the blood, and
leukocytosis. The appearance of leukocytosis is an indication of infection.
 Myeloid graft cells (granulocytes) in brief
- First cells to respond to infection by bacteria and fungi
- Phagocytosis in the circulation
- Short-lived cells
 Eosinophilic leukocytes - live in the blood for several hours, and then move to the tissues. Their phagocytosis
ability is weaker than neutrophils, they phagocytize free antigen-antibody complexes, thus removing them from
the body. Their number increases in allergic diseases, where they play a role in reducing the inflammatory
reaction. They also play a role in defense against parasites.
 Basophilic leukocytes - They are non-phagocytic cells whose role is to release active substances that play a role in
allergic reactions.
PHAGOCYTIC SYSTEM
 ОКСИДАТИВНИ И ХЕМИСКИ ПРОМЕНИ КОИ ВОДАТ ДО УНИШТУВАЊЕ НА
ПАТОГЕНИ

 a chemical weapon in macrophages and neutrophils is high levels of inducible nitric oxide synthetase
(iNOS), an enzyme that oxidizes L-arginine to give L-citrulline and nitro oxide (NO). Upon activation, mediated by
receptors such as TLRs or exposure to appropriate cytokines, phagocytes express high levels of the enzyme
 it is called inducible iNOS to distinguish it from other forms of the enzyme present in the body.
 Nitric oxide has powerful antimicrobial activity and can be combined with superoxide to give even more
powerful antimicrobial substances.
 Studies show that nitric oxide and substances derived from it are responsible for most of the antimicrobial activity
of macrophages against bacteria, fungi, parasitic worms, and protozoa.
EXAMININGTHE IMMUNE FUNCTION OF PHAGOCYTES

1.Chemotaxis and adherence of the microbe to the

phagocyte

2.Ingestion and phagosome formation

3.Fusion of phagosome with lysosome

4. Digestion of the ingested microbe

5. Disposal of waste material

1. EXAMINING THE IMMUNE FUNCTION OF PHAGOCYTES

 In case of bacterial or fungal aggression on the body, the polymorphonuclear cells react first, and then the
mononuclear phagocytes are also involved.
 Their immunological function is achieved through redox processes
THE MOST IMPORTANT TEST IS THE NITROBLUE TETRAZOLIUM (NTB) TEST
 А) qualitative
 Б) quantitative
 PHAGOCYTIC ACTIVITY

• One of the most important mechanisms involves the generation of hydrogen peroxide and superoxide
radicals, which lead to an increase in the pH of the phagosomes and thus allow the enzymes that are released to
attack the ingested microorganisms.

• The complex enzyme, NADPH oxidase, catalyzes the initial reaction, which leads to the generation
of superoxide radicals.
• NADH+ 2О2 → NADP+ + 2О2 - + H+
• Superoxide dismutase then catalyzes the synthesis of hydrogen peroxide:
• 2H+ + 2О2 - → H2 О2 + О2

• NADPH oxidase is a large enzyme complex in the phagosome membrane, which is composed of
membrane and cytosolic compartments, in response to phagocytic stimulation.

• Not all components of the multisubunit enzyme complex are clearly defined, but two of them, p47 and p67, are
located in the cytoplasm of unstimulated phagocytes, while two others are included in the cytochrome membrane
complex. The two chains of cytochrome b are: the heavy chain - gp91, and the light chain
CHRONIC GRANULOMATOSIS
 CASE CHRONIC GRANULOMATOUS DISEASE (CGD) :

• The genes that encode them, p47, p67, and p21, are located on autosome chromosomes, while gp91 is encoded by
the short arm of the X-chromosome.

• Four different genetic defects affect different components of the NADPH oxidase enzyme, but all result in a
disease with a similar form, called chronic granulomatous disease (CHD). The most common form of this disease is
X-linked CGB, which occurs as a result of a mutation in the gene encoding gp91.

• The diagnosis of CGD can be easily established using the metabolic defect of phagocytes. We need nitroblue
tetrazolium (NBT) paint, which is yellow and transparent. When it reduces, it becomes insoluble - cloudy and turns
purple.

• To test for CGD, a drop of blood suspended on a glass slide is taken, a phagocytic stimulus (eg, phorbol myristate
acetate (FMA), an oxidase activator) is added, and a drop of NBT dye is added. Neutrophils, which are the main
phagocytes in the blood, will engulf both the NBT dye and the FMA.

• In normal blood, the NBT dye is reduced to a dark purple, insoluble formazan, which is easily seen in
phagocytes, whereas in CGD patients no reduction of the color will occur.
QUALITATIVE NBT TEST

 The reduction of NBT is determined through superoxide anion, which occurs as a product of activated
oxidation. Reduced NBT has the characteristic of precipitating in the form of black formazan beads. There are
several modifications of the method, but the most used are the cytochemical ones that determine the
percentage of polymorphonuclear or monocytes that contain precipitated formazan grains.
 This reduction is not specific because NBT can be reduced by other reductases independently of oxidative
metabolism.
 Whole blood or already separated cells can be used.
 100 microliters of NBT solution (2 mg/mL) is added to 100 microliters of peripheral blood, incubated for 15
min/37oC, and
 then phagocytes are stimulated with endotoxin solution (15 μg/mL) or a suspension of killed bacteria.
 Incubate again for 15 min at 37oC and fix with formaldehyde to lyse the erythrocytes,
 and the other cells are placed on a glass slide using a cytocentrifuge.
 Contrast staining is performed and the percentage of cells containing precipitated reduced NBT is
determined microscopically.
QUANTITATIVE NBT TEST
 The addition of yellow NBT dye to plasma leads to the formation of an NBT-heparin or NBT-fibrinogen
complex that can be phagocytosed by the neutrophil.
 In normal neutrophils, phagocytic activity can be increased by adding (stimulating) endotoxin
 This method can be used to measure the degree of stimulation of unstimulated cells or their capacity for
phagocytosis after stimulation.
 Stimulated neutrophils internalize the dye complex into phagosomes, and after lysosomal fusion,
intracellular reduction leads to the formation of blue insoluble formazan crystals.
 The percentage of phagocytic cells can be determined by light microscopy, and the total color reduction can
be quantified spectrophotometrically after dioxane extraction.
QUANTITATIVE NBT TEST-SCHEMATIC

Nylon wool.
• Nylon wool is boiled in distilled water and the water is
changed at least three times, then combed and dried at room
temperature. 100 mg are measured and introduced into
Pasteur pipettes or into microcolumns (NEN) at the bottom of
which there is a filter disc or a small amount of glass wool

• The function of the nylon wool is to retain the phagocytic cells,

which can adhere (adhere) to the nylon wool fibers.

• It is preferable to use plastic test tubes, but as far as working

with glass vessels, they should be siliconized to prevent the
adherence of cells to their wall. It should be taken into account
that both neutrophils and monocytes can engulf
Extinction is measured on a spectrophotometer at
520nm for a blank test using dioxide
Laboratories have their own normal values and express them differently.
Most often, the results are expressed qualitatively in percentages.

• 50% NBT would mean that 50% of the phagocytic cells on a stained peripheral blood sample from the subject have blue
staining in the cytoplasm of the phagocytes, that is, there are positive phagocytic cells.

• Although the NBT test is used for many purposes, the most significant application is in the detection of
CHD.
• A diagnosis of CGD can be made as the most likely diagnosis of CGD or as a definitive diagnosis of CGD

MOST LIKELY DIAGNOSIS OF CGD: A patient has an (abnormal) NBT test or respiratory collapse in activated
neutrophils (less than 5% of control) and who has one of the following:
1) Chronic infection (in the liver; perirectal or lung abscess; adenitis or osteomyelitis) caused by staphylococci
2) Diffuse granuloma in the respiratory, gastrointestinal or urogenital tract
3) Failure (arrest) in development and/or hepatosplenomegaly or lymphadenopathy
FINAL DIAGNOSIS OF CGD: when the patient has an abnormal NBT test or respiratory collapse in activated neutrophils
(less than 5% of control) and has one of the following:
1) Mutation in gp91, p22, or p67
2) Absence of mRNA for one of the above genes by Northern blot analysis;
3) Maternal relatives, uncles, or nephews with an abnormal NBT test or respiratory collapse.

Diagnostic value of the NBT test: Since the lower limit of CGD is 5% of the value in healthy people for both the most
probable diagnosis and the final diagnosis for HGB, this means that from 0 to 5% relative ratio in the qualitative test, as
well as 0 to 6.2 femtomoles/ phagocytes in unstimulated NBT or 0 to 15 femtomoles/phagocytes in stimulated NBT
would be elements for chronic granulomatous disease.

DISEASE SPECTRUM: X-linked form of HGB (60-70% of patients), the disease occurs much earlier and in a stronger form
(more severe form) in contrast to patients with autosomal recessive inherited form.
 • Mutations result in absent or inadequate
CLINICAL CHARACTERISTICS hydrogen peroxide production
OF CGD • It leads to severe recurrent life-threatening
bacterial or fungal infections mostly of the lungs,
skin and GIT
• Frequent occurrences of granuloma abscesses, • Chronic inflammation
impossibility to phagocytose mo • Average survival is 20-25 years of age of persons
• Success in transplant treatment
• High antibody levels
• G interferon