Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

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Chapter | 11 Neutrophil Function
patients ( Cross et al. , 2000 ). Chronic granulomatous disease
is characterized by recurrent severe, but usually not fatal,
bacterial and fungal infections beginning in early childhood
( Malech and Nauseef, 1997 ). Neutrophils have an inability
to mount a respiratory burst because of an inability to activate
the NADPH oxidase system. A neutrophil bactericidal
dysfunction with decreased capacity to mount a respiratory
burst has been described in eight closely related Doberman
pinschers ( Breitschwerdt et al. , 1987 ). However, the defect
in oxygen radical generation was not as severe as in humans,
and it is not clear if the defect is in NADPH or in another
pathway such as myeloperoxidase deficiency.
Glutathione peroxidase deficiency is clinically similar
to chronic granulomatous disease ( Malech and Nauseef,
1997 ). The defects appear to result from a failure of the hexose
monophosphate pump to generate NADPH. Impaired
neutrophil bactericidal activity is also found in seleniumdeficient
cats and cattle and in copper-deficient cattle. Because
selenium is required for glutathione metabolism, selenium
deficiency probably causes a defect in the oxygen-dependent
pathway of bacterial killing ( Serfass and Ganther, 1975 ).
Myeloperoxidase deficiency has also been documented
as the most common of the inherited neutrophil defects in
humans ( Malech and Nauseef, 1997 ). However, the condition
produces few clinical signs. The number of individuals
that develop severe infections is small. Although killing of
bacterial organisms is delayed, killing is eventually complete.
Myeloperoxidase deficiency has been described in
cyclic hematopoiesis of gray collie dogs. Affected dogs
have defective bactericidal activity. These dogs have an
insertion of an adenine nucleotide in AP3B1 leading to
a frame shift with premature termination ( Horwitz et al. ,
2004 ). This is equivalent to the mutation in Hermansky-
Pudlak syndrome type II in humans but is unlike the ELA2
mutation seen in cyclic neutropenia in humans.
Abnormal neutrophil chemiluminescence responses have
been described in a group of Weimaraner pups with recurrent
fevers, diarrhea, pneumonia, pyoderma, lymphadenopathy,
stomatitis, and osteomyelitis ( Couto et al. , 1989 ). Decreased
chemiluminescence in response to phorbol esters was
the major neutrophil function alteration noted in these dogs.
Defects in humoral or cellular immune responses were not
detected.
2 . Oxygen-Independent Mechanisms
Nonoxidative mechanisms may be more important in killing
of microorganisms than previously suspected. Some
investigators hypothesize that the major role of the NADPH
oxidase system is to adjust phagosomal pH and to pump
electrons into the phagocytic vacuole ( Roos et al. , 2003 ;
Segal, 2005 ). This movement of ions across the phagosome
membrane produces conditions that are conducive to oxygenindependent
organism killing. For example, the initiation
of superoxide production is accompanied by phagosomal
alkalinization because of the proton-acceptor function of the
superoxide anion. This pH change may be essential for activation
of enzymes within the phagolysosome. Additionally,
gp91 phox of NADPH oxidase functions as a proton channel.
This proton pump appears to play a critical role in shuttling
hydrogen ion and several other ions across the cell and
phagosomal membranes ( Geiszt et al. , 2001 ). An influx of
potassium appears to be important in liberating proteases
such as elastase and cathepsins from their acidic proteoglycan
matrix within granules. NADPH oxidase also is thought
to induce depolarization that inhibits calcium influx into
neutrophils.
Neutrophil-independent bacterial killing mechanisms
are present within primary, secondary, and tertiary granules
and lysosomes ( Klebanoff and Clark, 1978 ). The primary
granules contain a variety of cationic proteins including
defensins, cathepsin G, azurocidin, and BPI protein.
Primary granules also contain hydrolases including collagenase,
elastase, β -glucuronidase, and lysozyme. The secondary
granules contain much of the lysozyme, lactoferrin,
adhesin, and chemotactic receptors, and gelatinase. A
third type of granule has been identified in humans, cows,
sheep, goats, horses, dogs, and rabbits ( Bertram, 1985 ). In
some species these granules contain gelatinase and some
acid hydrolases. However, tertiary granules of bovine neutrophils
appear to be distinct in that they contain a potent
antimicrobial system ( Gennaro et al. , 1983 ).
Defensins are a family of low-molecular-weight, antimicrobial
peptides that have been isolated from neutrophils
and heterophils of humans, rats, guinea pigs, and rabbits
( Evans and Harmon, 1995 ). They contain six invariant cysteine
residues and are arginine rich. The cysteine residues
form disulfide bonds, and pairing of the first and sixth
residues creates a cyclic structure. Similar peptides have
been isolated from bovine and equine neutrophils and from
chicken heterophils. These defensin-like peptides have
a broad spectrum of antimicrobial activity being active
against both Gram-positive and Gram-negative bacteria, as
well as some fungi, enveloped viruses, protozoa, and cells.
Cathepsin G is rich in cysteine and has microbicidal
activity against Gram-positive and Gram-negative bacteria
as well as some fungal organisms. Cathepsin G inhibits
bacterial respiration and energy-dependent transport systems,
as well as protein, DNA, and RNA biosynthesis.
BPI is a large, lysine-rich protein that is highly active
against E. coli and Salmonella typhimurium ( Weiss et al. ,
1978 ). However, it lacks antimicrobial activity against
Gram-positive bacteria and fungi. BPI appears to bind to
the outer membrane of susceptible Gram-negative bacteria
and increases membrane permeability to hydrophobic
molecules. BPI also activates enzymes that degrade envelope
phospholipids and peptidoglycans. Neutrophils from
neonatal children were reported to have three- to four-fold
less BPI per neutrophil compared to adults. This correlated
with a decreased antibacterial activity against E. coli