MOD2 Leukocytes

leukopoiesis

production of leukocytes WBC

where are leukocytes produced

partially in bone marrow — neutro, eosino, baso, mono, few lymphos

partially in lymphatic tissue — lympho, plasma cells

synergy

when substances combine/act together their action is different/greater than the sum of their individual actions

what cytokines and hormones help w leukopoiesis 

interleukins 1 to 19 — IL-1 to IL-19

GM-CSF — granulocyte/monocyte - colony stimulating factor

G-CSF — granulocyte - colony stimulating factor

M-CSF — macrophage - colony stimulating factor 

major function of leukocytes

protection against foreign agents

phagocytosis — antigens labelled by antibodies

lysis — direct killing mechanisms

overall functions of immune system

• encounters — lymphatic circulation, blood and tissues

• recognition — receptor cells

• activation — produce abs or chemical mediators

• deployment — unable to destroy by themselves, cooperate in deployment

• discrimination — btwn self and nonself antigens

• regulation — shuts down immune response when cleared

B lymphocytes

primary source of humoral immune response (adaptive immunity)

transforms into plasma cells — produces antibodies

T lymphocytes 

responsible for cellular immune responses (adaptive immunity)

regulation of antibody reactions — helping or suppressing activation of B lymphs

null cells 

lacks B/T cell surface markers — innate immunity 

1. killer cells/K

2. natural killer cells/NK

killer cells (K cells)

function via antibody-dependent cell-mediated cytotoxicity

recognize antibody-coated target cells via Fc receptors — induces lysis

dont require prior sensitization to antigen

natural killer cells (NK cells)

use direct cytotoxic mechanisms

recognize and kill virus-infected cells and tumor cells without prior sensitization

can also participate in antibody-dependent cell-mediated cytotoxicity

how do lymphs differ from leukos

• resting cells, undergoes mitosis when stimulated to produce memory/effector cells

• recirculate from blood to tissues back to blood

• B cells can rearrange antigen receptor gene segments for antibodies — variety of abs

• T cells can rearrange T cell receptor genes — variety of surface receptors

• T cells develop and mature outside of bone marrow in thymus 

which interleukins influence lymphocyte maturation

stem cells under influence of IL1 and IL6 differentiate into lymphoid stem cell CFU-L

primary lymphoid tissue

bone marrow and thymus

produces lymphocytes, promotes differentiation, doesnt require antigenic stimulation

stem cells in thymus > T lymphocytes

stem cells in bone marrow > B lymphocytes

secondary lymphoid tissue 

lymph nodes, spleen, mucosal associated lymphoid tissues (peyers patches, tonsils) 

main storage areas of already differentiated lymphocytes 

stages of development of lymphocytes

1. lymphoblast

2. prolymphocyte

3. lymphocyte

characteristics of maturation in lymphocytes

decrease in size

chromatin condensation

lose nucleoli

cytoplasm changes from dark to light blue/clear

few azurophilic granules may be present (mature)

whats observed when theres a shift to the left with lymphocytes 

first see increased number of lymphocytes 

blasts/prolymphs — hard to differentiate under mic

what can be used to differentiate between lymphocytes

have specific antigenic receptors/markers on membrane and cytoplasm —help differentiate by type/maturity 

flow cytometry is used to test the blood to determine what type of cell they are 

what expresses CD marker 2

all T cells

what expresses CD marker 3

all peripheral (post-thymic, mature) T cells

what expresses CD marker 4 

T helper cells

what expresses CD 5

all T cells

what expresses CD 8

cytotoxic T cells

what expresses CD 10

developing B cells

what expresses CD 19

developing and mature B cells

what expresses CD 20

developing and mature B cells

adult normal value of lymphocytes in the peripheral blood

20-44% 

approx 60-80% are T cells, 20-35% are B cells

morphological characteristics of plasma cells

typically not seen in peripheral blood — maybe one

has bluish cytoplasm

nucleus is typically on the side/edge of cell

has perinuclear halo

lymphocytosis

increase in lymphocytes

lymphopenia 

deficiency in lymphocytes 

variant/reactive lymphocytes

account for approx 5-6% of lymphs in peripheral blood

represent normal immune system

inc numbers may be found in viral disorders

represent stimulated lymphs w inc DNA and RNA activity

how to differentiate from reactive lymph and monocytes

nucleoli — sometimes present in lymph, absent in monos

chromatin — variable clumping

granules — few prominent azurophilic granules

neutrophils 

most numerous leukocyte in blood and marrow — 50-70% of all circulating WBC

not most numerous in body 

stays in blood for 8-10 hours, 3-5 days in tissue — cant go back into blood like lymphs

function: locate and destroy through phagocytosis 

neutrophilia

increased number of neutrophils (greater than 70% in PBS)

seen when acute bacterial infections

neutropenia

deficiency of neutrophils

seen with leukemia

what cells shift to the left most commonly 

neutrophils

maturation series of neutrophils

myeloblast > promyelocyte > myelocyte >metamyelocyte > band > segmented neutrophil

first stage of neutrophil maturation where we can differentiate that its a neutrophil

myelocyte (neutrophilic)

granules throughout neutrophil maturation

myeloblast — no granules

promyelocyte — azurophilic (nonspecific) granules

myelocyte — both azurophilic and neutrophilic (specific) granules

rest — abundance of fine violet-pink neutrophilic granules, non specific still there but harder to see through the neutro grans

what stage do nucleoli disappear in neutrophil maturation

metamyelocyte 

changes in cell size during neutrophil maturation

gets smaller as matures EXCEPT promyelocytes (2) can be larger than myeloblasts (1)

contents and functions of primary/azurophilic/nonspecific granules in neutrophils

lysozyme — enzyme breaks down the peptidoglycan in bacterial cell wall; acts more against GPO

myeloperoxidase — produces acid from H2O2 > makes bleach, cytotoxic to org

acid phosphatase — provides acidic environment to kill org

elastase — enzyme breaks down outer membrane of GNO

contents and functions of secondary/specific granules in neutrophils

lysozyme

NADPH oxidase — byproduct of O2, used in respiratory burst

cytochrome b — generates ATP

lactoferrin — antimicrobial

tertiary granules 

can only see using electron microscopy 

contain plasminogen activator, alkaline phosphate and gelatinase 

neutrophil membrane

contains receptors for most common opsonins

has coating of glycoproteins — adherence

high concentrations of cytoskeletal proteins — actin, myosin, tubulin for movement, shape changes, engulfing bacteria

antigenic receptors (markers)

antigenic receptors/markers on neutrophils 

CD 13 — granulocytes and monocytes

CD 15 — granulocytes

CD 33 — myeloid precursors and monocytes 

migration sequence 

1. margination, adherence, anchoring

2. diapedesis 

3. migration (directed or random)

margination

about 50% of neutros in blood at all times

cells roll along in contact with endothelial cells while patrolling inside walls of blood vessels

adherence 

flattening of the neutrophil against endothelial wall

response to chemical mediators released from area of inflammation

if small amounts of mediators, may be temporary 

anchoring

result of adherence when high concentrations of chemical mediators are present in vessels

diapedesis

movement of neutrophils from bloodstream to tissues through junctions in btwn cells of vessel wall in response to chemotaxins from area of inflammation

assisted by vasodilators released from infected site

migration

movement of neutrophils towards area of infection in the tissue

chemotaxins

induces chemotaxis — directed movement of a cell or organism in response to a chemical stimulus

naturally occurring substances produced by body’s activated coag, kinin, and complement systems or that are released from infected/injured tissues from lymphocytes and other leukocytes

when do neutrophils have vacuoles

after phagocytosis, neutro is degranulating (exocytosis of undigested material)

leaves behind holes — shows that the neutro is actively working

killing cascade (phagocytosis)

1. immune adherence (recognition)

2. endocytosis (engulfment)

3. lysosome fusion

4. killing and digestion

5. exocytosis

when does the phagocytic process begin

when neutrophil arrives at site of inflammation by chemotaxis

endocytosis 

extension of actin-rich pseudopods of membrane 

surrounds bacterium and forms phagocytic vacuole (phagosome) 

lysosome fusion

activated azurophilic and neutrophilic granules (lysosomes) attach to walls of phagosome and empty their contents into vacuole

respiratory burst

peroxide/peroxidase/halide system — major killing mechanism used by neutrophils

activated by NADPH oxidase, converts O2

what are some non-oxygen dependent methods for neutrophilic killing

• acidic pH 5.7 in phagosome kill pneumococcus

• microbicidal enzymes

• H+ ions on cationic granular proteins kill e.coli

• lysozyme hydrolyzes mucopolysaccharide wall in some bact

what happens after neutrophilic killing takes place 

dead microorg is digested by hydrolytic enzymes from lysosomes

useful components are absorbed into cytoplasm of neutrophil

waste components in phagosome ejected from cell via exocytosis 

what happens after neutrophils die and lyse

converted into pus — resulting release of lysosomal enzymes and pyrogens amplifies inflammatory response

maturation stages of monocytes

1. monoblast

2. promonocyte

3. monocyte

characteristics of monoblasts

nucleus is round, oval, slightly folded or convoluted, with fine, thread-like pale, red-purple chromatin and up to 5 nucleoli

cytoplasm is moderately basophilic with greyish colouration, no granules

* non motile

* non phagocytic

characteristics of promonocytes

nucleus is folded or convoluted with chromatin creases, red-purple

chromatin and 0-5 nucleoli

cytoplasm is abundant, blue-grey, often with pseudopods and vacuoles

* slightly motile

* can phagocytize, but rarely

monocyte granules

only azurophilic (primary) granules

monocytic maturation in blood and tissues 

red marrow — monoblast > promonocyte

blood — monocyte for 36-72hr; immature cells, relatively inactive

tissue — immature macrophage > macrophage

macrophage vs monocyte

• inc energy level

• inc metabolic rate

• produces hydrolases (lysozymes) in ER, package in lysosomes until cytoplasm filled with small azurophilic granules

• inc size — up to 50um diameter

function of macrophages

• phagocytic response

• immune recognition

• secretory effector cell

• iron metabolism

• preservation of youthful/healthy cell population

monocyte phagocytosis 

responsible for phagocytosis of dead neutrophils and body tissue in aftermath of acute infections 

predominate in chronic infections 

interleukin 1

best known monokine

activation of CD4 T cells by foreign antigen — activates tissue macrophages

stimulates hepatocytes to secrete acute phase proteins

stimulates GM-CSF production

acts as endogenous pyrogen

difference in response to inflammation btwn neutrophils and macrophages

macs arrive slower in smaller numbers

takes longer to exert cumulative effects

macs designed to survive combat w foreign microorganisms

functions of mono/macrophage secretions

• removal of old blood cells

• stimulation of self-defense against tumor cells

• modulation of immune function

• regulation of hematopoiesis

• stimulation of inflammatory reactions

• removal of infectious organisms by phagocytosis 

where are there alot of eos and basos

little in peripheral blood, lots in tissues

eosinophils 

0-4% of peripheral blood

have numerous specific eosinophilic cytoplasmic granules

eosinophilia

increase in eosinophils

seen in allergic reactions

40% or more when parasitic infections

eosinophilic granules

released to kill parasites and during hypersensitivity reactions

has major basic protein — enzyme against parasitic worms and bacteria

has eosinophil peroxidase

stimulates release of histamine

degranulates/deactivates mast cells and basophils

eosinophil membrane receptors

H1 histamine receptor 

H2 histamine receptor

IgE receptor

H1 histamine receptor

produces symptoms of allergic reactions

acted against by antihistamines

H2 histamine receptor

negative feedback — turns off inflammatory reaction caused by basophils/mast cells

IgE receptor 

binds IgE antibodies to cell — activates response from eosinos and basos

what stage of granulopoiesis can you determine the difference between granulocytes

myelocytes

basophils

0-2% of peripheral blood

immature form when in blood > turns into mast cells in tissues

basophilic granules 

• sulphated glycosaminoglycans 

• histamine

• enzymes

• eosinophil chemotactic factor ECF-A 

histamines in basophils

almost all synthesized and stored in mast cells

eosinophil chemotactic factor ECF-A

specific tetra peptides which attract eosinophils — induces eosinophils to go to site 

what happens as basophils use their granules

can synthesize more, causes anaphylaxis symptoms

basophil function

capable of ingesting foreign particles and produces heparin and histamine (induce inflammation)

what happens to parasites that are too big to phagocytose 

eosinophils release cytotoxic substances from their granules onto surface of parasite 

what cell increases during viral infections

lymphocytosis

what cell increases during acute bacterial infections

neutrophilia

what cell increases during chronic bacterial infections

monocytosis 

what cell increases during allergies and parasitic infections

eosinophilia

what cell increases during inflammation and allergies 

basophilia 

whats the most chemotactic chemical factor that signals neutrophil activation

leukotriene B