Page 1034 - Williams Hematology ( PDFDrive )

1008 Part VII: Neutrophils, Eosinophils, Basophils, and Mast Cells Chapter 66: Disorders of Neutrophil Function 1009

C3bi (a proteolytic fragment of C3b). Of particular importance is that GRANULES
both Fcγ receptors and GPI-coupled receptors appear to be localized
to lipid rafts. Lipid rafts are important, but elusive structures that facil- Nomenclature of Neutrophil Granules
itate signal transduction leading to phagocytosis by promoting several The neutrophil is known for its granules. When Paul Ehrlich introduced
membrane protein interactions. Initially the rafts were conceptionally aniline dyes in histochemistry and discovered the different subsets of
associated with caveolae, which are structures identified on endothelial leukocytes, the neutrophil granules were divided into those that took up
cells and thought to be important for transendothelial cell traffic. The the azure dye, the azurophilic granules, and the others, the specific gran-
73,74
caveolae were identified by their high content of cholesterol lipids and ules. When the peroxidase reaction was introduced, the azurophil
the presence of the structural protein, caveolin. Rafts were subsequently granules were found to be peroxidase-positive as a result of the pres-
59
identified on neutrophils, but these cells are devoid of caveolin. Rafts ence of the major myeloid cell protein, myeloperoxidase (MPO), and
75,76
are perhaps best viewed as patches of surface membrane that attract the specific granules were thus named peroxidase-negative granules.
many hydrophobic proteins including signaling molecules such as Because the azurophil granules are formed first, in the promyelocyte,
tyrosine kinases and phosphatases. Other membrane protein receptors and the specific granules later, in the myelocyte, these are also termed
that are not normally associated with rafts may change their conforma- primary and secondary granules, respectively. A tertiary granule subset
77
tion and subsequently associate with rafts upon binding their ligands. was identified in human neutrophils and shown to contain gelatinase,
This is particularly true for the Fcγ and GPI-coupled receptors. but the ultrastructure was not determined until the issue of the neu-
trophil gelatinase (matrix metalloproteinase [MMP]-9) as a possible
complex with neutrophil gelatinase-associated lipocalin (NGAL) was
SECRETORY VESICLES identified. 78,79
Granules were initially viewed of as small bags that emptied their
Secretory vesicles are small intracellular vesicles that were discovered content of bactericidal substances onto the ingested microorganisms
during the search for the structural basis for upregulation of a variety of when granules fuse with the phagocytic vacuole during phagocyto-
surface molecules on neutrophils in response to nanomolar concentra- sis, but it later became clear that granules are not only important for
tions of fMLP and other chemotactic stimuli. They were initially identi- their cargo, but also for their membranes, as they contain proteins that
fied by “latent” alkaline phosphatase. Secretory vesicles of neutrophils become incorporated into the membrane of the phagocytic vacuole
60
should not be confused with the vesicles that carry cargo from endo- and into the surface membrane when the granules are mobilized. 80,81 If
plasmic reticulum and Golgi in the constitutive secretory pathway of granules were classified by their content, both of matrix proteins and
other cells and that are sometimes also named secretory vesicles. Secre- membrane proteins, the number of different granule subsets that exists
tory vesicles of neutrophils are specialized endocytosis vesicles that in neutrophils would be meaninglessly high. Yet nature has provided a
are formed in the final stages of neutrophil maturation in the marrow. beautiful setting that allows the neutrophil to fine tune its response to
They contain plasma proteins, seemingly without any selectivity. Albu- a specific task. A priori, there would be two reasons for having different
min thus serves as a marker for secretory vesicles and has allowed the subsets of granules: One would be to ensure that proteins, which can-
identification of these as small intracellular vesicles that are scattered not coexist, are segregated; that is, protease-sensitive proteins are sepa-
throughout the cytoplasm of neutrophils as is true for neutrophil rated from proteases. The other reason would be to have proteins whose
granules. The plasma proteins inside secretory vesicles show no sign service is needed at one time separated from proteins whose service is
of degradation, thus no fusion takes place with lysosomal structures. needed at a different time.
61
Secretory vesicles behave like the traditional neutrophil granules. They
require a specific signal for mobilization. Secretory vesicles are not
62
important for their cargo (plasma proteins), but for their membrane Heterogeneity of Neutrophil Granules
which becomes fully incorporated into the plasma membrane of the Among the peroxidase-positive granules, subsets can be identified that
neutrophil upon stimulation. 61,63–66 Secretory vesicles host most of the are rich in defensins as well as some that are not. 82,83 Functionally, no
neutrophil chemotactic and GPI-coupled receptors, TLRs, and one of difference has been identified in terms of the regulation of exocytosis of
the early acting downstream effectors, phospholipase D. They enrich these peroxidase-positive granule subsets. Other constituents include
84
67
the plasma membrane with receptors for adhesion and signaling, and the serine proteases elastase, cathepsin G, proteinase 3, and neutrophil
can be seen as the structural basis for transition of neutrophils from serine protease 4 (NSP4), and the inactive serine protease azurocidin
circulating quiescent cells that do not respond well to stimuli such as (aka CAP 37), the antimicrobial proteins BPI (bacterial permeabili-
chemoattractants and objects to be phagocytosed, to highly responsive ty-increasing protein), lysozyme, and the α defensins, which are the
cells capable of establishing firm contact with endothelium. The signals dominating species. Defensins are also named HNPs (human neu-
80
generated by tethering of selectins or PSGL-1 to the endothelium are trophil peptides). The membrane of the azurophil granules contains
sufficient to mobilize secretory vesicles. Secretory vesicles are com- CD63 (granulophysin) and CD68, but their role in neutrophil func-
pletely mobilized in vivo during neutrophil diapedesis. 21,66 tion remains unclear. 85,86 Many of the proteins present in peroxidase
The first identified marker of secretory vesicles, latent alkaline granules are proteolytically processed both at the N-terminus and the
phosphatase, is known to be elevated in chronic myeloproliferative dis- C-terminus to the active mature forms, which are stored in the granule
orders except for chronic myelogenous leukemia (CML), but the content matrix.
of secretory vesicles in neutrophils from patients with chronic mye- Peroxidase-negative granules can be divided into three subsets
loproliferative disorders is not different from normal neutrophils. 68–70 based on the distribution of the two marker proteins lactoferrin and gel-
The best marker for secretory vesicles is CD35, a transmembrane pro- atinase: granules that contain lactoferrin, but no gelatinase (15 percent
tein of 160 to 250 kDa that binds complement components C3b and of peroxidase-negative granules), granules that contain both proteins
C4b, because CD35, in contrast to alkaline phosphatase, is absent from (60 percent), and granules that are rich in gelatinase, but low (or absent)
the plasma membrane of unstimulated neutrophils, and because it is in lactoferrin (25 percent). The latter are named gelatinase granules or
87
absent from granules (in contrast to α β ). 32,65, 71 It is not known whether tertiary granules, whereas those that contain lactoferrin are called spe-
M 2
secretory vesicles contain lipid rafts, but most GPI-linked proteins are cific or secondary granules. It is a characteristic of peroxidase-negative
raft-associated and are localized to secretory vesicles in neutrophils. granules that the proteins present in their matrix are not proteolytically
72

Kaushansky_chapter 66_p1005-1042.indd 1009 9/21/15 10:47 AM