Leukocyte Classification

Examination of a stained blood smear reveals many different types of leukocyte. Those that have a cytoplasm filled with granules are called granulocytes (Figure 4-2). Granulocytes also have a characteristic lobulated, irregular nucleus, so that they are described as “polymorphonuclear” (as opposed to the single rounded nucleus of “mononuclear” cells such as macrophages). Granulocytes are classified based on the staining properties of their granules. Cells whose granules take up basic dyes such as hematoxylin are called basophils; those whose granules take up acidic dyes such as eosin are called eosinophils; and those that take up neither basic nor acidic dyes are called neutrophils. All three populations play important roles in the defense of the body.

Neutrophils

The predominant blood leukocyte is the polymorphonuclear neutrophil granulocyte, otherwise called the neutrophil (Figure 4-3). About two thirds of the hematopoietic activity of the bone marrow is devoted to neutrophil production. Neutrophils are formed by bone marrow stem cells at a rate of about 8 million per minute in normal humans; they migrate to the bloodstream and about 12 hours later move into the tissues. They live for only a few days and must therefore be constantly replaced. Neutrophils constitute about 60% to 75% of the blood leukocytes in most carnivores, about 50% in the horse and 20% to 30% in cattle, sheep, and laboratory rodents. Normally, however, circulating neutrophils account for only 1% to 2% of the total population. The vast majority are sequestered in capillaries within the liver, spleen, lungs, and bone marrow. During bacterial infections, the numbers of circulating neutrophils may increase 10-fold as the stored cells are released from the bone marrow and other organs.

The production of neutrophils is regulated by a cytokine called granulocyte colony-stimulating factor (G-CSF) and their loss by their rate of apoptosis. Normal neutrophils migrate to tissues, where they eventually become apoptotic and are then phagocytosed by macrophages. The production of G-CSF is regulated by their rate of apoptosis. Thus dying neutrophils are removed by macrophages. These macrophages produce interleukin-23 (IL-23) so that, as neutrophils die, IL-23 production increases. IL-23 promotes IL-17 production by lymphocytes (Th17 cells; Chapter 20). IL-17 stimulates G-CSF production and stem cell activity. As a result, the rate of neutrophil production matches the rate of their removal by apoptosis. Toll-like receptors (TLRs) are also expressed on myeloid stem cells. During microbial infections, pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides bind to these TLRs and trigger the stem cells to produce more neutrophils. TLRs thus provide a mechanism whereby neutrophil availability increases rapidly in response to infection.

Structure

Neutrophils suspended in blood are round cells about 10 to 20 µm in diameter. They have a finely granular cytosol at the center of which is an irregular sausage-like or segmented nucleus (Figure 4-4). The chromatin within the nucleus is condensed and compacted so that neutrophils do not divide. Electron microscopy shows three major types of enzyme-rich granules in the cytosol (Figure 4-5). Primary (azurophil) granules contain enzymes such as myeloperoxidase, lysozyme, elastase, β-glucuronidase, and cathepsin B. Secondary (specific) granules lack myeloperoxidase but contain lysozyme and collagenase and the iron-binding protein lactoferrin. Tertiary granules contain gelatinase. These granules are synthesized at different stages in the cell’s development. Thus primary granules are synthesized at the promyelocyte stage, secondary granules at the myeloid stage, and tertiary granules late in the development process. Granules formed early in development are rarely exocytosed, whereas tertiary granules are readily exocytosed. Because neutrophil granules contain a complex mixture of bactericidal molecules, the cells may regulate their release at inflammatory sites to ensure that they are appropriate. Neutrophil granule proteins enhance monocyte adherence to vascular endothelium, trigger macrophages to secrete cytokines, and activate dendritic cells, thus promoting antigen presentation. Neutrophil secretory vesicles and granule membranes also serve as storage sites for receptors and other membrane-integrated proteins. Mature neutrophils have a small Golgi apparatus, some mitochondria, a few ribosomes, and a little rough endoplasmic reticulum.

Emigration from the Bloodstream

Circulating neutrophils are normally confined to the bloodstream so that in normal tissues, neutrophils are simply carried along by the flow. In inflamed tissues, however, molecules released by dead and dying cells cause these fast moving cells to slow down, stop, bind to blood vessel walls, and emigrate into the tissues. This emigration is triggered by molecules that affect both the endothelial cells that line blood vessel walls and the neutrophils themselves.

Changes in Endothelial Cells

In aggregate, the endothelial cells that line all blood vessels collectively have a huge surface area (estimated at 4000 square meters in humans) and thus serve as a broad sensor of microbial invasion. When bacterial products such as LPS or damage-associated molecular patterns (DAMPs) from damaged tissues such as thrombin or histamine reach the capillary endothelium, they stimulate the cells to express a glycoprotein called P-selectin (CD62P) on their surface. P-selectin is normally stored in cytoplasmic granules but can move to the cell surface within minutes after cell stimulation. Once expressed, the P-selectin can bind a protein called L-selectin (CD62L) on the surface of passing neutrophils. At first, this binding is transient because the neutrophils readily shed their L-selectin. Nevertheless, the neutrophils gradually slow, roll along the endothelial cell surface as they lose speed, and eventually come to a complete stop (Figure 4-6). This mainly happens in venules where the vessel wall is thin and the diameter sufficiently small to permit the neutrophils to make firm contact with the endothelium.

Changes in Neutrophils

As neutrophils roll along the endothelial surface, the second stage of adhesion occurs. Platelet-activating factor (PAF), secreted by the endothelial cells, triggers the rolling neutrophils to express an adhesive protein called CD11a/CD18 or leukocyte function–associated antigen-1 (LFA-1). LFA-1 is an integrin that binds to an intercellular adhesion molecule-1 (ICAM-1 or CD54) on the endothelial cells (Figure 4-7). This strong binding brings the neutrophil to a complete stop and attaches it firmly to the vessel wall despite the shearing force of the blood flow. Adherent neutrophils also secrete small amounts of elastase. The elastase removes CD43 (leukosialin), an antiadhesive protein, from the neutrophil surface, permitting the cells to bind even more strongly.

After several hours, endothelial cells activated by IL-1, IL-23, or tumor necrosis factor-α (TNF-α) express the strongly adhesive E-selectin (CD62E). IL-1 and IL-23 also induce endothelial cells to produce the chemokine CXCL8, and this attracts still more neutrophils. TNF-α stimulates endothelial cells to secrete IL-1. It also promotes vasodilation, procoagulant activity, and thrombosis and increases the expression of adhesion proteins and chemotactic molecules.

Neutrophils themselves increase vascular permeability and open up gaps between endothelial cells as a result of endothelial cell contraction and disruption of intercellular junctions. They secrete the chemokines CXCL1, 2, 3, and 8 in response to the binding of LFA-1 to endothelial ICAM-1. Neutrophil phospholipase A2 releases arachidonic acid, which is converted to leukotriene A₄. This is subsequently processed by endothelial cells to produce permeability-inducing thromboxane A₂ and leukotriene C₄. Neutrophil-derived oxidants also increase vascular permeability.