Myeloid cells

Neutrophils

Neutrophil differentiation in the marrow can be roughly divided into six morphologically distinctive stages: myeloblast, promyelocyte, myelocyte, metamyelocyte, band, and mature neutrophil ( Fig. 10.2 ). Differentiation begins in the marrow with the myeloblast, a relatively large immature cell with oval nucleus, prominent nucleolus, and scanty agranular basophilic cytoplasm. Myeloblasts typically express immature markers CD34 and CD117 and can usually be differentiated from other types of blasts by expression of myeloid cell surface proteins CD13 and CD33 and the granule enzyme myeloperoxidase (MPO) . Cytoplasmic differentiation of the myeloblast gives rise to the promyelocyte , a large cell with an oval nucleus, small nucleolus, and abundant light blue cytoplasm containing numerous lavender-colored (azurophilic) primary granules . Further differentiation yields the myelocyte , a medium-sized cell with an oval nucleus, absent nucleolus, and abundant pale cytoplasm with an admixture of azurophilic primary and light pink secondary granules . The myelocyte is the last stage in leukocyte differentiation capable of mitotic division. Further differentiation of the myelocyte yields the metamyelocyte , a small- to medium-sized cell with an oval, indented nucleus and abundant pale-stained cytoplasm with numerous secondary and tertiary granules, followed by the smaller band neutrophil with an elongated nonsegmented nucleus and ending with the mature segmented neutrophil ( polymorphonuclear leukocyte ) with a segmented nucleus (typically three or four segments). As myeloid cells mature, secondary and tertiary granules accumulate, and primary granules decline in number.

As mentioned previously, neutrophils contain two major types of granules, primary (azurophilic) and secondary (specific) granules, as well as tertiary granules and secretory vesicles ( Fig. 10.3 ). Primary granules are medium-sized, lavender-colored (i.e., azurophilic) cytoplasmic granules on Wright-stained smears. Primary granules first become apparent at the promyelocyte stage and contain MPO, lysozyme, elastase, and defensins. The enzyme MPO catalyzes the reaction of hydrogen peroxide and chloride ion to form the microbicidal agent hypochlorous acid (“bleach”). Lysozyme is an enzyme that hydrolyzes peptidoglycans that make up the cell wall of primarily gram-positive bacteria. Elastase is an enzyme that not only cleaves elastic tissue (allowing for movement of neutrophils through connective tissue) but also degrades bacterial membrane proteins. Defensins are small, cationic, microbicidal proteins that kill microbes by binding to and forming porelike membrane defects, leading to osmotic lysis.

Secondary granules first become apparent at the myelocyte stage as small, indistinct, light pink cytoplasmic granules on Wright-stained smears. Secondary granules in neutrophils contain lactoferrin, collagenase, histaminase, and lysozyme. The antimicrobial properties of the iron-binding protein lactoferrin result from interference with iron bioavailability for bacterial growth, direct damage to microbial cell membranes, and interference with virus binding to cells. Secondary granules in eosinophils also contain major basic protein (MBP) , which is toxic to helminthic parasites. Meanwhile, secondary granules in basophils (and mast cells) contain histamine , which acts to increase microvascular permeability, allowing for leukocyte migration into inflamed tissues. Tertiary granules become most apparent at the metamyelocyte stage and contain gelatinase (matrix metalloproteinase), an enzyme that may play a role in neutrophil migration through connective tissue. Neutrophils also contain secretory vesicles that contain leukocyte alkaline phosphatase (LAP) , an enzyme for which a distinct function has yet to be determined ( Box 10.1 ).

BOX 10.1

Technical Note

Differentiation of granulocytes and precursors from monocytes and precursors on blood and bone marrow smears can be accomplished by staining for either myeloperoxidase (MPO) or chloroacetate esterase (CAE) activity with a soluble colorless substrate converted to an insoluble colored product that is deposited within the cytoplasm and can easily be seen by light microscopy ( Fig. 10.4 ). Whereas granulocytes are positive for MPO and CAE, monocytes are negative. Monocytes can be positively identified by staining for alpha naphthyl butyrate esterase (ANBE) .

Most neutrophils remain in reserve within the marrow storage pool , ready to enter the circulation when triggered by infection or inflammation. Blood neutrophils themselves spend little time in the circulation and instead rapidly enter inflamed tissues. Neutrophils are also found loosely adherent to vascular endothelium, especially in the pulmonary capillary bed and in the marginal pool . Neutrophils in the marginal pool rapidly enter the circulation by demarginating in response to physical or emotional stress.

Circulating neutrophils enter inflamed tissues by first rolling along and firmly binding to activated endothelium and then passing through postcapillary venules into inflamed tissues ( Fig. 10.5 ). The process begins with inflammatory cytokine-induced expression of leukocyte adhesion receptors (E-selectin, P-selectin, intercellular adhesion molecule 1 [ICAM-1]) on endothelial cells. Passing neutrophils first loosely “roll along” the endothelium via neutrophil L-selectin interaction with endothelial E- and P-selectins. This is followed by firm binding of neutrophils to endothelium via neutrophil integrin (leukocyte function-associated antigen 1 [LFA -1]) interaction with endothelial ICAM-1. Firmly bound neutrophils then migrate through platelet endothelial cell adhesion molecule ( PECAM) –modified endothelium in response to a variety of chemotactic signals (complement C5a and IL-8) arising from inflamed or damaged tissues. Leukocyte response to external signals is mediated by binding of ligands to cell surface receptors for cell adhesion, phagocytosis, chemotaxis, and cell growth ( Fig. 10.6 ). Adhesion to endothelium is mediated by receptors for LFA-1 and ICAM-1. Movement of leukocytes to sites of tissue damage or infection is mediated by binding of inflammatory chemokines to leukocyte chemokine receptors. Leukocyte maturation and growth signals are generated by binding of cytokines to cytokine receptors. Neutrophils that collect at sites of infection release netlike complexes of chromatin and antimicrobial agents (including myeloperoxidase, elastase, and defensins) that entrap and kill extracellular pathogens. Formation of these netlike extracellular traps (NET) is dependent on peptidyl-arginine deiminase (PAD4) –mediated nuclear disintegration.

Neutrophilia is defined as an increased absolute blood neutrophil count in excess of 11,000 neutrophils per microliter in adults (somewhat higher in children) ( Fig. 10.7 ). Neutrophilia is associated with a range of conditions, including bacterial infections, tissue damage (e.g., myocardial infarction or thermal burns), chronic inflammatory diseases (e.g., rheumatoid arthritis or ulcerative colitis), malignancy, myeloproliferative neoplasms, metabolic disorders (e.g., thyrotoxicosis), drugs (e.g., corticosteroids or lithium), and severe emotional stress ( Table 10.2 ). Although neutrophilia is commonly seen with gram-positive bacterial infection, neutropenia sometimes occurs with gram-negative bacteremia or septic shock. Rarely, ingested bacteria may be seen within neutrophils on peripheral smear ( Fig. 10.8 ). Extreme reactive neutrophilia accompanied by increased immature myeloid cells (myeloid left shift) is referred to as a leukemoid reaction because these findings may mimic chronic myeloid leukemia. Leukemoid reactions may be seen in acute infections (especially in children), hemolysis, and a variety of solid tumors. Leukemoid reactions can be differentiated from chronic myeloid leukemia by staining for LAP. Whereas leukemoid neutrophils express high LAP, neutrophils in CML are LAP weak to negative ( Fig. 10.9 ). Left-shifted neutrophilia accompanied by numerous nucleated RBCs is termed leukoerythroblastosis ( Fig. 10.10 ). This condition usually indicates the presence of an abnormal infiltrative process in the bone marrow, such as metastatic cancer, leukemia, or myelofibrosis.

Several neutrophil function disorders lead to increased susceptibility to infection, most often superficial pyogenic bacterial infections. Neutrophil dysfunction can arise from defects in adhesion, chemotaxis, degranulation, and bactericidal activity ( Fig. 10.11 ). Adhesion defects , such as the autosomal recessive disorder leukocyte adhesion deficiency caused by β2-integrin deficiency, prevent the transmigration of circulating neutrophils through vascular endothelium into infected tissues (diapedesis). Patients with leukocyte adhesion deficiency typically present with neutrophilia and recurrent nonsuppurative bacterial infections (without pus formation).

Chemotaxis defects , such as those caused by ethanol intoxication, opsonin (immunoglobulin, complement) deficiency, and immune complex disease , prevent neutrophil migration to areas of infection by interfering with neutrophil response to complement C5a and IL-8 elaborated by inflamed tissue.

Degranulation defects prevent release of bactericidal substances. In the rare autosomal recessive disorder Chediak-Higashi syndrome , abnormal lysosomal fusion leads to huge neutrophil granules noted on peripheral smear. Patients with Chediak-Higashi syndrome are usually susceptible to pyogenic infections (especially Staphylococcus aureus ) because of both neutropenia and various neutrophil defects in chemotaxis, degranulation, and microbicidal activity. The neutropenia appears to result from reduced marrow neutrophil production, that is, ineffective granulopoiesis.

Microbicidal defects lead to recurrent bacterial and fungal infection. Mutations of enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase lead respectively to deficiency of neutrophilic superoxide or hypochlorous acid, tyrosyl radical, and peroxynitrite ( Fig. 10.12 ). Chronic granulomatous disease (CGD) is caused by mutations in NADPH oxidase , the enzyme responsible for conversion of molecular oxygen (O ₂ ) to microbicidal superoxide (O ₂ ⁻ ). Most cases CGD are X-linked and thus are most common in males. However, up to one-third of cases follow an autosomal pattern of inheritance. Superoxide-deficient neutrophils and monocytes in CGD ingest but do not kill catalase-positive microorganisms, including S. aureus, Aspergillus fumigatus, and Candida albicans. Under normal circumstances, ingested microbes are killed within phagolysosomes by toxic oxidizing agents, including hydrogen peroxide and superoxide. However, by neutralizing hydrogen peroxide, catalase-positive microbes are especially resistant to killing. The diagnosis of CGD can be established by the nitroblue tetrazolium (NBT) test . In this test, normal NADPH oxidase–positive neutrophils oxidize the colorless NBT substrate to form an insoluble, dark blue reaction product. In contrast, a negative to weak positive NBT test is seen in CGD.

Acquired neutropenia (absolute neutrophil count <1500/μL) is often seen after viral infections, especially in childhood. Other causes of acquired neutropenia include acquired immunodeficiency syndrome (AIDS), overwhelming sepsis, drugs, autoimmunity, and aplastic anemia ( Table 10.3 ). Patients with an absolute neutrophil count below 500/mL because of marrow failure or chemotherapy have a significantly increased risk of systemic bacterial infection and should be managed as inpatients with intravenous antibiotics. On the other hand, there is poor correlation between neutrophil count and risk of infection in patients with adequate marrow reserve. For example, children with chronic benign neutropenia and many adults with immune neutropenia show no increased risk of serious infection. Chronic neutropenia of any cause may lead to pyoderma caused by S. aureus, Escherichia coli, or Pseudomonas aeruginosa and otitis media caused by Streptococcus pneumoniae or P. aeruginosa.