Electron micrograph of a human mature neutrophil. The nucleus shows the typical multilobed configuration with the heterochromatin at the periphery and the euchromatin more centrally located. A small Golgi apparatus (G) is present; other organelles are sparse. The punctate appearance of the cytoplasm adjacent to the convex aspect of the nuclear profile is due to glycogen particles. Adjacent to the concave aspect of the nuclear profile are numerous granules. Specific granules appear less dense and more rounded than azurophilic granules. The latter are fewer in number and are extremely electron dense, x 22,000. (Courtesy of Dr. Dorothea Zucker-Franklin.) For comparison, the inset shows a neutrophil from a blood smear observed in the light microscope. XI,800.

• Azurophilic granules (primary granules) are larger and less numerous than specific granules. They arise early in granulopoiesis and occur in all granulocytes, as well as in monocytes and lymphocytes. The azurophilic granules are the lysosomes of the neutrophil and contain myeloperoxidase, which appears as a finely stippled material with the TEM. Myeloperoxidase helps to generate highly reactive bactericidal hypochlorite and chlor-amines. In addition to a variety of the typical acid hydrolases, azurophilic granules also contain cationic proteins called defensins, which in function are analogous to antibodies.

• Tertiary granules in neutrophils are of two types. One type contains phosphatases and is sometimes called a phosphasome. The other type contains metallopro-teinases, such as gelatinases and collagenases, which are thought to facilitate the migration of the neutrophil through the connective tissue.

Aside from these granules, membrane-bounded organelles are sparse. A small Golgi apparatus is evident in the center of the cell, and mitochondria are relatively few in number (see Fig. 9.7).

Neutrophils are motile cells; they leave the circulation and migrate to their site of action in the connective tissue

An important property of neutrophils and other leukocytes is their motility. Neutrophils are the most numerous of the first wave of cells to enter an area of tissue damage. Their migration is controlled by the expression of adhesion molecules on the neutrophil surface that interact with corresponding ligands on endothelial cells (Fig. 9.8).

The initial phase of neutrophil migration occurs in the postcapillary venules and is regulated by a mechanism involving neutrophil-endothelial cell recognition. Selectins on the surface of the circulating neutrophil (CD62L) interact with receptors (GlyCAM-1) on the surface of the endothelial cells. The neutrophil becomes partially tethered to the endothelial cell as a result of this interaction, which slows the neutrophil and causes it to roll on the surface of the endothelium. In the second phase, another group of adhesion molecules on the neutrophil surface, called integ-rins (VLA 5), are activated by chemokine signals from the endothelial cells. In the third phase, integrins and other adhesion molecules from the immunoglobulin superfamily (e.g., ICAM, VCAM) expressed on the neutrophil surface engage with their specific receptors on the endothelial cells, attaching the neutrophil to the endothelial cell. The neutrophil then extends a pseudopod to an intercellular junction. Histamine and heparin released at the injury site by perivascular mast cells open the intercellular junction, allowing the neutrophil to migrate into the connective tissue. With the TEM, the cytoplasmic contents of a neutrophil pseudopod appear as an expanse of finely granular cytoplasmic matrix with no membranous organelles (see Fig.

9.7). The finely granular appearance is due to the presence of actin filaments, some microtubules, and glycogen, which are involved in the extension of the cytoplasm to form the pseudopod and the subsequent contraction that pulls the cell forward. Once the neutrophil enters the connective tissue, further migration to the injury site is directed by a process known as cbemotaxis, the binding of chemoattractant molecules and extracellular matrix proteins to specific receptors on the surface of the neutrophil.

Neutrophils are active phagocytes at the site of inflammation

Once at the site of tissue injury, the neutrophil must recognize any foreign substance before phagocytosis can occur. Neutrophils can recognize some bacteria and foreign organisms that have had no modifications made to their surfaces, whereas others must be opsonized (coated with antibodies and/or complement) to make them more attractive to the neutrophil. After recognition and attachment, the antigen is engulfed by extended pseudopods of the neutrophil and internalized to form a phagosome (Fig. 9.9). Specific and azurophilic granules then fuse with the phagosome membrane, and the lysosomal hydrolases of the azurophilic granules digest the foreign material. After digestion, the degraded material is stored in residual bodies or exocytosed. Most neutrophils die in this process; the accumulation of dead bacteria and dead neutrophils constitutes the thick yellowish exudate called pus.

Neutrophils also secrete interleukin-'l (IL-1), a substance known as a pyrogen (fever-inducing agent). IL-1 induces synthesis of prostaglandins, which in turn act on the thermoregulatory center of the hypothalamus to produce fever. Fever is therefore a consequence of acute inflammation involving a massive neutrophilic response.

Inflammation and wound healing also involve monocytes, lymphocytes, eosinophils, basophils, and fibroblasts

Monocytes also enter the connective tissue as a secondary response to tissue injury. At the site of tissue injury they transform into macrophages that phagocytose cell and tissue debris, fibrin, remaining bacteria, and dead neutrophils. Normal wound healing depends on the participation of macrophages in the inflammatory response; they become the major cell type in the inflammatory site after the neutrophils are spent. At the same time that the macrophages become active at the site of inflammation, fibroblasts near the site and undifferentiated mesenchymal cells in the adventitia of small vessels at the site begin to divide and differentiate into fibroblasts and myofibroblasts that will secrete the fibers and ground substance of the healing wound. Like neutrophils, monocytes are attracted to the inflammatory site by chemo-taxis. Lymphocytes, eosinophils, and basophils also play a role in inflammation, but they are more involved in the immunologic aspects of the process (see Chapter 13).


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