Lines of Defense and Neutrophil Characteristics

Homeland Security - Lines of Defense

• The first line includes mechanical and chemical barriers
• The second line consists of leukocytes, including phagocytes like granulocytes and monocytes/macrophages, and natural killer cells, inflammation, fever and antimicrobial substances.
• The third line has cell-mediated immunity (T-cells) and Humoral immunity (B-cells)

Neutrophils

• The primary function is phagocytosis
• Neutrophils are the predominant WBC in dogs, cats, and horses
• Ruminants and some lab animals have more lymphocytes than neutrophils

Neutrophil Characteristics

• Neutrophil granules do not particularly pick up either acidic (red) or alkaline (blue) stains
• Identification is based on nuclear morphology (polymorphonuclear)
• Four varieties are observed in peripheral blood: bands, segs, hypersegs, and toxic

Band Neutrophils

• Also known as stab cells
• They are immature neutrophils
• The narrowest portion of nucleus is at least 2/3rds as wide as the widest portion of nucleus
• Small numbers are normally in circulation
• Elevated in response to inflammation or infection
• Increased numbers of band cells is called a left shift

"Seg" Neutrophils

• Known as PMN
• They are mature neutrophils
• The nucleus is segmented, with two to five segments joined by a thin strand of chromatin
• The chromatin can be difficult to see and may appear as separate segments

Hypersegmented Neutrophils

• These are very mature neutrophils, and have more than five lobes
• This pathological condition may prevent neutrophils from leaving the circulation
• Hypersegmentation may be seen within one day of blood draw
• Smears made from old blood will make smears right away and the whole blood will continue to age in vitro

Toxic Neutrophils

• These immature neutrophils contain morphologic abnormalities due to decreased maturation time
• Characteristics include foaminess or vacuoles in the cytoplasm and cytoplasmic basophilia
• Dohle bodies may be present and the nuclear chromatin is often less condensed
• Cells may be larger than normal and have toxic granules
• Toxic neutrophils are associated with any inflammation severe enough to intensely accelerate its production

Neutrophil General Information

• Neutrophils are polymorphonuclear neutrophilic granulocytes
• Production in bone marrow takes three to six days
• They leave the bone marrow and do not return
• They remain in circulation for six to 10 hours, and are replaced twice daily or more
• Neutrophils migrate to tissues and/or areas of disease to remove bacteria and debris
• They may live two to three days or only a few hours in tissues
• Low numbers are normal in respiratory, intestinal, and urinary tracts

The Pools

• The proliferation pool, in the bone marrow, is where cells are undergoing development
• Cells are either in the storage pool (spleen or bone marrow), the circulating pool (peripheral blood), or the marginal pool (stuck along the vessel walls)
• Eventually, they move into the tissue pool where they perform their functions

More on the Marginal Pool

• Marginated neutrophils will move into the circulating pool during stress, exercise, trauma, epinephrine exposure, corticosteroid treatment, and infection
• Dogs, horses, and cows have a 1:1 ratio whereas cats have a 1:3 ratio within the circulating pool.
• Cells will return to the marginal pool if not needed in the tissues

Neutrophil Functions

• Neutrophils are phagocytes that engulf microorganisms
• They are the first to defend against invaders that bypass mechanical and chemical defenses, and can respond very quickly
• Neutrophil granules (lysosomes) contain digestive enzymes that destroy bacteria
• Once neutrophils leave circulation to enter the tissues, they do not return

Leukocyte Movement Into Tissues

• Diapedesis is the outward passage of white blood cells through intact blood vessel walls
• Neutrophils must leave blood vessels to enter tissues via emigration
• More inflammation or infection causes more cells to undergo diapedesis
• Neutrophils remain in tissues for a few hours or up to two to three days, and then are phagocytized by macrophages

Leukocyte Movement and Job Performance

• Attraction occurs via chemotaxis (cytotaxins)
• Adherence is attachment due to opsonins
• Ingestion occurs as the pseudopod engulfs the invader
• Digestion occurs via lysosomal enzymes

Stimulating Leukocyte Movement Into Tissues

• Attraction: Cytotaxins are inflammatory chemicals produced from the interaction between microorganisms and the tissues they are invading
• This includes blood clot byproducts, viral and bacterial products, immunoglobulin products, mast cell products, or prostaglandins
• Chemotaxis refers to how neutrophils are attracted to tissues and are stimulated to emigrate by cytotaxins

Leukocyte Recognition of "Foreign Invaders

• Adherence: Opsonins are plasma proteins (usually a specific antibody) that coat microorganisms and cells, identifying them as foreign
• Opsonization is the process of coating microorganisms and cells with opsonins so they will be phagocytized

How Neutrophils and Phagocytes Destroy Foreign Invaders

• Ingestion occurs when the neutrophil advances its pseudopod to entrap a microorganism and internalize it
• A vacuole will be formed around it
• Neutrophilic granules (lysosomes) utilize digestive enzymes and move to the edge of the vacuole to fuse with its membrane
• Digestion takes five seconds as the lysosomes degranulate to kill ingested bacteria or dissolve foreign material with enzymes
• Following ingestion of five to 25 bacteria, acid wastes accumulate in the neutrophil, and it dies
• Pus is dead tissue cells, neutrophils, and other WBCs

Neutrophil Numbers in Peripheral Blood

• Numbers are maintained within normal limits in healthy animals by balancing escape rate from peripheral blood to tissues with replacement of used cells from marginal or storage pool

Neutrophil Response to Inflammation or Infection

• Massive, acute infection can cause total neutropil population to undergo diapedesis within hours
• This may result in temporary and short-lived neutropenia
• Neutropenia triggers the release of mature segs from the storage pool in the bone marrow to replace lost cells
• Cells shift to increased numbers of PPSCs to enter production, which takes 3-6 days to mature
• The bone marrow releases many new and immature cells into circulation, resulting in neutrophilia
• The need for segs can be overshot, resulting in dramatic neutrophilia lasing several days even after disease is under control

Neutropenia and Leukocytopenia

• Neutropenia occurs when neutrophils decrease as nearly all enter tissues with no storage pool of cells in the bone marrow to replace them
• This typically happens when infection is out of control and reserves of neutrophils are depleted
• Decreased numbers of neutrophils will decrease total WBCs, which is leukopenia
• Prognosis is poor when the body is losing the battle against invading microorganisms and/or stem cell production has ceased
• Decreased bone marrow production of WBCs can be caused by viruses, drugs, or cancers

Neutrophilia and Leukocytosis

• Neutrophilia occurs with any increase above the normal numbers of neutrophils in circulation
• Increased demand for neutrophils causes the bone marrow to release the storage pool
• Increased numbers of neutrophils will increase total WBCs, which is leukocytosis
• Leukocytosis with accompanying neutrophilia typically indicates infection

Left Shift

• This is an increase in the number of immature neutrophils in circulation in response to inflammation, infection, and/or tissue damage
• The degree may be graded by the number of immature cells seen; grading is from 1+ to 4+ with 1+ being bands only

Regenerative Left Shift

• Total WBC count is elevated due to neutrophilia
• Number of immature neutrophils may remain equal to or less than that of mature neutrophils
• This indicates a proper bone marrow response with sufficient time and ability to respond to increased demand

Degenerative Left Shift

• The total WBC and neutrophil count is normal or falling
• The number of immature neutrophils is greater than that of mature neutrophils
• The marginal and storage pools have been depleted and the bone marrow cannot meet demand, leading to a poor prognosis

Right Shift

• The presence of excessive numbers of hypersegmented neutrophils in peripheral blood
• Often observed when neutrophils remain in circulation longer than normal due to increased levels of corticosteroids from stress or treatment

Toxic Changes

• Results from severe infection, toxicity, endotoxins, or acidosis
• Often accompany a degenerative left shift and have a negative prognostic sign unless observed in felines

Epinephrine Response

• Physiologic leukocytosis results from fear, pain, exercise, or restraint
• The marginated cells enter circulation
• The response is transient, and will return to normal within 20 to 30 minutes after the animal returns to a calm state

Corticosteroid Response

• Referred to as a "stress leukogram"
• Steroids speed up the release of neutrophils from the marginal and storage pool
• Corticosteroids also inhibit diapedesis
• Can be endogenous or exogenous in origin
• A single dose of oral cortisone can increase the neutrophil count two to three times above normal for up to eight hours

Exploration of the body's defense mechanisms, including mechanical and chemical barriers, leukocytes such as neutrophils, and cell-mediated immunity. Focus on neutrophil characteristics, identification, and the varieties observed in peripheral blood.