Administration of recombinant bovine granulocyte colony stimulatory factor to periparturient dairy cows was evaluated as a method to prevent periparturient im-.
PHYSIOLOGY AND MANAGEMENT Effects of Granulocyte Colony-5tlmulating Factor Administration to Periparturlent Cows on Neutrophlls and Bacterial Shedding MARCUS E. KEHRLI, JR., JESSE P. GOFF, MARK G. STEVENS,! and THOMAS C. BOONE2 Metabolic Diseases and Immunology Laboratory NaIIonaJ AnImal Disease Center. Agriwl1uraJ Research service. USDA
Ames, Iowa 50010 ABSTRACT
Administration of recombinant bovine granulocyte colony stimulatory factor to periparturient dairy cows was evaluated as a method to prevent periparturient immunosuppression. Eleven of 21 cows were experimentally infected with Staphylococcus aureus in one mammary quarter prior to the study. Cows were randomly assigned to four groups in a 2 x 2 factorial design to evaluate the effects of placebo or recombinant bovine granulocyte colony stimulatory factor administration on chronic, subclinically infected and uninfected cows during the periparturient period. Blood neutrophils were isolated and evaluated for phagocytic activities 5 wk before expected parturition through 7 wk postpartum. Administration of recombinant bovine granulocyte colony stimulatory factor (5 j.1g/kg body weight or placebo subcutaneously beginning 14 d prepartum through 10 d postpartum) resulted in a prepartum and postpartum leukocytosis of 35,600/J1l and 53,500/J11, respectively. This was attributed to a mature neutrophilia of 24,01O/J11 during prepartwn and 38,080/ J1l during postpartwn treatment periods (pretreatment baseline = 2330/J1l). Mononuclear cell counts averaged 761O/J1l during prepartum and 9830/J11 during postpartum treatment periods (baseline = 3450/J1l). Neutrophil random and directed migration were reduced during recombinant bovine granulocyte colony stimulatory factor treatment compared with placebo or baseline levels. Ingestion
Received October 26, 1990. April 10. 1991. IBruceUosis Research Laboratory. 2Amgen, Inc., Thousand Oaks, CA 91320. ~ed
1991 J Dairy Sci 74:2448-2458
of bacteria and cytotoxicity by neutrophils was increased during recombinantbovinegranulocytecclonystimul~
tory factor therapy compared with placebo or baseline levels. Shedding of S. aureus in lacteal secretions was unaffected by recombinant bovine granulocyte colony stimulatory factor treatment. In summary. administration of recombinant bovine granulocyte colony stimulatory factor increased the nwnber and functional activity of neutrophils and prevented some aspects of periparturient immunosuppression in dairy cows. (Key words: mastitis, hematology, cytokine) Abbreviation key: G-CSF = granulocyte colony stimulatory factor, IMI = intramammary infection, rb = recombinant bovine, ADNC = antibody-dependent neutrophil-mediated cytotoxicity, AINC = antibody-independent neutrophil-mediated cytotoxicity. INTRODUCTION
Native defenses of the bovine mammary gland are continually challenged by environmental exposure to bacteria, and many factors affect the outcome of this challenge. Once the teat canal barrier is penetrated by bacteria, defense mechanisms in lacteal secretions (lactoperoxidase, complement, lactoferrin, and resident immune cells) determine the outcome of bacterial presence in the udder (eradication, subclinical infection, or clinical disease). In lacteal secretions. neutrophils ingest milk fat and casein. resulting in degranulation and a loss of pseudopodia, which reduces the neutrophil's microbicidal potential (27). The b0vine mammary gland is most susceptible to infection during the periparturient period, which correlates well with immunosuppression observed during this period (19, 23, 30). Neutropenia and impaired immune cell fimction 2448
GRANULOCYTE COLONY SllMULATORY FACfOR
during the periparturient period are suspected to increase the susceptibility of the bovine mammary gland to infection and clinical mastitis (9, 11). Impaired neutrophil microbicidal mechanisms (13, IS, 20), impaired lymphocyte blastogenesis (10, 12, 13, 32), and decreased serum complement, conglutinin, and immunoglobulin levels (14) may contribute to a cumulative deficit in defenses against mastitis. Milk. leukocytes derived from impaired blood leukocytes may shift the balance between bacterial clearance and intramammary infection (lMI) in favor of bacteria establishing a new IMI or the ultimate progression of an existing IMI into clinical mastitis. Immunomodulation may provide a new tool for management of infectious diseases of periparturient dairy cows. Colony-stimulating factors are growth factors that regulate the survival, growth, proliferation, and differentiation of hematopoietic progenitor cells from the bone marrow. The potential clinical utility of recombinant human granulocyte colony stimulatory factor (G-CSF) has been reported for various neutropenic conditions (5). Both growth and differentiation of normal human granulocyte progenitors are supported and induced by recombinant human G-CSF. Availability of large quantities of recombinant bovine (rb) G-CSF has made it possible to conduct the study reported here, which evaluated the immunomodulatory effects of IbG-CSF in periparturient dairy cows. MATERIALS AND METHODS RecombInant BovIne G-CSF Preparation
Recombinant bovine G-CSF expressed in
Escherichia coli was purified (33). The homogeneous protein was adjusted to a concentration of 2 mg/ml in water (pH 3.5). Endotoxin levels were ~.5 endotoxin units/ml as determined by limulus ameobocyte assay. The colony-stimulating activity of the protein (1 x lOS U/mg) was determined by a mouse bone marrow assay as described (2). AnImals and Experimental Design
Twenty-one multiparous Holstein cows were evaluated during the periparturient period ranging from 5 wk prepartum to 7 wk postpar-
2449
tum. Gestation periods were synchronized to minimize the calving period duration. Eleven cows were experimentally infected with Staphylococcus aureus in one mammary quarter prior to the experiment. Cows were assigned in a randomized complete block design to allow for simultaneous evaluation of rbG-CSF on both infected and wtinfected cows during the periparturient period. Beginning 14 d prior to the projected 278-. g
G-CSF
Prq>.1um Tl PoSlpltlUm Tl
'8
..2
40000
CQ
~ '". g
30000
~
40000
30000
~
:::l
::>
anwn T. PostpartUm T. POOl G-CSF
Neutrophil Iodination
a.seliDe
I'rq>anwn Tl Pos",utum T. POOl G-CSF
Figure 3. Effect of recombinant bovine granulocyte colony stimulatory factor (G-CSF) or placebo administration on neutrophil functions in periparturient cows with or without Staphylococcus aureus intramammary infections (IMI). Time of parturition was assigned a zero time value, and the periods represent baseline, prepartum treatment (Tx), postpartum treatment, and posttreatment periods.
sues from the blood stream. A closely related cytokine, human granulocyte-macrophage colony stimulatory factor, has been found to increase surface expression of neutrophil and macrophage adherence proteins (1) while at the same time suppressing migration of neutrophils when measured by under agarose techniques similar to those employed in the present study (7). The most logical explanation of the findings in this experiment is that decreased neutrophil random migration may be due to increased expression of neutrophil surface adherence proteins; conversely, increased random migration associated with decreased stickiness of neutrophils is known to occur (4). The decrease in directed migration was also likely due to increased stickiness of the neutrophil cell surface, thus resulting in slower chemotactic migration under agarose. In vivo, such a phenomenon would result in retention of extravascular neutrophils in infected tissues. Neutrophilic Fc receptor-mediated bacterial ingestion and cytotoxic activities were mark-
edly enhanced by rbG-CSF administration. Purified G-CSF enhances the cytotoxic activities of neutrophils in vitro in homologous mouse systems, in heterologous systems with human neutrophils and murine G-CSF, and in homologous human systems (17, 24). In cows infected with S. aureus during wk 2 of lactation, minor enhancement of chemiluminescence activity and basal production of superoxide anion were observed. Cows with the highest blood capacity for generation of reactive oxygen species (defined as superoxide anion production capacity per neutrophil times the number of circulating neutrophils) have less milk loss following experimental intramammary challenge with E. coli (8). Therefore, cows receiving rbG-CSF, as a result of having 10 to 15 times more circulating neutrophils, should be more resistant to the detrimental effects of any new IMI. Previous studies with cows found no change in SCC when 1 or 3 J.lglkg of recombinant human G-CSF were given daily (6); howJoumal of Dairy Science Vol. 74, No.8, 1991
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KEHRU, JR, ET AL.
TABLE 2. Effects of recombinant bovine granulocyte colony stimulatory factor (rbG-CSF) administration to periparturient Holstein cows on neutrophil functions. Neutrophils isolated dairy from four Holstein steers were used as internal laboratory standards to determine the daily mean for each assay. Data are mean percentage of steer standards (±SEM) of all placebo-injected (n 11) or rbG-CSF-injected cows (n 10) for each neutrophil function evaluated.
=
=
treatment period
Postpartum treatment period
After rbG-CSF treatment period
X
X
X
Prepartum
Baseline period
Neutrophil function
Group
Random migration
Controls rbG-CSF
Directed migration
Controls rbG-CSF
101 lOS 101 100
Antibody-independent neutrophil cytotoxicity
Controls rbG-CSF
169 323
63 117
Antibody-dependent neutrophil cytotoxicity
Controls rbG-CSF
102 97
Bacterial ingestion
Controls rbG-CSF
106
6 9 4 4
Controls rbG-CSF
92
Stimulated superoxide anion production
Controls rbG-CSF
92
Stimulated chemiluminescence
Controls rbG-CSF
1I0 99
Staphylococcus aureus killing
Controls rbG-CSF
102
X
Iodination
lID 89 95
94
SEM 5 7.5 1.2 1.0
3 4 2.5 2.4
SEM 4103 65* 3b .3100 97* .3b 470 328 1124 311 7IlO 4b 126* 4113 130* 6b 577* 64* 3b 1.4 99 101 1.2
4 8
127* 119*
3.9 2.9
102* 91*
4 5 3.52.5 b
SEM 8 7
96 90 100 97* 155 809*
1.0 .7 81 245
64* 139*
loab
1I6 149* 62* 55* 90* 95*
SEM
99 108 101 101
7 34b
2.3 4 .7 .7
97 171
8 30
75 89
7 11
III
4 4
Il5
6 3 2.2 1.0
67* 69*
3 2.4
93
I.5 2.1
94
103* 108*
7 5
112 104
97 100
3.7 2.9
103 101
2.0 3 1.9 2.8
""Indicates mean significantly different (P < .05) from placebo-injected controls for any particular column of data. *lndicates mean significantly different (P < .05) from a previous time period for that group of cows.
ever, repeated daily injections of 10 J.l.g/kg recombinant hwnan G-CSF increased baseline SCC values of :QOO,OOO/ml to 300,000 to 4OO,OOO/ml (unpublished data, J. S. Cullor and T. C. Boone, personal communication). Based upon these previous studies, a 5-J.l.g/kg dose of rbG-CSF was used in the present experiment in an attempt to achieve a significant neutrophilia and an increase in SCC to about 500,OOO/ml. The 500,000 SCC target was based upon earlier studies on what affords protection against intramammary bacterial challenge (29). Milk sce were increased in cows receiving rbG-CSF at 5 J.l.g/kg by almost 50%, but this effect was not significant statistically. We speculate that higher doses of rbGeSF (e.g., 7 to 8 J.l.g/kg) and larger experimental group sizes would likely be needed to observe a statistically significant increase in milk see. An earlier study with recombinant human G-CSF given daily at 3 J.l.g/kg demonstrated a 10% increase in sec, which was not significant although a significant increase of Journal of Dairy Science Vol. 74, No.8, 1991
Shedding of Staphylococcus aureus •
=
:E
~
'5 ~
IJ
Controls G-CSF Treated
6
5
