Phagocytes

Overview of Phagocytes

(1) Polymorphonuclear Leukocytes (aka: granulocytes, neutrophils, polys, PMNs, segmented forms or "segs") – capable of rapid movement and contain diverse and potent cytotoxins.

(2) Eosinophils and Basophils – primarily tissue cells involved in atopic processes and parasitic infections, and function largely by extracellular release of toxins.

(3) Monocytes and Macrophages – longer-lived and tend to ingest particles and digest them as a part of tissue healing.

Phagocytes circulate in an inactive state under physiologic conditions. Activation Þ release cytotoxic agents induces significant damage to normal tissue as well as damaging invading organisms, so selective cell activation is a protective mechanism for the host.

Myeloid Series of Maturation: The Neutrophil

myeloblast Þ promyelocyte Þ myelocyte Þ metamylocyte Þ band Þ Granulocyte (either PMN, Eosinophil, or Basophil)

PMNs develop in the bone marrow from the pluripotent stem cell which is induced into cell division and then encouraged to develop along granulocytic lines by G-CSF and/or GM-CSF made by monocytes.

The earliest granulocytic precursor which is morphologically recognizable is the myeloblast. These cells are capable of cell division (mitotic stage) through the myelocyte stage, and divide their granule contents among daughter cells. From the metamyelocyte stage on, the cells do not divide (post-mitotic stage). Eosinophils are recognizable at the myelocyte stage.

Secretory Products of the Neutrophil

Primary (azurophilic) granules

Arise from proximal (concave) surface of the golgi at the promyelocyte stage
Contain lysosomes which have pH optima in the acid range, including: myeloperoxidase, lysozymes, lysosomal hydrolases, cathepsins (cationinc proteins), elastase

Secondary (specific) granules

Arise from the distal (convex) surface of the golgi at the myelocyte stage
Contain enzymes with pH optima in the neutral range: alkaline phosphatase, lactoferrin (chelate iron as to prevent it from being grabbed and used by bugs), collagenase (mediator of host tissue damage by destroying normal collagen), lysozymes

Tertiary (Microvesicular) Granules

Contain: Plasminogen activator, Alkaline phosphatase

The Neutrophil membrane

Membranes of phagocytes contain both specific receptors and enzymes which can be activated to enhance or permit cell killing. The receptors enable the cells to respond specifically to certain stimuli.
The neutrophil has receptors for a bunch of different things (including C3b, Fc portions of Igs, Histamin, etc etc etc).
The receptors are linked to several effector mechanisms via G-protein and phospholipase C. These include a membrane-bound, NADPH-dependent oxidase, and calciosomes which activate cell movement and ingestion by affecting actin gel-sol interactions. In addition, the cells are capable of generating and responding to arachidonic acid metabolites, prostaglandins and leukotrienes, which affect cell movement and activation state.

Distribution of PMNs:

STORAGE POOL – PMNs in the BM; contains 7 times as many neutrophils as the intravascular pool.

INTRAVASCULAR POOL – consists of:

Marginal Pool (PMNs adherent to endothelium)

Circulating Pool (PMNs floating around in the blood, measured by peripheral blood counts. Circulate for no more than 7 hours, then migrate into tissues where they die as end-stage cells)

TISSUE POOL

The Neutrophil Life Cycle

PMNs take 10-14 d to mature from myeloblast to a mature PMN (can happen faster under stress conditions), are then released into the blood by crawling out of the BM and into the blood.

Process of exiting blood into tissues: activation Þ rolling Þ adhesion (ie margination) Þ diapedesis Þ basement membrane penetration Þ direct migration (chemotaxis)

Quick description of other myeloid cells

Eosinophil- Bilobed nucleus, red appearing granules containing basic material (major basic protein and eosinophil peroxidase which converts O₂ into toxic radicals).

Basophil - Bilobed nucleus, blue appearing granules containing acidic material (histamine), have on their surface abundant number of receptors for IgE, IgE mediates degranulation of these cells

Function of Phagocytes

(1) ROLLING along endothelial surfaces - governed by shear forces generated by laminar flow and by selectins interacting with endothelial carbohydrates.

(2) STICKING - interaction between integrins on surface of phagocytes and endothelial cell receptors.

(3) MIGRATION - Chemotaxis, leukotaxis. Phagocytic cells crawl along surfaces. Motion, whether random or in response to chemical gradients, is accomplished by activation of surface contact or receptors, with cytoplasmic contractile protein transformations which enable the cell to move in a directed fashion. Chemotactic factors - C5a, fMLP, leukotrienes and prostaglandins - are stimuli for directed movement. Cells must adhere to the wall of blood vessels before they can exit from the circulation. Production of pus requires directed movement of large numbers of neutrophils.

(4) RECOGNITION - binding to surface opsonins on particles. Ingestion is highly specific. Opsonization ("to prepare for dining") with surface deposition of C3b and IgG enables phagocytes to recognize pathogens or damaged host tissues.

(5) INGESTION - phagocytosis. The cell membrane binds to particles with surface characteristics which stimulate ingestion. Pseudopodia extend around the particle, and the pseudopodia merge at the distal end of the particle. This results in production of a PHAGOSOME, an organelle bound by external plasma membrane containing to ingested particle.

(6) DEGRANULATION - Once the phagosome is formed, the lysosomal granules move in and their membranes fuse with the plasma membrane around the phagosome to form a PHAGOLYSOSOME. The enzymes in the primary granule have acid pH optima, and the secondary have neutral pH optima - the phagolysosome acquires first a neutral and then an acid pH, so enzymes are activated in sequence.

(7) OXIDATIVE KILLING - The membrane-bound NADPH-dependent oxidase is activated by a variety of stimuli, and sets in process a 4-electron reduction of molecular oxygen, generating toxic oxygen species capable of lipid peroxidation, oxidant damage to amino acids and to carbohydrates. OXYGEN is reduced to SUPEROXIDE ANION, which in turn is reduced under the influence of superoxide dismutase and acid to HYDROGEN PEROXIDE. Superoxide anion and peroxide interact in the presence of iron to form HYDROXYL RADICAL (OH.), which is short-lived but highly toxic. Under the influence of myeloperoxidase (or eosinophil peroxidase), contained in primary granules, peroxide is transformed into HYPOCHLOROUS ACID, or bleach, which is also a strong oxidant with antibacterial activity. Peroxide is detoxified by catalase to water and oxygen.

RESPIRATORY BURST:

Form chloramines
Activate latent metalloproteinases
Inactivate antiproteinases

ANTIOXIDANTS which protect normal tissue and some organisms include CATALASE, INTRACELLULAR THIOLS (especially reduced glutathione), ASCORBIC ACID, and VITAMIN E.

Clinically Significant Neutrophil Counts

1,500 – normal

1,000-1,500 – no significant propensity to infection; fevers can be managed on outpatient basis

500-1,000 – some propensity to infection; occasionally fever can be managed on outpatient basis

>500 – significant propensity to infection; fevers should be managed on inpatient basis with antibiotics; few clinical signs of infection

Neutropenia – a condition of decreased numbers of neutrophils.

Normal neutrophil count is 1500-4000/mm³. Absolute neutrophil count (ANC) is calculated by multiplying the white count by the fraction of the cells which are neutrophils on the differential.
Susceptibility to serious infection is increased when ANC < 200.
Neutropenia (congenital or acquired) may be due to:

(1) Increased number of PMNs in marginal pool (Pseudo-Neutropenia) – occurs as a result of adherence to endothelium, usually under the influence of endogenous or exogenous substances, such as endotoxin, GM-CSF, which increase neutrophil membrane adherence.
(2) Decreased production in the bone marrow - occurs as a result of bone marrow disease - effects of drugs, infiltration with tumor, bone marrow failure (aplastic anemia, ineffective hematopoieses (myelodysplastic processes), certain viral infections (CMV, EBV), severe folate and B-12 deficiency.
(3) Increased rate of destruction - seen with autoimmune diseases, drug reactions, many infectious processes (bacterial, viral), hypersplenism.

Neutrophilia – a condition of increased numbers of neutrophils (>6,000/mm³)

usually seen as a response to acute infection, especially bacterial and fungal, and immature forms (bands and less mature forms) may circulate in response to stress.
Other causes are myeloproliferative disorders (CML, polycythemia vera, e.g.), bone marrow replacement with extramedullary hematopoieses (myelofibrosis with myeloid metaplasia), and substances which decrease neutrophil adherence (corticosteroids, epinephrine).

EOSINOPHILIA is seen with allergic reactions, parasitic infections, some tumors, and myeloproliferative processes.

BASOPHILIA is seen with myeloproliferative processes, some parasitic infections.

Disorders of Phagocyte Function

Deficiency of Igs and complement produce difficulties with recognition and chemotaxis. These may be inherited or acquired, and may result from liver failure or protein-losing enteropathy. Many immune complex disorders may consume complement components, and result in defective recognition. A variety of drugs (corticosteroids, non-steroidal anti-inflammatory agents, alcohol) may impair phagocyte function.
Several inherited disorders have lead to greatly enhanced understanding of normal phagocyte function. They include:

(1) CHEDIAK-HIGASHI SYNDROME – Autosomal recessive, with fused primary and secondary granules, impaired chemotaxis and platelet function, partial occulo-cutaneous albinism, and defects in natural killer cell function which lead to uncontrolled infection with Epstein-Barr virus which is usually fatal for these patients. Marked by presence of abnormal white blood cells which have abnormal microtubule formation, affecting movement and membrane fusion of lysosomes.
(2) PHAGOCYTE Leu-CAM, or Mo-1, DEFICIENCY – Autosomal recessive defect in leukocyte-cell adhesion molecules, or iC3b receptor, with abnormal beta chain of CR3. Patients have marked leukocytosis due to inability of cells to marginate, with susceptibility to multiple pathogens due to defective chemotaxis and phagocytosis.
(3) CHRONIC GRANULOMATOUS DISEASE OF CHILDHOOD – X-linked. Deficient activity of the NADPH oxidase. Marked by phagocytic cells that ingest but do not kiill certain bugs.

Catalase-positive organisms (S. aureus, etc) – are ingested but not killed. These bugs can destroy H₂O₂ generated by their own metabolism. Because enzyme-deficient PMNs can’t produce H₂O₂ and bug H₂O₂ is destroyed by bug catalase, H₂O₂ is not available as a substrate for myeloperoxidase. Thus, the myeloperoxidase-halide system of bacterial killing fails.

Catalase-negative organisms – are ingested and killed. These bugs (Strep, etc) produce sufficient H₂O₂ to permit oxygen-depndent microbicidal mechanisms to proceed. In effect, the substrate for myeloperoxidase is produced by the bug, and the bug in a sense kill themselves.

(4) MYELOPEROXIDASE DEFICIENCY – is sometimes associated with recurrent infections but often is of little clinical consequence.
(5) SEVERE G6PD deficiency – results in increased susceptibility to infection due to inability to mount a sustained respiratory burst because of depletion of NADPH in the absence of G6PD.
(6) ASPLENIA – whether congenital or acquired, results in increased susceptibility to encapsulated organisms (Streptococcus pneumoniae and Hemophilus influenzae type b) and some other pathogens due to loss of splenic macrophages, which are very sensitive to small amounts of opsonins on the surface of invading bacteria.