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Phagocytes are cells which ingest particles. The process of eating particles is called "phagocytosis," a process which is one of the distinguishing features of eukaryotic cells, found even in such primitive organisms as the amoeba, Chaos chaos, which hunt, ingest, and kill microbes for nutritional purposes. Many cells in our body are capable of phagocytosis, but only two are good enough to be considered professional: neutrophilic polymorphonuclear leukocytes (PMN, neutrophils) and macrophages, which are derived from monocytes.
Our professional phagocytes work in concert with fluid phase factors -- notably complement and antibodies -- to hunt, ingest, and kill microbes in defense of the multicellular being. At sites of typical local infection, the neutrophil dominates early (acute) responses (30 min); however, macrophages takeover in longstanding (chronic) conditions. This takeover is generally observed within 48 h (begins in 6h). This is not always true (as you will see, the neutrophil remains the predominant cell throughout all phases of periodontal disease in the gingival crevice and the junctional epithelium).
The immunological clearance of most pathogenic microbes requires phagocytic cells. Fluid phase factors are simply not good enough, in fact, such microbes are pathogenic because they can usually evade neutralization by complement and antibody. Additionally, phagocytes are efficient at degrading and removing debris (such as dead bacterial bodies) and providing a locally responsive means of orchestrating inflammatory processes. These additional functions cannot be performed by fluid phase factors alone.
Herein, we will discuss neutrophils as our "prototypical" phagocyte. Monocytes behave very much like neutrophils, but have several additional functions which we will discuss in the following chapter.
The neutrophil has been referred to as a first line of defense, meaning that it is the first defensive cell type to be recruited to a site of inflammation; however, there are other inflammatory cells already present (such as the mast cell). The neutrophil possesses an awesome mix of antibacterial weaponry, both within its granules and by virtue of its ability to reduce dioxygen to hydrogen peroxide (described below). Its primary mission is to find bacteria or fungi and neutralize them by phagocytosis. Neutrophils are much smaller than tissue macrophages (22 µm) but about the same size as monocytes.
Maturation of the neutrophil. The neutrophil "begins" its 2-week lifespan in the bone marrow, with the commitment of a hematopoietic stem cell to myeloblastic differentiation. Even before this cell ceases proliferation, during the "promyelocytic stage" of the "mitotic phase," it begins to produce storage granules called azurophil granules, which contain certain enzymes (such as myeloperoxidase, MPO and elastase). Because these are the first granules to appear in differentiation, they are also called "primary granules." Also, the formation of granules prior to the cessation of proliferation is a fairly unusual commitment (usually, cells terminate proliferation prior to such differentiation). Interestingly, promyelocytes are not fully committed to neutrophilic differentiation, and can be driven in the monocytic direction. This reinforces the close kinship between neutrophils and monocytes, and I tend to think of neutrophils as monocyte-like cells which have undergone terminal differentiation in the bone marrow.
Specific granules are made in the "myelocyte" stage (near the end of the mitotic phase), and continue to be produced for some time during the post mitotic phase. Because these granules appear second, they are also known as "secondary granules." The specific granules eventually outnumber the azurophil granules (60-70 azurophil granules per cell, and 120-140 specific granules per cell) because cell division dilutes out the azurophil granules and specific granules continue to be synthesized throughout the mitotic phase. Note the time sequence of mRNA transcript appearance in the developing cells (Figure 2). Clearly, there are successive waves of mRNA transcription. It is felt that different granule types simply reflects the time at which the different proteins (such as MPO, elastase, lactoferrin, etc) were packaged together by the golgi. In other words, the difference between specific and azurophil granules is an artificial one, and in real life, things are not so simple. At the end of the myelocytic stage, the chromatin condenses and signals the end of the mitotic phase. After that, the neutrophil matures rather slowly (4-5 days).Fate of the neutrophil. Most of the neutrophils formed in the bone marrow never enter the blood, instead they are phagocytosed by bone marrow macrophages. The bone marrow contains about 30 times more neutrophils than blood. This excess of bone marrow neutrophils provides a buffer against neutrophil depletion and enables the body to respond to various challenges with a massive out-pouring of neutrophils. The most well-known causes of neutrophil release into the blood are infections.
Most of the neutrophils which manage to enter the blood circulation never see the external environment. One per cent escapes through the oral cavity of dentulous individuals. Many neutrophils die within the tissues and may provide some nutrition to these tissues (remember the yolk sac origin of the hematopoietic cells?). In this capacity, they act as "trephocytes," or cells which bring nutrition. This may explain why vigorous muscular exercise (football, childbirth, running, epileptic fits) results in neutrophilia (elevation of blood neutrophils up to 8 fold; ie., 20-30 x 106 cells/ml). This form of neutrophilia is regulated by epinephrine. By dying within the tissues, neutrophils may also release cytosolic antifungal agents which confer protection against mucocutaneous fungal infection. In this capacity, they have been referred to as "necrocytes." Most of the circulating blood neutrophils are eliminated by elements of the "reticuloendothelial system -- a system comprised of monocytes and macrophages which are neither reticular nor endothelial."
Interaction of neutrophils with chronic immune cells. Without a doubt, the functions outlined above represents the primary importance of neutrophils. Nevertheless, we also know that neutrophils are capable of regulation chronic inflammatory activities via both preformed granule components (Lala, A., et al., 1992. Oral Microbiol. Immunol. 7: 89-95) and inducible molecules such as TNFa, IL-1b, IL-1ra, IL-8, and TGFb1 (Cassatella, 1995. Immunol. Today 16: 21-26). The neutrophil releases relatively modest levels of cytokines (compared to mononuclear cells), but this varies with the stimulus and the measured cytokine. For example, the neutrophil produces just as much TGFb1 as monocytes if stimulated by LPS.
The adherence of neutrophils to tissues is a carefully regulated process. Adherence to the wrong tissue can lead to severe damage (Kishimoto. 1991. J. NIH Res. 3:75-77).
Adherence to vascular endothelium. Adherence (margination or pavementing) to the lumenal surface of the vascular endothelium is an important first step in the biological function of phagocytes. When phagocytes can't do this properly, severe recurrent infections occur. There are at least two phases of adherence: the selectin-dependent phase and the integrin-dependent phase. The phases are in reference to the two types of adhesion molecules which play a role leukocyte adherence to endothelium: (i) selectins - carbohydrate-binding (lectins) molecules once referred to as "LEC-CAMs" and (ii) leukocyte b2 integrins - LFA-1 and Mac-1(CR3).
Leukocyte b2 integrins include three related transmembranous glycoproteins consisting of a noncovalently associated heteroduplex structure (the larger subunit, a = 180-150 kdal; the smaller b2 subunit = 95 kdal; Fig. 4B). They all share a common b2 subunit and differ in the a subunit. The a subunit genes are all clustered on chromosome 16 and the b2 subunit is encoded on chromosome 21. The three leukocyte integrins are: LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18 or CR3), and p150,95 (CD11c/CD18 or CR4). The distribution of LFA-1 (leukocyte function-associated molecule) is greater than that of the other two leukocyte b2 integrins, found on virtually all leukocytes (including lymphocytes) except some tissue macrophage.
The adherence of phagocytes to vascular endothelium is a dynamic process which is initiated by a phenomenon called "rolling." The neutrophil or monocyte allows itself to be pushed along by the blood, occasionally rolling on the lumenal surface of the capillaries by weak, reversible interactions. Rolling permits the leukocyte to probe the endothelium, possibly using L-selectin (leukocyte-endothelial cell adhesion molecule-1, LECAM-1) which is constitutively expressed on "unstimulated leukocytes." L-selectin is expressed by a number of cells, including lymphocytes. It binds to certain extended, sialylated, fucosylated, sulfated, O-linked carbohydrates present on certain endothelial glycoproteins, 50 and 90 kdal, which have been designated "glyCAMs," or "vascular addressins." The 90 kdal glyCAM has been identified as sialomucin CD34 (Baumhueter et al., 1993. Science 262: 436-438). In addition, rolling may be mediated by ICAM-1 expressed on the endothelial cells and leukocyte LFA-1 in a low affinity state. However, inasmuch as certain specific carbohydrates can impede the rolling reaction, it would appear that the most prominent role (kind of a pun!) in rolling is played by a lectin-ligand interaction, ie., L-selectin and the vascular addressins.
Interendothelial cell-junction diapedesis is the migration of the leukocyte between the endothelial cells (as opposed to going through the cells) and egress of the leukocyte through the capillary wall. Actually, it is still debated whether leukocytes go between or through the endothelial cells, and some people believe neutrophils go through the endothelial cells as well as between them. At any rate, diapedesis is initiated by the integrin-dependent phase of adherence.
Interleukin-8 (IL-8) is a cytokine produced by stimulated endothelial cells (Fig. 5c) and monocytes/macrophages. In transendothelial migration, the endothelial cells release IL-8 which causes the leukocyte to rapidly shed its L-selectin (Fig. 5d). In fact, treatment of neutrophils with IL-8 brings about a rapid loss of ability to bind activated endothelial cells (IL-8 was once called "leukocyte adhesion inhibitor"). Other hormones, including GM-CSF and platelet activating factor (PAF), also are elaborated by excited endothelial cells and may cause the shedding of L-selectin. The shedding of L-selectin is required for transendothelial egress.
As a result of IL-8 and PAF stimulation (they are secretagogues), the phagocyte also reveals the leukocyte b2 integrins (Huber et al., 1991. Science. 254:99-102), Mac-1 (CR-3) and LFA-1 (high affinity) which, in the case of the neutrophil, have been sequestered within the specific granules. These two molecules assume a higher affinity state (probably as a result of phosphorylation) and co-associate reversibly with cytoskeletal elements. Reversible binding --
LFA-1 binds the immunoglobulin superfamily molecule, ICAM-1 (and ICAM-2) and is crucial in mediating reversible leukocyte-endothelial adherence (leukocytes referring to both myeloid and lymphoid white blood cells). The phosphorylation of the cytosolic domains of LFA-1 may be important in dictating the reversibility of the process. Both the cytosolic domain of the a and the the b2 subunit of LFA-1 are phosphorylated, but only the cytosolic domain of the b2 subunit is phosphorylated in an inducible, reversible manner (Figdor et al., 1990. Immunol. Today 11:277-280). The ligand affinity of LFA-1 increases with phosphorylation and decreases with dephosphorylation. In this state, the leukocyte begins to search for the boundaries between two endothelial cells.
Leukocyte adhesion deficency, type 1 (LAD-1)
The congenital absence (in homozygotes) or deficiency (in heterozygotes) of the b2 subunit (CD18) is referred to as "leukocyte adhesion deficiency, type 1." This results in the absence/deficiency of the leukocyte b2 integrins LFA-1, Mac-1, and p150/95. Severe pyogenic infections are seen in such individuals primarily because the neutrophils cannot easily leave the blood.
Another immunoglobulin superfamily cell adhesion molecule, CD31 (Platelet-Endothelial Cell Adhesion Molecule-1, PECAM-1, EndoCAM) congregates at the intercellular borders of endothelial cells (Fig. 5e). It is also found on all leukocytes. CD31 is a 130 kdal transmembrane glycoprotein which serves as a homophilic glue; that is, it's ligand appears to be another CD31. The binding of CD31 on endothelium with CD31 on leukocytes may guide leukocytes to the boundaries between endothelial cells and also appears to increase the binding avidity of the b2 integrins (as well as the b1 integrins). Increasing integrin binding avidity has been referred to as "integrin activation." Two domains of CD31 appear to be important in this homophilic interaction (the domains have been designated "C1" and "C2") Blocking CD31 interaction blocks greatly impairs transendothelial migration.
The interendothelial transmigration of phagocytes (Fig.5f) is rapid. It is initiated by the indentation of the endothelium. Neutrophils show distinct polarization, extending a pseudopod between the endothelial cells while maintaining the nuclei and granules on the lumenal side. Transmigration across the endothelial boundary does not require a chemotactic gradient (see "chemotaxis" below). Initially, the neutrophil extends a pseudopod virtually free of both granules and nucleus between two adjacent endothelial cells. The leukocyte forms very close contact with the endothelial cell. This is important because it makes a tight seal which prevents the extravasation of fluids outside of the vessel. Finally, the leukocytes accumulate briefly between the basement membrane and the endothelial cell before entering the connective tissues. It is unknown how the leukocyte penetrates the basement membrane, it had been classically felt that proteases faciltated the process. However, a variety of protease inhibitors (ie., methoxysuccinyl-alanyl-alanyl-valyl-prolyl-chloromethyl ketone [MSAAVPCK], a fairly specific one for elastase) do not seem to block the the migration of leukocytes across the basement membrane. CD31 (via one of its domains called "C6") also appears to assist the leukocyte in traversing the basement membrane.
The process the leukocyte uses to locate its target and subsequently migrate to the target location is known as "chemotaxis." Chemotaxis is the directed movement of a cell along a chemical concentration gradient. About 1 nM chemoattractant (such as formyl-methionyl peptide) is required. The cell senses a chemical gradient (about a 1% change in concentration) across its body. Chemokinesis is the increased motility of a cell in response to a chemical that does not involve the sensing of a concentration gradient or any directed movement. Random migration is the non-directional movement of a cell without any apparent external stimulation. Mature neutrophils are capable of moving at a rate of 400 µm/h. Immature neutrophils move more slowly at 60 µm/h. In fact, many important activities are depressed in the immature neutrophils.
Phagocytes respond to chemical signals (chemotaxins) from the external environment. The neutrophil recognizes signals from a variety of sources, as shown in table 1. These chemical signals can direct the movement of the phagocyte if provided as a gradient and stimulate secretion (secretagogue). Secretion results in the release of specific granule components and the "up-regulation" of such stored membrane molecules such as Mac-1 (CR-3) and cytochrome b.
IL-8 is a multifunctional, homodimeric "chemokine" of the a subfamily (ie., it possesses a Cys-X-Cys motif and is encoded on chromosome 4) which attracts both neutrophils and T-cells. Several factors (TNFa and IFNg) in table 1 "prime" the phagocyte rather than act as chemotaxins. Priming is a vague term, but refers to the process of heightening the responsiveness of the phagocyte to other chemotaxins. Formyl-methionyl peptides such as N-formyl-methionylleucylphenyl-alanine (FMLP) are important chemotaxins generated by bacteria as a biproduct of protein synthesis. Bacteria often initiate protein translation with N-formyl-methionine. Bacterial LPS (endotoxin) occasionally acts in an opposite fashion to factors which "prime"; it reduces chemotaxis induced by IL-8, but not chemotaxis induced by N-formyl-methionyl peptides (Bignold et al., 1991. Infect. Immun. 59:4255-4258). This may be a "positive" mechanism of immobilizing of leukocytes once they are in the vicinity of a bacterial target (as opposed to simple immobilization in an area with the highest concentration of chemotaxins).
As the cell moves toward its target, its receptors are internalized and re-expressed (in the lingo of cell biologists, receptors divide time between the membrane and endosomal compartments). On the neutrophil, receptors are swept backward (tail-lighting) prior to ingestion. Receptors are then re-expressed either as a result of the secretion of specific granules (where receptors are sequestered) or by some other process. This process is referred to as "receptor cycling."
The most well-studied chemotaxin receptor is the receptor for formylmethionyl peptides, known as the "formylmethionyl peptide receptor," or "FPR" (Figure 6B). The normal human neutrophil expresses about 50,000 FPR/cell. The FPR binds to formylated hydrophobic peptides derived from bacteria. The FPR is a transmembrane glycoprotein (about 32 kdal, unglycosylated) which undergoes affinity state changes modulated by a cytosolic, 40 kdal GTP/GDP-binding "G protein." The G protein is sensitive to cholera and pertussis toxin. Chemotaxis and high affinity binding of FMLP are inhibited by those toxins.
The effects produced by stimulation of the FPR and other chemotaxin receptors are transient (order of minutes). There are likely to be several mechanisms of terminating the chemoattractant signal, including destruction of the chemoattractant, a task which has been assigned variously to metalloproteases (ie., CD10, see appendix) and myeloperoxidase. A chymotrypsin-like membrane serine protease ("mAb 1-15 enzyme") has also been shown to bind FMLP, but no functional significance has been given to this observation (King et al., 1991. J. Immunol. 146:3115-3123) except that it may be involved somewhere in the response of neutrophils to FMLP (either as a receptor or as a ligand inactivator).
Complement receptors. The leukocyte b2 integrins play a fundamental role in the binding of the leukocyte to its target. Important in target-binding are Mac-1 (CR3) and p150,95 (CR4). Mac-1 is found on granulocytes, monocytes, macrophage, large granular lymphocytes, and some immature B-cells. The closely-related p150,95 is associated almost exclusively with phagocytic myeloid cells (neutrophils and monocytes). Mac-1 and p150,95 recognize iC3b. These are the two most important complement receptors for phagocytosis. In monocytes, antibodies against CR4 are more effective at blocking phagocytosis than antibodies against CR3, suggesting that for monocytes, CR4 is more important in phagocytosis than CR3.
Fc Receptors (FcR). Phagocytic adhesion to targets may also be mediated by antibody using surface receptors collectively known as "Fc receptors." "Fc" refers to a portion of the antibody to which the receptor binds. The student should bear in mind that phagocytes cannot bind all immunoglobulin types ("isotypes" will be discussed in chapter 8), but instead will bind only those types for which it possesses receptors. Importantly, there are no Fc receptors for IgM, which means that antibody-mediated phagocytosis (complement-independent) requires a secondary immune response involving a switch in immunoglobulin isotype from IgM to IgG.
In contrast to interendothelial transmigration, epithelial transmigration by phagocytes requires a gradient and is slower. Both neutrophils and monocytes can cross endothelium readily; and in vitro, both are capable of making indentations in endothelial cells. Neither neutrophils nor monocytes can indent epithelium; and in the absence of a chemical gradient, no monocytes and only a few neutrophils can traverse an epithelial barrier (Cramer. 1992. In: Gallin, Goldstein, and Snyderman (eds). Inflammation: Basic Principles and Clinical Correlates, Second Edition. Raven Press, NY. p341-351).
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