Veterinary Research Communications, 27 (2003) 453^461

# 2003 Kluwer Academic Publishers. Printed in the Netherlands

A Low-molecular-weight Fraction of Bovine Colostrum and

Milk Enhances the Oxidative Burst Activity of
Polymorphonuclear Leukocytes
H. Sugisawa1, T. Itou1, M. Saito1, T. Moritomo2, Y. Miura1 and T. Sakai1*
1
Department of Preventive Veterinary Medicine and Animal Health, Nihon University
School of Veterinary Medicine, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan;
2
Laboratory of Fish Pathology, Nihon University School of Veterinary Medicine, Japan
*Correspondence: E-mail: sakai@brs.nihon-u.ac.jp

Sugisawa, H., Itou, T., Saito, M., Moritomo, T., Miura, Y. and Sakai, T., 2003. A low-molecular-weight
fraction of bovine colostrum and milk enhances the oxidative burst activity of polymorphonuclear
leukocytes. Veterinary Research Communications, 27(6), 453^461

ABSTRACT

Bovine colostrum and milk contain many immunomodulatory components. The low-molecular-weight
fraction (510 kDa) was separated from colostrum and milk by gel ¢ltration chromatography, and its
e¡ect on the oxidative burst of bovine polymorphonuclear leukocytes (PMNL) was investigated in vitro.
The oxidative burst activity induced by Staphylococcus aureus was considerably enhanced when
PMNLs were incubated with this low-molecular-weight fraction. However, phorbol 12-myristate 13-
acetate did not trigger a burst after priming with this fraction. The oxidative burst activity enhanced by
this fraction was reduced after heating. These results con¢rmed that a low-molecular-weight sub-
stance(s) of less than 10 kDa, present in bovine milk and colostrum, enhances the oxidative burst
activity of PMNL.

Keywords: colostrum, milk, oxidative burst, immunomodulation, polymorphonuclear leukocytes

Abbreviations: cfu, colony-forming units; GM-CSF, granulocyte^macrophage colony-stimulating

factor; HBSS, Hanks balanced salt solution; IL, interleukin; LCDL, luminol-dependent chemilumines-
cence; LMW, low-molecular-weight; PBS, phosphate-bu¡ered saline; PMA, phorbol 12 myristate 13
acetate; PMNL, polymorphonuclear leukocyte(s); ROS, reactive oxygen species; SDS-PAGE, sodium
dodecyl sulphate^polyacrylamide gel electrophoresis; TNF-a, tumour necrosis factor a

INTRODUCTION

Bovine colostrum and milk contain many bioactive substances, such as interleukin 1
(IL-1b), IL-6 and tumour necrosis factor a (TNF-a) (Goto et al., 1997; Hagiwara et al.,
2000). Exposing neutrophils to these cytokines ampli¢es the magnitude of the
oxidative burst to an activating agonist. We have shown that bovine colostrum
activates phagocytosis by bovine polymorphonuclear leukocytes (PMNL) (Sugisawa
et al., 2001) and that bovine milk enhances the oxidative burst activity (Sugisawa et al.,
2002). In addition, it has been shown that an ultra¢ltered product from bovine whey
enhances neutrophil function (Roth et al., 2001) and that the phagocytic activity of
PMNL is enhanced in breast-fed infants (Menge et al., 1998). These results all suggest

453
454

that constituents of colostrum and milk enhance the function of PMNL.

To determine which components of bovine milk and colostrum activate PMNL, we
fractionated colostrum and milk samples by gel ¢ltration chromatography. The activity
of each fraction was then evaluated from the amount of reactive oxygen species (ROS)
released from PMNL.

MATERIALS AND METHODS

Colostrum and milk samples

Bovine colostrum and milk samples obtained from 13 clinically healthy Holstein cows
were centrifuged at 40 000g for 30 min at 48C. The lipid and cellular layers were
removed and the aqueous layer was pooled and stored at ^808C.

Isolation of PMNL

Peripheral blood collected from three clinically healthy Holstein cows into heparinized
vacutainers was diluted with a 1.6-fold volume of the phosphate-bu¡ered saline (PBS)
(8.0 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4, 0.2 g KH2PO4, pH 7.2, in 1 L distilled water),
routinely used in this study. Diluted blood (4 ml) was layered onto the same volume of
Lymphoprep (Nycomed Pharma, Oslo, Norway). After centrifugation at 750g for 20
min, the fraction containing the PMNL and all the erythrocytes was isolated. The
erythrocytes were lysed by twice adding 10 ml of lysis bu¡er (8.26 g NH4Cl, 1.19 g
NaHCO3, 0.0378 g EDTA2Na, pH 7.3, in 1 L distilled water). The PMNL were
pelleted by centrifugation at 430g for 10 min, washed twice with cold PBS, stained with
Tu«rk solution (Wako, Osaka, Japan) and counted using a haemocytometer. The ¢nal
cell density was adjusted to a concentration of 16107 cells/ml in Hanks balanced salt
solution without Ca2+ and Mg2+ (HBSS(^)) (Gibco, Gaithersburg, MD). The cell
viability, as assessed by the trypan blue dye exclusion technique, was always over 95%.

Gel ¢ltration chromatography

Colostrum was diluted 10-fold with PBS, and insoluble particles were removed by
centrifugation at 1600g for 10 min. Thereafter, the clari¢ed colostrum and undiluted
milk samples were passed through a 0.45 mm ¢lter and samples (2 ml) were
fractionated by gel ¢ltration in PBS through a column containing Superdex 200pg
(Amersham Pharmacia, Cambridge, UK). The protein concentration in the elution
was assessed from the UV absorption at a wavelength of 280 nm using a GeneQuant
pro spectrophotometer (Amersham Pharmacia). The four fractions that separated from
colostrum or milk were resolved by SDS-PAGE and stained using a Silver Stain kit
(Atto, Tokyo, Japan) according to the manufacturer's instructions. For analysis of the
low-molecular-weight fraction by SDS-PAGE, the electrophoresis was performed in
 455

Tricine bu¡er (cathodic side: 12.1 g Tris, 17.9 g Tricine, 1.0 g sodium dodecyl sulphate
in 1 L distilled water; anodic side: 24.2 g Tris-HCl in 1 L distilled water, pH 8.9).

Opsonization of bacteria

Staphylococcus aureus (ATCC 25923), cultured in brain-heart infusion broth (Nissui,

Tokyo, Japan) at 378C for 12 h, were killed by heating at 708C for 30 min and then
centrifuged at 8000g for 30 min. After three washes with PBS, the bacteria were
adjusted to approximately 161010 cfu/ml in HBSS(^). Bacterial suspensions (1 ml)
were mixed with 9 ml of normal pooled bovine serum, diluted 1:3 (v/v) with PBS,
incubated at 378C for 30 min, washed three times with PBS, adjusted to 161010 cfu/ml
in HBSS(^) and stored at ^808C.

Assay of oxidative burst

The oxidative burst of PMNL was assessed as indicated by luminol-dependent

chemiluminescence (LDCL). Luminol (¢nal concentration, 80 mg/ml; Wako Pure
Chemical Industries, Osaka, Japan), PMNL (16105 cells) and 100 ml of each fraction
(OD280 0.357) or phorbol 12-myristate 13-acetate (PMA; ¢nal concentration 25 pg/ml;
Sigma Chemical Co., St Louis, MO, USA) were incubated for 30 min in a total volume
of 400 ml of HBSS(^). The PMNL suspension was stimulated with 100 ml of the
suspension of opsonized S. aureus (¢nal concentration 56108 cfu/ml) or PMA (¢nal
concentration 100 ng/ml) and the LDCL was measured for 30 min in a LB953
luminometer (EG&G Berthold, Wildbad, Germany) at 378C.

RESULTS

Gel chromatography and SDS-PAGE

Figure 1 shows gel exclusion chromatograms for 2 ml volumes of undiluted bovine

milk and for colostrum, diluted 10-fold. The molecular weights of the components of
the four fractions (fractions 1, 2, 3 and 4), obtained from both milk and colostrum,
were determined by SDS-PAGE (Figure 2). Bands were detected from fractions 1, 2
and 3. No bands were observed from fraction 4, except for a 60 kDa band, which
probably resulted from tailing.

Assay of oxidative burst

The oxidative burst activities of PMNL stimulated with S. aureus were assessed in the
presence of fractions 1, 2 3 or 4. Fraction 4 signi¢cantly enhanced the oxidative burst
activity of PMNL compared to the control or the other three fractions (Figure 3).
456

Figure 1. Exclusion chromatograms of bovine colostrum (A) and milk (B)

Three subfractions (4-1, 4-2 and 4-3) were observed in fraction 4 of both colostrum
and milk. All these subfractions from both milk and colostrum enhanced the oxidative
burst activity, but both fractions 4-2 were the most e¡ective. Fraction 4-2 from milk
potentiated the oxidative burst enhancement with S. aureus but not with PMA (Figure
4). The oxidative burst-promoting activity of milk fraction 4-2 was reduced by heating
(Figure 5).

DISCUSSION

This study examined which constituents of bovine milk and colostrum stimulated
PMNL. It appeared from the SDS-PAGE that fractions 1, 2, 3 and 4 contained
substances with molecular weights (kDa) of at least 200 (e.g. IgM, IgA), 30^200 (e.g.
IgG, BSA), 10^30 (e.g. a-lactalbumin, b-lactoglobulin) and less than 10, respectively.
 457

Figure 2. Analyses of bovine colostrum (A) and milk (B) using SDS-PAGE. Lane 1, molecular
weight marker; lanes 2 and 7, bovine colostrum and milk; lanes 3 to 6, bovine colostrum
fractions 1 to 4, respectively; lanes 8 to 11, bovine milk fractions 1 to 4, respectively

Figure 3. Oxidative burst induced by S. aureus in PMNL exposed to each fraction from
colostrum and milk. The graph shows the integral of chemiluminescent intensity for 10 min
after initiation. Results are expressed as means+SD from three individuals. The values were
signi¢cantly di¡erent from the chemiluminescent responses of untreated PMNL (*p50.05)
458

Figure 4. Oxidative burst induced by PMA or S. aureus in PMNL exposed to milk fraction 4-2
or PMA. The graph shows the integral of the chemiluminescent intensity for 10 min after
initiation. Results are expressed as mean+SD from three individuals. Values were signi¢cantly
di¡erent from the chemiluminescent response of untreated PMNL (#p50.05 compared with
PMA-induced value without fraction 4-2; *p50.05 compared with S. aureus-induced value
without fraction 4-2)

Figure 5. Oxidative burst induced by S. aureus in PMNL exposed to heated milk fraction 4-2 of
milk. The results were expressed as the percentage of the positive control (treated with
unheated fraction 4-2). Results are expressed as means+SD from three individuals. All the
values were signi¢cantly di¡erent (p50.05) from the chemiluminescent response of the positive
control
 459

However, as shown in Figure 2, although the nominal minimal molecular weight of the
contents of fraction 1 (lane 3) was 200 kDa, there were proteins in it which migrated at
about 18, 25 and 60 kDa. This is probably because they were subunits of the
quaternary structures of the proteins that had been adherent during gel ¢ltration
chromatography but had separated following heating with SDS and mercaptoethanol.
From Figure 1, it can be seen that the proportion of the LMW fraction in milk was
higher than that in colostrum, but fraction 4 from both sources activated the oxidative
burst activity of bovine PMNL. There was a large molecule protein band contaminant
in fraction 4 of both milk and colostrum, probably caused by tailing. However,
although bands of the same molecular weight were seen from fractions 2 and 3, they
did not possess the priming activity. The constituents of the LMW fractions of bovine
colostrum and milk are not well understood but they may contain proteose peptones
(Merin et al., 2001). Bovine colostrum and milk casein digests have various bioactiv-
ities (Shah, 2000). Ultra¢ltered bovine whey activates PMNL (Roth et al., 2001) and
bovine lactoferricin, a digestion product of lactoferrin, stimulates the phagocytic
activity of human neutrophils (Miyauchi et al., 1998). A substance with a molecular
weight of about 5 kDa in bovine colostrum activates the proliferation of CKT-1 cells
(Kishikawa et al., 1996). Therefore, our LMW fraction must contain many bioactive
substances. Fraction 4-2 of colostrum and milk promoted the greatest oxidative burst
activity, but resolution of this fraction (molecular mass range 1423^26 625 kDa) by
SDS-PAGE in Tricine bu¡er did not generate a clear band. We speculate that a clear
band was not formed because the active substance is a small molecule or because the
active substance is not dyed by the silver stain.
Exposing neutrophils to substances that amplify their responses to activating
agonists is known as priming. Tumour necrosis factor a (TNF-a), granulocyte-
macrophage colony-stimulating factor (GM-CSF), IL-1b, INF-g, IL-8, platelet-
activating factor and lipopolysaccharide can all prime the oxidative burst of neutro-
phils (Gay, 1990; Tennenberg et al., 1993; Mikami et al., 1998; DeLeo et al., 1998). The
oxidative burst was enhanced in PMNL primed by milk fraction 4-2, when stimulated
with S. aureus, but it was not enhanced when stimulated with PMA. Primed PMNLs
exposed to sequential homologous stimuli become desensitized (McPhail et al., 1984).
However, as the oxidative burst was less in PMNL primed by milk fraction 4-2 and
stimulated with PMA than in PMNL not primed and stimulated with PMA, it is clear
that the activity in milk fraction 4-2 di¡ers from that of PMA. The LMW fraction of
peritoneal dialysis e¥uent reduces the oxidative burst induced by PMA (Daniels et al.,
1993). Thus, the priming e¡ect of milk fraction 4-2 may antagonize PMA. Since this
activity of the LMW fraction of milk was unstable after heating, its active site may be
degenerated.
The present study showed that only the LMW (510 kDa) of bovine colostrum and
milk enhanced the oxidative burst activity of PMNL in vitro. The molecular weights of
pro-in£ammatory cytokines, such as TNF-a, IL-1b, IL-6 and INF-g, which have
considerable priming activity (Swain et al., 2002), are within the range 10^30 kDa
(Cerretti et al., 1986; Maliszewski et al., 1988; Cludts et al., 1993; Ebrahimi et al., 1995).
Therefore, fraction 3 from colostrum and milk should contain large amounts of these
cytokines. However, whereas fraction 4 had a powerful priming e¡ect, fraction 3 did
460

not. These results indicated that the major priming agent in colostrum and milk is a
LMW substance(s) that is not a cytokine, contrary to the suggestion by Roth and
colleagues (2001). This substance(s) may participate in the immune response of the
mammary gland or enhance PMNL activity in the calf following ingestion in
colostrum and milk. Such components of LMW would be absorbed from the jejunum
in the newborn calf. Phagocytes, especially neutrophils, play an important role in
innate immunity (Smith, 1997; Goldman, 2000; Burg and Pillinger, 2001). Young
animals take a long time to acquire immunity (Siegrist, 2001), whereas innate
immunity, such as that provided by PMNLs, does not require an immunological
memory, and activation of such innate immunity may be e¡ective in newborn animals.
On the other hand, extreme enhancement of PMNLs damages tissue (Babior, 2000), so
substances that activate PMNLs may participate in mastitis. Accordingly, the
substances should be puri¢ed and characterized and the mechanism by which they
activate PMNL elucidated.

REFERENCES

Babior, B.M., 2000. Phagocytes and oxidative stress. American Journal of Medicine, 109, 33^44
Burg, N.D. and Pillinger, M.H., 2001. The neutrophil: function and regulation in innate and humoral
immunity. Clinical Immunology, 99, 7^17
Cerretti, D.P., McKereghan, K., Larsen, A., Cosman, D., Gillis, S. and Baker, P.E., 1986. Cloning,
sequence, and expression of bovine interferon-gamma. Journal of Immunology, 136, 4561^4564
Cludts, I., Cleuter, Y., Kettmann, R., Burny, A. and Droogmans, L., 1993. Cloning and characterization of
the tandemly arranged bovine lymphotoxin and tumour necrosis factor-alpha genes. Cytokine, 5, 336^
341
Daniels, I., Lindsay, M., Porter, C., Haynes, A.P., Fletcher, J. and Morgan, A.G., 1993. E¡ect of peritoneal
dialysis e¥uent on superoxide anion production by polymorphonuclear neutrophils. Nephron, 64, 382^
387
DeLeo, F.R., Renee, J., McCormick S., Nakamura, M., Apicella, M., Weiss, J.P. and Nauseef, W.M., 1998.
Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly. Journal of
Clinical Investigation, 101, 455^463
Ebrahimi, B., Roy, D.J., Bird, P. and Sargan, D.R., 1995. Cloning, sequencing and expression of the ovine
interleukin 6 gene. Cytokine, 7, 232^236
Gay, J.C., 1990. Priming of neutrophil oxidative responses by platelet-activting factor. Journal of Lipid
Mediators, 2, S161^175
Goldman, A.S., 2000. Back to basics: host responses to infection. Pediatric Reviews and Communications,
21, 342^249
Goto, M., Maruyama, M., Kitadate, K., Kirisawa, R., Obata, Y., Koiwa, M. and Iwai, H., 1997. Detection
of interleukin-1 beta in sera and colostrum of dairy cattle and in sera of neonates. Journal of Veterinary
Medical Science, 59, 437^441
Hagiwara, K., Kataoka, S., Yamanaka, H., Kirisawa, R. and Iwai, H., 2000. Detection of cytokines in
bovine colostrum. Veterinary Immunology and Immunopathology, 76, 183^190
Kishikawa, Y., Watanabe, T. and Kubo, S., 1996. Puri¢cation and characterization of cell growth factor in
bovine colostrum. Journal of Veterinary Medical Science, 58, 47^53
Maliszewski, C.R., Baker, P.E., Schoenborn, M.A., Davis, B.S., Cosman, D., Gillis, S. and Cerretti, D.P.,
1988. Cloning, sequence and expression of bovine interleukin 1 alpha and interleukin 1 beta
complementary DNAs. Molecular Immunology, 25, 429^437
McPhail, L.C., Clayton, C.C. and Snyderman, R., 1984. The NADPH oxidase of human polymorpho-
nuclear leukocytes. Evidence for regulation by multiple signals. Journal of Biological Chemistry, 259,
5768^5775
 461

Menge, C., Neufeld, B., Hirt, W., Schmeer, N., Bauerfeind, R., Baljer, G. and Wieler, L.H., 1998.
Compensation of preliminary blood phagocyte immaturity in the newborn calf. Veterinary Immunology
and Immunopathology, 62, 309^321
Merin, U., Bernstein, S., Bloch-Damti, A., Yagil, R., Creveld, C.V., Lindner, P. and Gallop, N., 2001. A
comparative study of milk serum proteins in camel (Camelus dromedarius) and bovine colostrum.
Livestock Production Science, 67, 297^301
Mikami, M., Llewellyn-Jones, C.G. and Stockley, R.A., 1998. The e¡ect of interleukin-8 and granulocyte
macrophage colony stimulating factor on the response of neutrophils to formyl methionyl leucyl
phenylalanine. Biochimica et Biophysica Acta, 1407, 146^154
Miyauchi, H., Hashimoto, S., Nakajima, M., Shinoda, I., Fukuwatari, Y. and Hayasawa, H., 1998. Bovine
lactoferrin stimulates the phagocytic activity of human neutrophils: identi¢cation of its active domain.
Cellular Immunology, 187, 34^37
Roth, J.A., Frank, D.E., Weighner, P. and Weighner, M., 2001. Enhancement of neutrophil function by
ultra¢ltered bovine whey. Journal of Dairy Science, 84, 824^829
Shah, N.P., 2000. E¡ects of milk-derived bioactives: an overview. British Journal of Nutrition, 84(supple-
ment 1), S3^10.
Siegrist, C.A., 2001. Neonatal and early life vaccinology. Vaccine, 19, 3331^3346
Smith, J.A., 1997. Exercise immunology and neutrophils. International Journal of Sports Medicine,
18(supplement 1), S46^55
Sugisawa, H., Itou, T. and Sakai, T., 2001. Promoting e¡ect of colostrum on the phagocytic activity of
bovine polymorphonuclear leukocytes in vitro. Biology of the Neonate, 79, 140^144
Sugisawa, H., Itou, T., Ichimura, Y. and Sakai, T., 2002. Bovine milk enhances the oxidative burst activity
of polymorphonuclear leukocytes in low concentrations. Journal of Veterinary Medical Science, 64,
1113^1116
Swain, S.D., Rohn, T.T. and Quinn, M.T., 2002. Neutrophil priming in host defense: role of oxidants as
priming agents. Antioxidants and Redox Signaling, 4, 69^83
Tennenberg, S.D., Fey, D.E. and Lieser, M.J., 1993. Oxidative priming of neutrophils by interferon-
gamma. Journal of Leukocyte Biology, 53, 301^308

(Accepted: 24 December 2002)