Page 351 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPtEr 22 Phagocyte Deficiencies 331

in the cytosol cleave the ester linkage, trapping the fluorescent lactate dehydrogenase should be monitored simultaneously. The
probe in the cytosol. The labeled neutrophils are added to each release of azurophilic granules can be assessed by determination
well and incubated in the absence or presence of phorbol myristate of β-glucuronidase activity. Supernatant fluids or cell extracts
acetate (PMA) to promote adherence through activated integrins. obtained from stimulated neutrophils are incubated with
At the end of the incubation, the wells are washed to remove 4-methylumbelliferyl-β-d-glucuronide. Alternatively, myeloper-
nonadherent cells. The fluorescence of each well is determined oxidase can be determined by using commercially available
with a fluorescent microplate reader and compared with the enzyme-linked immunoassays. CD63 is also found in the
fluorescence of a control well with a fixed number of fluorescent membrane of azurophilic granules and migrates to the neutrophil
cells. Under control conditions, fewer than 10% of the neutrophils surface after stimulation with fMLF in the presence of cytochalasin
adhere to plastic or to plastic coated with fetal bovine serum. b. The release of specific granules can be assessed by determination
Slightly more neutrophil adherence is observed on wells coated of lactoferrin levels using an enzyme-linked immunoassay. The
with fibrinogen. Treatment of normal neutrophils with PMA carcinoembryonic antigen CD66b (formerly CD67) is found on
for 30 minutes results in the adherence of 100% of the neutrophils the neutrophil surface and in the specific granules, and its
under all conditions. This adherence is abnormal in patients expression on the surface of the neutrophils is increased after
with LAD. Neutrophils isolated from patients with typical LAD-1 stimulation with fMLF or LPS. Detection of the constituents of
generally exhibit markedly reduced adherence under both secretory granules can be assessed by flow cytometric analysis
unstimulated and PMA conditions. of the change in expression of surface proteins, such as adhesion
molecules, and cytochrome b 558 of the NADPH oxidase.
Neutrophil Chemotaxis
Neutrophil chemotaxis in vivo can be evaluated by using skin Generation of Reactive Oxygen Species
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windows. Skin blisters are gently raised on the volar surface of The release of reactive oxygen intermediates, such as O 2 and
the forearm using a vacuum pump and a blister device, with H 2 O 2 , is an important component of the neutrophil bactericidal
little hemorrhage or vascular damage. The roof of the blister is machinery. Neutrophils isolated from patients with CGD are
removed, and the exposed dermis is bathed with autologous unable to generate superoxide, leading to their oxygen-dependent
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serum with the use of a skin window chamber. In 24 hours, bactericidal defect. The production of O 2 can be detected by
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exudative neutrophils accumulate in the autologous serum bathing using the reduction of cytochrome c. Because O 2 causes a
the skin lesion. The skin chamber provides a mechanism for one-to-one stoichiometric reduction of ferricytochrome c to
characterizing the immune cells as well as the soluble immune ferrocytochrome c, the resultant increase in the absorption
mediators that accumulate in the autologous serum during the spectrum at 550 nM can be used to quantitate the production of
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evolution of the inflammatory response. Chemotaxis in vitro is O 2 . Superoxide dismutase is added to an identical tube to control
generally measured by using a Boyden chamber. The Boyden for the nonspecific reduction of cytochrome c. However, since
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chamber includes three components: a lower (chemoattractant) cytochrome is not permeable to the cells, the detection of O 2 is
chamber, a nitrocellulose or polycarbonate filter layer, and an limited to that released into the extracellular milieu. Neutrophils
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upper cell chamber. The lower compartments of the Boyden isolated from patients with CGD produce little O 2 in response
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chamber are filled with a chemoattractant, such as fMLF; 10 to PMA in 10 minutes. However, some patients with forms of
M) or IL-8 (10 ng/mL). Recently an improved rapid fluorescence- CGD associated with residual superoxide production have low,
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based measure of neutrophil chemotaxis, which uses a new 96-well but detectable, O 2 production in 60 minutes. Neutrophils isolated
disposable chemotaxis chamber that can be read in a fluorescence from X-linked heterozygous carriers of CGD can yield a full
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microplate reader, has been developed. spectrum of O 2 production, whereas neutrophils from autosomal
recessive carriers of CGD generally yield a normal response.
Expression of Surface Antigens Although the detection of O 2 by reduction of cytochrome c is
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The expression of cell surface antigens on neutrophils relies on useful in the diagnosis of patients with CGD, it cannot be used in
labeled mAbs analyzed by flow cytometry. The panel may include the diagnosis of carriers because of the wide spectrum of responses
the β 2 integrins (CD11a, CD11b, CD11c, and CD18), selectins that result from the degree of X-chromosome lyonization.
(CD62L), Fcγ receptors I, II, and III (CD64, CD32 and CD16), The extracellular release of H 2 O 2 can be measured by using
leukosialin (CD43), the common leukocyte antigen (CD45), and horseradish peroxidase–induced oxidation of either phenol red
markers for the specific granules (CD67), and azurophilic granules or Amplex red. PMN suspensions in the presence of horseradish
(CD63). The expression of surface antigens can be used to assess peroxidase and one of the chromophores are exposed to either
the responsiveness of neutrophils to particular ligands, such as PMA or buffer alone. Changes in optical density of phenol red
fMLF and lipopolysaccharide (LPS). at 600 nm can be determined with a standard microplate reader.
Amplex red is a much more sensitive fluorescent chromophore,
Neutrophil Degranulation and H 2 O 2 -dependent changes in fluorescence can be determined
The proteases, acid hydrolases, and inflammatory mediators with a fluorescence microplate reader.
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released from storage granules in neutrophils can mediate bacterial The NBT test is a qualitative assay of O 2 production. Either
killing, tissue damage, healing, and immune regulation. Lactoferrin whole blood or isolated neutrophils are mixed with NBT in a
from specific granules can chelate iron, resulting in a bactericidal chamber slide and stimulated with PMA for 15–30 minutes at
or bacteriostatic effect. Stimulation of neutrophils with various 37°C. The slide is counterstained with 0.1% safranin and examined
secretagogues can release granular enzymes into the extracellular under a microscope. The NBT test yields a visual record of the
fluid. Treatment of the neutrophils with cytochalasin b (5 µg/ reduction of the NBT dye to the insoluble, blue-black deposits
mL) disrupts microfilament assembly and facilitates the release of formazan. Normal neutrophils, but not neutrophils from
of both specific and azurophilic enzymes. To differentiate patients with CGD, reduce the yellow dye to black-brown-blue
degranulation from cell lysis, release of the cytosolic enzyme aggregates in the cells. The NBT test can be used to diagnose