Page 1311 - Clinical Immunology_ Principles and Practice ( PDFDrive )

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Assessment of Neutrophil Function

Debra Long Priel, Douglas B. Kuhns

Neutrophils, also known as polymorphonuclear neutrophils destruction of the ingested microorganism. Excess production
(PMNs; because of their multilobed nucleus) or granulocytes of ROS and release of lysosomal hydrolases into the extracellular
(because of the numerous granules found in the cytoplasm), are milieu can lead to tissue damage and inflammation.
major contributors to innate host defense against invading Neutrophils can also contribute extracellular microbicidal
microorganisms, particularly bacteria and fungi. Neutrophils activity through the formation of neutrophil extracellular traps
are bone marrow–derived, terminally differentiated cells that (NETs), a matrix of DNA and granular enzymes that is purported
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are incapable of further cellular division but may be also be to entrap bacteria and promote their killing. During NET forma-
1
derived from progenitor populations in the spleen. Early studies tion, the nucleus loses its lobular shape, the nuclear membrane
indicated that neutrophils have a short life span in the circulation disintegrates into a chain of vesicles surrounding the DNA, and
(t 1/2 = 6–8 hours) and then survive an additional 1–2 days in the granular integrity is lost. The nuclear material fills most of the
2
surrounding tissue; more recent data suggest that neutrophils cell, mixing with the granular contents. The cells round up and
may survive 10 times longer in circulation, up to 5.4 days. 3 DNA is forcibly extruded from the cell, conveying with it granular
Neutrophils, with a diameter of 10–15 µm and a volume of enzymes trapped within the DNA matrix. Mitochondria in
3
346 µm , have a unique morphology. The nucleus of the mature neutrophils are few in number and exhibit relatively little oxidative
neutrophil is segmented into 3–5 lobes with chromosomes phosphorylation but may play an important role in NET forma-
randomly distributed among the lobes. Neutrophils also have tion. Activation of neutrophils by immune complexes results in
an extensive array of storage granules prepackaged with specific marked depolarization of the mitochondria, increased mitochon-
4
proteins defined by the differentiation stage during maturation. drial ROS production, and redistribution of the mitochondria
Granules are classified into four distinct populations: azurophilic, to the periphery of the neutrophil. A low-density subset of
specific, gelatinase, and secretory granules. Azurophilic granules neutrophils may be more prone to NET formation. 10
contain myeloperoxidase, lysozyme, antimicrobial peptides, Neutrophils display a diverse array of cellular functions.
defensins, proteases, and the lysosomal acid hydrolases. The Abnormalities in these functions can severely compromise host
specific granules contain lactoferrin, lysozyme, and vitamin defense, leading to recurrent bacterial and fungal infections
B 12 –binding protein and also serve as storage pools for CD11b/ (Chapter 22). To localize specific deficiencies of neutrophil
CD18 and cytochrome b 558 of the superoxide anion radical function, assays have been developed that mimic these functions
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(O 2 )–generating enzyme, nicotinamide adenine dinucleotide both in vivo and in vitro. Often, a preliminary screening of several
phosphate (NADPH) oxidase, or NOX2. The gelatinase granules neutrophil functions is performed to localize deficits and then
are a subset of the specific granules that have a high content of more vigorous testing of specific function is performed. Assays
gelatinase. The secretory granules are highly mobilizable intracel- to assess neutrophil function should address several limitations—
lular vesicles that contain alkaline phosphatase and other surface the number of cells required for the assay, the type of cell
antigens. preparation needed (isolated neutrophils versus whole blood),
The primary function of neutrophils is the ingestion (phago- the overall incubation time for the assay, the complexity of
cytosis) and subsequent killing of microorganisms. This process the assay, and the rapidity of data collection. These issues
requires the assembly of NOX2, a multiphagocyte oxidase (phox) become more critical if multiple functional assays are planned
component enzyme complex consisting of at least three cytosolic concurrently. Since neutrophils cannot be stored or frozen and
components—p47 phox , p67 phox5 , and p40 phox6 —and two membrane maintain viability, neutrophils from a normal subject are generally
components—p22 phox and gp91 phox —that constitute cytochrome assayed in parallel to validate the results, doubling the number
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b 558 . This enzyme reduces molecular O 2 to O 2 using NADPH of assays to be performed. Additionally, isolation of neutrophils
generated by the oxidation of glucose through the pentose can take 1–2 hours, limiting the time available for functional
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phosphate pathway; O 2 either spontaneously or enzymatically assays. Fluorescent probes have increased the sensitivity of many
converts to hydrogen peroxide (H 2 O 2 ). In the presence of a metal, of the assays and eliminated the need for radioactive probes.
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2+
such as iron (Fe ), H 2 O 2 and O 2 can react to form the highly The use of multiwell microplates and microplate readers has
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reactive hydroxyl radical, OH . Alternatively, the azurophilic reduced the number of cells required and has facilitated the
granule constituent, myeloperoxidase, catalyzes the formation collection of data. Experience in the handling of neutrophils
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of hypochlorous acid from H 2 O 2 and chloride (Cl ). The combined and the time constraints of assays can limit the availability of
activities of these reactive O 2 species (ROS), antimicrobial this testing to laboratories that specialize in assessment of
peptides, and lysosomal hydrolases result in the ultimate neutrophil function.
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