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Neutrophil lactoferrin contentVariation among mammals.

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THE ANATOMICAL RECORD 221567-515 (1988)
NeutrophiI Lactoferrin Content:
Variation Among Mammals
JAMES C. BARTON, RICHARD T. PARMLEY, THOMAS W. BUTLER,
SUE WILLIAMSON. SANDRA MAcKENZIE, DAVID B. CHANDLER,
WARREN BLACKBURN, AND LOUIS W. HECK, JR.
Veterans Administration Medical Center (J.C.B., S. W ,D.B.C., WB., L W H.), and
Department of Medicine, Comprehensive Cancer Center, and Multipurpose Arthritis Center,
University ofAlabama at Birmingham, Birmingham, Alabama 35233 (J C.B., II WB.,
D.B.C., WB., L WH.);Department of Pediatrics, University of Texas Health Science Center at
San Antonio, San Antonio, Texas 78229 (R.TP.);and Birmingham Zoo,
Birmingham, Alabama 35223 (S.M.)
ABSTRACT
Lactoferrin (LfJ in blood andor marrow neutrophils was semiquantified using indirect immunofluorescence technique in nine mammalian species.
Neutrophil iron-binding reactivity (NFeBR), which corresponds primarily to Lf, was
also visualized and semiquantified using functional cytochemical (FeNTA-AF)technique at the light microscopic level in these nine and in a n additional fifteen
mammalian species, and in selected species at the ultrastructural level. Neutrophil
immunoreactive Lf was positively correlated with total cellular and granule content
of NFeBR among these nine species, and with previously reported concentrations of
neutrophil Lf quantified by radioimmunoassay. Relative levels of Lf in neutrophil
extracts from rat, hamster, and human were confirmed using SDS-polyacrylamide
gel electrophoresis and immunoblotting. Relatively high levels of immunoreactive
neutrophil Lf andor NFeBR were observed in carnivores (ten species) and primates
(six species). Among rodents (five species), the levels were variable, and the artiodactyls (four species) studied had low levels. These results demonstrate that neutrophi1 Lf levels vary widely among mammalian species. In addition, FeNTA-AF
technique provides a rapid means of evaluating animals for relative quantities of
neutrophil Lf.
Lactoferrin (LO functions defined primarily in vitro
include bacterial killing, feedback inhibition of granulopoiesis, and nonheme iron transport (Broxmeyer, 1984).
Some functions of neutrophil Lf have also been inferred
from observations in vivo, e.g., the increased frequency
of bacterial infections among rare patients with congenital neutrophil Lf deficiency. However, because the neutrophils of these patients possess other anatomic,
functional, and biochemical anomalies (Boxer et al.,
1982), a n isolated neutrophil Lf deficiency state remains
unknown among humans. Alternatively, animals with
naturally low neutrophil Lf levels could be useful for
studies of Lf function. The evaluation of prospective
subjects by established means would require a n immunologic assay of neutrophil Lf, procedures for efficiently
extracting neutrophil Lf, which may vary from species
to species (Masson et al., 1969; Baggiolini et al., 1970;
Kinkade et al., 19761, and, possibly, the production of
species-specific, anti-Lf antibodies. By staining the blood
cells of a variety of mammals with iron nitrilotriacetateacid ferrocyanide (FeNTA-AF) method and by using a
cytochemical scoring technique, we visualized and semiquantified Lf as neutrophil iron-binding reactivity
(NFeBR) (Parmley et al., 1982; Barton and Parmley,
1986; Barton et al., 1987). In mammals, NFeBR scores
could be positively correlated with granule content of
NFeBR at the ultrastructural level, with indirect im0 1988 ALAN R. LISS, INC.
munofluorescence staining of neutrophil Lf, and with
quantification of total neutrophil Lf by radioimmunoassay and immunoblotting in the respective species. The
present methods demonstrate the interspecies variability in neutrophil Lf, and permit the rapid evaluation of
animals for their relative quantities of neutrophil Lf.
MATERIALS AND METHODS
Blood and Marrow Collection
Human blood and marrow specimens were acquired
according to the Declaration of Helsinki and with the
approval of the Human Use Committee of the University of Alabama a t Birmingham. Peripheral blood
smears from humans were prepared at the time of finger
puncture. Morphologically normal marrow was obtained
from the posterior iliac crests of six patients undergoing
marrow aspiration and biopsy for staging of malignancy; all other human and animal subjects were apparently healthy at the time of specimen collection.
Principles of laboratory animal care as promulgated by
the National Research Council were observed. Labora-
Received August 19, 1987; accepted November 16, 1987.
Address reprint requests to Dr. James C. Barton, Division of Hematology/Oncology, University of Alabama at Birmingham, University Station, Birmingham, AL 35294.
568
J.C. BARTON ET AL.
tory animals were acquired from Southern Animal
Farms, Prattville, AL (rats),Jackson Laboratories, Bar
Harbor, ME (mice), Charles River Breeding Laboratories, Raleigh, NC, and Kingston, NY (guinea pigs and
hamsters), and Myrtle's Rabbitry, Thompson Station,
TN (rabbits). Blood was obtained from the retroorbital
venous sinus from laboratory rodents under general
anesthesia with a heparinized microhematocrit tube.
Femoral marrow was collected from rats, rabbits, mice,
and hamsters immediately after the animals had been
sacrificed by cervical dislocation. In other animals, blood
smears were prepared from fresh heparinized blood obtained by phlebotomy or heart puncture. Zoo animals
were maintained under conditions specified by the Animal Welfare Act and the American Association of Zoological Parks and Aquariums. For these subjects, blood
was obtained by venipuncture a t the time of routine
health maintenance examinations; other specimens were
acquired through veterinarians' clinical practices.
Functional Cytochemical Technique
For light microscopy, NFeBR was visualized in blood
and marrow neutrophils as previously described in detail (Barton and Parmley, 1986). Briefly, thin smears
prepared on standard glass slides were air-dried at room
temperature overnight, fixed with freshly prepared buffered formol acetone (BFA) for 6 minutes, and rinsed in
H2O. The smears were then incubated in freshly prepared 1%saponin (Fisher Scientific Co., Fairlawn, NJ)
a t 50°C for 1 hour, and rinsed with 0.9% NaC1. The
slides were incubated in freshly prepared 3 mM FeC13:
4 mM nitrilotriacetate (NTA) in 0.9% NaC1, pH 7.4
(FeNTA), at room temperature for 1 hour (Barton and
Parmley, 1986). The slides were then rinsed in 0.9%
NaC1, pH -5.5. In certain experiments, the slides were
rinsed instead in 0.9% NaCl a t pH 4.0 or 8.0 after FeNTA
incubation to determine whether there were cytochemically demonstrable pH-dependent differences in ironprotein binding affinity. The slides were then incubated
in 1%acid ferrocyanide (AF) for 10 minutes, and rinsed
in water (Barton and Parmley, 1986). The smears were
counterstained in 1%Gurr's Nuclear Fast Red (Esbe
Laboratory, Toronto, Canada) in 5% A12(S04)3.18 H20
for 5-10 minutes, rinsed in water, routinely dehydrated,
cleared, and mounted. Normal and abnormal human
control slides were included in each staining run. Blood
smears similarly fixed with BFA were stained with AF
technique alone for the assessment of native ferric iron
in the cells, and to serve as a negative control for FeNTAAF staining. A blood smear from each subject was
stained with WrightIGiemsa stain. One hundred consecutive mature neutrophils or heterophils (having two or
more nuclear lobes) in the thin edge of each smear were
rated 0 to 5 + on the basis on the distribution, color
intensity, and granularity of their cytoplasmic stain deposits by light microscopy at 1,000 x magnification (Table 1, Fig. 1). A rating of 4 was defined as that
appearance and quantity of FeBR visualized most commonly in the neutrophils of normal human subjects. The
ratings were added to yield a NFeBR score (0-5001,
which is almost linearly related to human neutrophil Lf
content determined by immunoassay over a wide range
of values (Barton et al., 1988).
We also visualized NFeBR in ultrastructural specimens using a previously described method (Parmley et
al., 1982). Briefly, venous blood samples were collected
in heparinized tubes from six rats, four cows, four rabbits, four mice, and four humans. The samples were
centrifuged at 1,500 x g for 3 minutes, and the buffy
coat was removed with a pipette. The cells were resuspended in 3% glutaraldehyde in 0.1 M cacodylate buffer,
pH 7.35, for 1 hour. After rinsing in 0.1 M cacodylate7% sucrose buffer, the cells were resuspended for 1hour
in 1% saponin at 50-55°C. The cells were then rinsed
twice in cacodylate-sucrose buffer and once in 0.9% NaC1,
exposed to FeNTA solution for 1hour at room temperature, rinsed three times in cacodylate-sucrose buffer, and
incubated for 30 minutes in 1% AF. After three additional rinses in cacodylate-sucrose buffer, the cells were
postfixed in 1% 0 ~ 0 4routinely
,
dehydrated, and embedded in S p u r low viscosity medium. Thin sections (5070 nm thick) were examined without counterstains with
a Zeiss 109 electron microscope a t a n accelerating voltage of 50 kV.
TABLE 1. Scoring criteria for NFeBR'
Individual cell rating
0
Cytoplasmic pattern
-
Granularity
-
Color
-
Granules
-
Relative FeBR,
quantityicell2
1+
2+
3+
Diffuse +
2 25% focal
Weak
Weak
Weak
+ strong
Pale grayish Pale grayish Pale grayish
blue
blue + medium
blue
blue
None
Rare
Rare-few
Focal (any)
Undetectable Severely
deficient
Diffuse
Moderately
deficient
Low normal
4+
Diffuse
Strong
Medium blue
5+
Diffuse +
2 25% focal
Strong +
very strong
Medium blue
+ blue-black
Few-moderate Moderate, many
overlying
nucleus
Normal
High normal,
increased
'100 consecutive mature neutrophils (possessing 2 or more nuclear lobes) on the thin edge of a peripheral blood smear are scored after
normal and abnormal control slides stained simultaneously are reviewed. Total score is the sum of 100 individual cell ratings (range of
scores 0-500); see also Figure 1.
'In comparison with normal human peripheral blood neutrophils.
569
NEUTROPHIL LACTOFERRIN IN MAMMALS
0
1+
2+
3+
4+
5+
Fig. 1. Cartoons of mature blood neutrophils depicting the relationships of the intensity and distribution of cytoplasmic FeNTA-AF stain
deposits to individual cell rating; see also Table 1.
Indirect lmmunofluorescence Technique
fluoresceinated second antibody layer only. After a final
Fresh heparinized blood or suspended marrow was washing in PBS and mounting in Fluoromount-G
centrifuged at 1,500 x g for 15 minutes, and b a y coat (Southern Biotechnology Associates, Inc.), the preparaa Leitz orthoplan/*rthomat
cells were collected. After the cells were washed either tions were
in cold PBS, p~ 7.4, or in Hank’s balanced salt solution, microscope. Immunofluorescence reactivity was rated
pH 7.2 cytocentrifuged Smears were prepared on glass according to these criteria: 0 = no Staining; k = faint
5 minutes(cytospin
2; &andon staining in occasional cells; 1 + = faint; 2 + = weak;
slides gt 200 wm
Southern Instruments. Inc.. Sewicklev.
PA). The smears 3+ = moderate; and 4 + =
”,
were air-dried for 24 hours,’fixed in fresh cold BFA for 1
Purification of Human Neutrophil Lactoferrin
minute, and rinsed in water. The slides were then incuHuman
neutrophil Lf from normal donors was purified
bated in 1% saponin at 50°C for 1 hour, and rinsed
using
a
n
isolation procedure that included sequential
thoroughly with 0.9% NaCl and then with PBS to reduce
nonspecific, but not specific, Lf staining. The cells were sodium chloride extraction, heparin-Sepharose affinity
then overlayered with 10 pl of polyspecific rabbit anti- ‘chromatography, and AcA44 gel filtration chromatograhuman milk Lf (lot #011063; Calbiochem-Behring Corp., ,phy (Heck, L.W., unpublished results). Coomassie blue
La Jolla, CA) diluted 1:lO in 2% fetal calf serum/l% :staining of the gel after sodium dodecyl sulfate-polysodium azide, for 30 minutes at 4°C. After washing, the acrylamide gradient gel electrophoresis of the reduced
cells were overlayered with fluorescein-labeled goat an- purified protein demonstrated two polypeptides of Mr
tirabbit IgG (lot #J5A026; Southern Biotechnology As- 80,000 and 78,000.
sociates, Inc., Birmingham, AL), 1:lO dilution, for 30
Production of Antibodies to Human Neutrophil Lactoferrin
minutes a t 4°C. Similar procedures were employed for
immunostaining using MoAb-5B2, a mouse monoclonal
Murine monoclonal antibodies to neutrophil Lf were
IgG2B antihuman neutrophil Lf (used as clarified as- produced in the Hybridoma Core Facility a t the Univercites), and fluorescein-labeled goat antimouse IgGZB (lot sity of Alabama at Birmingham. AIJ mice were immu#F5X085; Southern Biotechnology Associates, Inc.). nized with a total of 600 pg of the purified human
Control slides were prepared using either a first layer neutrophil Lf according to the protocol of Lieberman and
antibody of isotype unrelated to the second layer, or the colleagues (1972). After the fourth injection of antigen
570
J.C. BARTON ET AL.
Fig. 2. FeNTA-AF (left) and indirect immunofluorescence (right) (A); cytoplasmic immunoreactive Lf staining was also weak (B). Pig
staining of Lf in neutrophils, arranged in increasing order of cyto- blood (C,D); hamster marrow (E,F); dog blood (G,H); and human blood
bar = 10 pm.
plasmic staining intensity. In the cow, the nuclei stained more in- (I,.J). ~1,400;
tensely with nuclear fast red than did the cytoplasm with FeNTA-AF
in saline, the popliteal, inguinal, subaxillary, and brachial lymph node lymphocytes were fused with the
nonsecretory myeloma Pcx63-Ag 8.653 (Kearney and
Lawton, 1975). Hybridomas secreting antibodies with
specificity for human neutrophil Lf were selected using
an ELISA assay (Engvall and Pearlmann, 1972). Eight
hybridoma positive wells containing antibodies against
human neutrophil Lf were identified and cloned in tis-
sue culture. Subsequently, three stable hybridoma [ Z
IgM and 1 IgG (y 2b-(5B2)]were injected into Pristaneprimed CAF mice. Ascites fluid was collected and the
antibodies purified by sequential DE-52 cellulose ionexchange chromatography followed by Sephadex G150
gel filtration chromatography. The specificity of these
antibodies has been verified by solid-phase binding assays and immunoblotting using iodinated antibodies
Fig 3. FeNTA-AF staining of neutrophils at the ultrastructural
level. A. Rat. B. Cow. C. Mouse. D. Human. N = nucleus. ~18,000;
bar = 1 Fm.
(Dunn et al., 19851, and immunofluorescence. A poly- trophoretic transfer of proteins to nitrocellulose and inclonal monospecific goat antibody to human milk lacto- cubation of nitrocellulose with iodinated antibodies were
ferrin was provided by Dr. Jiri Mestecky.
performed as previously described (Dunn et al., 1985).
lmmunoblotting Procedure
RESULTS
Samples of rat and hamster bone marrow, human
Functional Cytochemical Technique
peripheral blood neutrophils, and purified human neuNeutrophils or heterophils in blood films contained
trophil Lf were prepared to minimize epitope denatura- granular cytoplasmic FeNTA-AF stain deposits that vartion (Daniel et al., 1983) and separated using SDS- ied widely in relative amounts among species (Table 2,
polyacrylamide gradient gel electrophoresis. The elec- Fig. 2). Within the same species or individual, the distri-
572
J.C. BARTON ET AL.
TABLE 2. Interspecies comparison of neutrophil lactoferrin determined by functional cytochemical and
immunologic methods
NFeBR score'
(n)
Rat, albino Wistar
Cow, Holstein
Rabbit, New Zealand white
Pig, Sinelair miniature
Hamster, golden Syrian
Mouse, C57B1/6J
Dog, mixed breed
Cat, domestic shorthair
Human
40
160
169
176
264
320
371
385
403
k 0 (3)4,5
+ 27 (3)
k 14 (414
f 18(3)
54 (7)4
+ 0 (3I4z5
+ 44(5)
9(3)
k 3 (5114
+
+
Granule
NFeBR'
1
2
3
4
5
Immunostaining
Immunoassay
~ g / l O 7cells3
k
1+
2+
1+
2-3
3+
4+
4+
4+
34,35.8
+
20-40
77,100,178
'Values are expressed as the mean 5 standard deviation of the mean.
'Ratings of FeNTA-AF stain deposits in neutrophil cytoplasmic granules observed at the ultrastructural level in order of
increasing intensity.
3These data represent values obtained with different sources of neutrophils (peripheral blood, marrow, peritoneal exudate),
neutrophii isolation procedures, and immunologic assays. Each assay was performed with species-specific antibody. Rabbit
heterophil Lf was quantified by van Snick et al. (1974) and Boxer (1985), respectively; mouse neutrophil Lf by Segars and
Kinkade (1977); and human neutrophil Lf by de Vet and ten Hoopen (1978), Oseas et al. (19811, and Bennett and Kokocinski
(1978), respectively. The variation among values for human seems largely attributable to methodological differences. Assay
results in other species are unknown to the authors.
4Similar staining intensity was observed in mature blood and marrow neutrophils in these species.
5Similar values were obtained for other strains of these respective species, the hooded Lister rat and the WCBGF1 J + / +mouse.
TABLE 3. Neutrophil iron-binding reactivity (NFeBR) scores in other mammals
Order, species
NFeBR Score (n)'
Marsupiala
Didelphys virginiana (opossum)
Carnivora
Eira barbara (tayra)
Lutra canadensis (river otter)
Ursus americanus (American black bear)
Paradoxurus hermaphroditus (palm civet)
Suricata suricatta (meerkat)
Panthera tigris altaica (Siberian tiger)
Panthera onca (jaguar)
Felis concolor (cougar)
Artiodactyla
Capra hircus (domestic pygmy goat)
Odocoileus uirginianus (white-tailed deer)
Perissodactyla
Equus caballus (Tennessee walking horse)
Edentata
Dasypus nouemcinctus (armadillo)
Rodentia
Cauia porcellus (Guinea pig, Charles River strain I1 tricolor)
Dasyprocta cristata (golden-rumped agouti)
Primates
Cercopithecus patas (Patas monkey)
Macaca mulatta (macaque)
Papio sphinx (mandrill baboon)
Papio leucacephaeus (drill baboon)
Pongo pygmaeus (orangutan)
284
277 k 6 (3)
316
284
348
420
392
364
400,398
72
105,160
321 k 16(3)
236
112
110
396
385,394
378,425
398 & 18(5)
387 k 18(3)
'Values for three or more subjects are expressed as the mean f 1standard deviation of the mean.
bution of FeBR among neutrophils was very homogeneous. Stain deposits observed at the ultrastructural level
occurred in increasing order of intensity in the cytoplasmic granules of rat, cow, rabbit, mouse, and human
neutrophils (Table 2, Fig. 3). This order corresponded to
values of NFeBR scores in the respective species (Table
2). The positively stained granules were generally of an
intermediate size range and varied among species, with
the smallest population of positively stained granules
observed in cows and the largest in rabbits. Values of
NFeBR scores in the mouse, guinea pig, and human
were also positively related to previously published values of neutrophil Lf for these respective animals (Table
2). Staining with AF alone revealed no positivity in
neutrophils (Koszewski et al., 1967; Barton and Parmley, 1986). Values of NFeBR scores in 20 additional
573
NEUTROPHIL LACTOFERRIN IN MAMMALS
cytoplasmic FeNTA-AF positivity, similar to that observed with AF technique alone, was observed in some
species in some mid- and late erythroblasts, and is consistent with the presence of ferritin in these cells (Parmley et al., 1982; Barton and Parmley, 1986). In light and
electron microscopic specimens, eosinophils, basophils,
and lymphocytes had no staining; nuclear staining was
not observed. The severely FeBR-deficient neutrophils
present in some species were readily distinguished from
eosinophils and basophils at the light microscopic level
by the nuclear configuration and distinctive cytoplasmic
granules clearly visualized by the counterstain in the
latter cells. Blood leukocyte counts (both differential and
estimated absolute counts) and the morphologic and
tinctorial features of leukocytes, erythrocytes, and platelets observed on Wright/Giemsa-stainedblood films were
similar to those previously described for the same or
closely related species (Andrew, 1965; Schalm et al.,
1975; Archer et al., 1977; Smith, 1983).
Indirect lmmunofluorescence Technique
Fig. 4. Autoradiography of detergent-solubilized hamster and human neutrophils and purified human neutrophil Lf using iodinated
polyclonal anti-human neutrophil Lf (A) and MoAb-5B2 (B) run under
nonreducing conditions. The protein samples loaded on this gel are:
lane 1, extract of 7.5 x lo5 hamster neutrophils; lane 2, extract of 7.5
x lo5 human neutrophils; lane 3, 10 pg of purified human neutrophil
Lf. After electrophoresis, the proteins were transferred to nitrocellulose paper: lanes 1-3, (A), were incubated with iodinated goat antibody
to human neutrophil Lf; lanes 1-3 (B) were incubated with iodinated
MoAb-5B2.
species are displayed in Table 3. Of all animals studied,
carnivores (ten species) and primates (six species) had
relatively high cytochemical scores, whereas the artiodactyls (four species) studied had relatively low scores.
The neutrophil FeNTA-AF reactivity among rodent species (five species) was highly variable. No definite relationship could be found between the relative quantities
of neutrophil Lf estimated by functional cytochemical or
indirect immunofluorescence staining and the absolute
numbers of granulocytes normally present in the blood
of the respective species (Andrew, 1965; Schalm et al.,
1975; Archer et al., 1977; Smith, 1983). There are also
significant interspecies variations in the contents a n d
or activities of neutrophil enzymes (Jain, 1967, 1968;
Padgett and Hirsch, 1967; Prieur et al., 1974; Rausch
and Moore, 1975). However, the relative amounts of
NFeBR and immunoreactive Lf observed in the present
study were found to have no obvious relationship to
previously reported concentrations of these other
substances.
Occasional monocytes in some species had weak diffuse FeNTA-AF staining. This was particularly prominent in the human, the Patas monkey, and the mandrill,
suggesting the presence of surface andor cytoplasmic Lf
in these cells (Bennett and Kokocinski, 1978; Barton
and Parmley, 1986). One or two fine cytoplasmic granules similar to those observed with AF technique alone
were observed in occasional monocytes in many species,
suggesting the presence of cytoplasmic ferritin. Focal
Similar results were obtained with polyclonal and
monoclonal antibodies. Great variability in immunoreactive neutrophil Lf among species was demonstrable
by immunostaining, but there was a homogeneous distribution of staining among neutrophils within the same
species andor subject. Fluorescence intensity was positively related to values of NFeBR scores, to the rating
of neutrophil granule FeNTA-AF stain deposits assessed
at the ultrastructural level, and to concentrations of
neutrophil Lf quantified by radioimmunoassay in the
respective species studied (Table 2, Fig. 3).
lmmunoblotting Technique
Extracts of hamster neutrophils showed moderately
decreased immunoreactive Lf (Mr 76,000) in comparison
with human neutrophil extracts (Mr 78,000 and 80,000)
using polyclonal and monoclonal immunoblotting analysis (Fig. 4). Electrophoresis of rat neutrophil extracts
had a protein band corresponding to Lf (Mr 76,0001,
which required prolonged autoradiography for visualization; because of its faintness, this result is not displayed.
Differences among the estimated molecular weights of
these Lfs are probably attributable to differences in glycosylation of the polypeptide chains.
DISCUSSION
The results of this study demonstrate that substantial
differences in neutrophil Lf content exist among mammals. Neutrophil immunoreactive Lf and NFeBR semiquantified in the present study were positively related
for each species so studied. Both of these parameters
were positively related to concentrations of neutrophil
Lf previously determined by immunoassay in rabbits,
mice, and humans, respectively (van Snick et al., 1974;
Segars and Kinkade, 1977; Bennett and Kokocinski,
1978; de Vet and ten Hoopen, 1978; Oseas et al., 1981;
Boxer, pers. comm., 1985). Interspecies variation in values of NFeBR scores, particularly low values, could be
due to qualitative differences in Lf. Such alterations
could include the presence of a single iron-binding site
on the Lf molecule, by analogy to certain transferrins
(Martin et al., 19841, or other changes in Lf or in its
associated granules that simultaneously diminish chem-
5 74
J.C. BARTON ET AL.
ical stability, immunoreactivity, and/or iron binding of
Lf. However, all known Lfs are relatively thermostable
proteins with similar values of molecular weight and
charge that have interspecies immunologic cross-reactivity, possess a 2 : l molar ratio of iron:protein binding,
and maintain iron binding a t acid values of pH (Masson
et al., 1969; Baggiolini et al., 1970;Kinkade et al., 1976).
Our present immunoblotting results using polyclonal
and monoclonal antilactoferrins demonstrate substantial differences among species in quantities of neutrophil Lf. I n ongoing studies of human neutrophil Lf in a
variety of physiologic and pathologic states, we have
also found excellent correlations between NFeBR, immunoreactive Lf, and quantities of Lf determined by
radioimmunoassay (Barton et al., 1988). Therefore, the
corresponding intensities of NFeBR and immunoreactive Lf staining that varied among species are best explained by the occurrence of quantitative interspecies
differences in neutrophil Lf.
Marked interspecies differences have been observed
for a variety of neutrophil granule components, e.g.,
myeloperoxidase is markedly reduced in cattle (Gennaro
et al., 1983) and goats (Rausch and Moore, 1975); lysozyme activity is practically undetectable in Rhesus monkeys, cats, cows, goats, sheep, and hamsters (Rausch and
Moore, 1975); and alkaline phosphatase activity is very
low in Rhesus monkeys, cats, and mice (Rausch and
Moore, 1975). Not surprisingly, the interspecies differences of these enzymes cannot be correlated with the
interspecies differences of Lf observed in the present
study, suggesting different regulatory mechanisms for
synthesis of granule proteins. Carnivores and primates
appeared to have greater NFeBR and neutrophil Lf content than herbivores, but a similar correlation with phylogeny has not been observed for neutrophil
myeloperoxidase, lysozyme, or alkaline phosphatase
contentslactivities. Although the mechanisms that regulate Lf production are poorly understood, the control of
Lf synthesis in mammals may similarly affect the neutrophil and the breast glandular cell, because milk Lf
concentrations determined for the dog, goat, cow, mare,
rat, mouse, guinea pig, rabbit, and human (Masson and
Heremans, 1971) generally correspond to the relative
amounts of neutrophil Lf in these respective animals
observed in the present study.
ACKNOWLEDGMENTS
The authors recognize the following individuals for
their cooperation and efforts in acquisition of many of
the blood specimens studied for this project: Drs. Charles
R. Becker, Simon Gelman, Larry Boots, Larry Britt,
Kenneth Zuckerman, Richard T. Gualtieri, and Ken
Boschert, and Mr. Edward Dillard. Mr. Frank Denys
assisted in making our photographic results. Ms. Diane
Cerna in the Cell Identification Laboratory of the University of Alabama at Birmingham (under the direction
of Dr. C.E. Grossi) performed the immunofluorescence
staining. Dr. Grossi and Dr. Edgar F. Prasthofer provided advice concerning the immunofluorescence technique. Dr. Mary Ann Accivitti is director of the
Hybridoma Core Facility where the monoclonal antibodies were produced. This work was supported by Veterans
Administration Medical Research Funds and National
Institutes of Health Hematology Training Program
Grant 5T32-AMO7488(NIADDKD). T.W.B. is the reciuient of a Frommeyer Fellowship.
I
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