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PHYSIOLOGY AND MANAGEMENT Granulocyte Colony-Stimulating Factor Effects on Lymphocytes and Immunoglobulin Concentrations in Periparturient Cows JUDITH R. STABEL, MARCUS E. KEHRLI, JR., JOHN R. THURSTON, JESSE P. GOFF, and THOMAS C. BOONE' Immunology of Ruminant Perinatal Disease Project Metabolic Diseases and Immunology Laboratory National Animal Disease Center Agricultural Research Service, USOA Ames. IA 50010

alleviate immunosuppression in periparturient cows. (Key words: lymphocytes, cytokine, granulocyte colony-stimulating factor, immunoglobulin)

ABSTRACT

Immunomodulatory effects of recombinant bovine granulocyte colonystimulating factor were evaluated in periparturient dairy cows. Eleven of 21 cows were experimentally infected with Staphylococcus aureus in one mammary quarter prior to the study. Cows were assigned to four groups in a randomized complete block design to evaluate the effects of recombinant bovine granulocyte colony-stimulating factor (5 pg/kg of body weight or placebo injected subcutaneously once daily beginning 14 d prepartum through 10 d postpartum) on infected and uninfected cows during the periparturient period. Blood lymphocytes were isolated and evaluated from 5 wk before expected parturition through 7 wk postpartum. Lymphocyte function was evaluated using a blastogenesis assay, a mitochondrial methylthiazoltetrazolium cleavage activity assay, and an in vitro assay of IgM production. Serum concentrations of IgM, IgG1, conglutinin, and hemolytic complement were also determined. Injections of cows with recombinant bavine gImdcqk colatly-stimulating factor resulted in enhanced lymphocyte blastogenesis and mitochondrial methylthiazoltetrazolium cleavage activity in unstimulated cultures, higher serum IgM, and increased in vitro IgM production by B lymphocytes. These data provide support for the use of recombinant bovine granulocyte colony-stimulating factor to

Received January 10, 1991. Accepted M a y 6. 1991.

'AMGEN, Inc., Thousand Oaks, CA 91320, 1991 J Dairy Sci 743755-3762

Abbreviation key: Con A = concanavalin A, G-CSF = granulocyte colony-stimulating factor, GM-CSF = granulocyte-macrophage colony-stimulatingfactor, IL-1 = interleukin 1, IMI = intramammary infection, MTT = methylthiazoltetrazolium, PBMC = peripheral blood mononuclear cells, PWM = pokeweed mitogen. INTRODUCTION

Immunosuppression is highly correlated with stressors such as parturition, lactation, nutritional deficiencies, and environmental adaptation (4, 10, 11, 17, 18, 23, 27). Indeed, parturition and the immediate periparturient period impose conditions of extreme stress on the dairy cow. The bovine mammary gland is more susceptible to infection and clinical disease during this period than during the remainder of lactation or the dry period (20, 25, 29). Intrarnammary infections (IMI) may result from a reduced ability of the gland to prevent bacterial colonization or growth. This may be due in part to a loss in immune cell function during this critical period. A marked impairment in lymphocyte function as measured by lymphocyte blastogenesis and in vitro Ig production has been observed at parturition in the dairy cow (14, 21, 34, 37). Preventing adverse changes in immune cell function via administration of immunomodulators such as recombinant cytokines may potentially reduce the incidence of clinical disease in periparturient d a q cows. In a separate manuscript (16), we reported on experiments in which recombinant bovine

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granulocyte colony-stimulatingfactor (G-CSF) was administered with the objective of preventing periparturient impairment of neutrophil function and postpartum neutropenia. In vivo administration of G-CSF induces differentiation of hematopoietic progenitor cells to neutrophilic granulocytes (9, 35). In addition, GCSF enhances effector functions of mature neutrophils by increasing phagocytic and cytotoxic activities as well the secretion of superoxide anion (2). Activated neutrophils have the capability to secrete interleukin-1 (IL-1), an important regulatory cytokine involved in proliferation and differentiation of T and B lymphocytes (3, 7, 12). It is not known whether G-CSF induces production of IL-1; however, experimental evidence indicates that g r a n u l o q k m m m ~ ecolony-stimulating fador (GM-CSF), a glycoprotein in the same class as G-CSF, will invoke expression and secretion of I L 1 by human neutrophils (19). Therefore, recombinant bovine G-CSF may be capable of indirectly enhancing various aspects of humoral and cell-mediated immunity by upregulation of cytokines such as IL-1. We report here the effects of recombinant bovine G-CSF on selected lymphocyte functions, plasma calcium, and serum Ig, complement, and conglutinin concentrations in penparturient dairy cows. MATERIALS AND METHODS

0-CSF Preparatlon

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 G-CSF on both infected and uninfected cows during the periparturient period. Five cows with S. aureus IMI and 5 uninfected cows were given 5 pg of G-CSF/kg of body weight injected subcutaneously once daily, beginning 14 d prior to the projected calving date to 10 d postpartum. Six cows with S. aureus IMI and 5 uninfected cows were injected with carrier alone following the same schedule. Initially, sampling was twice a week beginning an estimated 5 wk before the expected calving time. Frequency of sampling was increased to a Monday, Wednesday, Friday schedule about 4 wk before expected parturition and continued at that frequency for at least 3 wk postpartum. Animals were then sampled twice a week for the next 4 w k Establishment of Experimental S. w e u s IMI

Eleven cows were experimentally infected by intracisternal administration of approximately 50 cfu of S. aurew (Newbould strain 305, ATCC 29740) in one mammary quarter at least 1 mo prior to these studies. Staphylococcus aureus IMI persisted in all infected cows throughout the duration of these studies as was assessed by daily determination of bacterial shedding.

Recombinant bovine G-CSF expressed in Plasma Calcium Determinatlons Escherichia coli was purified as described Plasma calcium concentrations were deter(39). The homogeneous protein was adjusted to a concentration of 2 m g / d in water (pH mined by atomic absorption spectrophotometry 3.5). Endotoxin levels were 12.5 endotoxin (26). units/ml as determined by limulus amoebocyte assay. The colony-stimulating activity of the Leukocyte Preparatlon protein (1 x 108 U/mg) was determined by a Peripheral blood mononuclear cells mouse bone marrow assay as described @. R. (PBMC) were isolated by buoyant density Avalos et al., 1991, unpublished data). centrifugation (40 min at 400 x g) over a colloidal polyvinylpyrolidone-coated silica Anlrnals and Experlmental Deslgn gradient (specific gravity = 1.084 g/ml) and Twenty-one multiparous Holstein cows washed in phosphate-buffered saline as previwere evaluated during the periparturient peri- ously described (15). This isolation procedure od, ranging from 5 wk prepartum to 7 wk typically yields 88 to 92% lymphocytes and 8 postpartum. Gestation periods were synchro- to 12% monocytes in our laboratory (13). nized to minimize the calving period duration. Lymphocyte Blastogenesis. Peripheral blood Eleven cows were experimentally infected with mononuclear cells were plated at a density of 2 Journal of Dairy Science Vol. 74, No. 11, 1991

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x 1 6 cells per well in 96-well tissue culture d of incubation, Supernatants were harvested plates. Tissue culture media (.2 ml of RPMI- and frozen at -70°C until analyzed by ELISA.

1640, GIBCO Laboratories, Grand Island, NY, Number 380-2400, with 25 mM HEPES buffer and 2 mM L-glutamine) was supplemented with the following: heat-inactivated fetal bovine serum (A-ll 15-L, Hyclone Laboratories, Inc., Logan, UT) to a 10% fiial concentration; and an antibiotic, antimycotic solution (A9902, Lot 76F-6832, Sigma Chemical Company, St. Louis, MO) resulting in 100 units of penicillin G, .1 mg of dihydrostreptomycin, and 250 ng of amphotericin B/ml of medium. Media for mitogenic stimulation contained .5, 1, or 5 pg of concanavalin A (Con A, C-5275, Sigma Chemical Company)/ml of medium. Cells were incubated for 72 h at 39'C in 5% C02. then pulsed with 14.8 kBq per well of [methyl-3KJthymidine (Amersham, Arlington Heights, E)for 18 h. Cells were then harvested onto glass fiber filters (PHD cell harvester, Cambridge TechnoIogy. Inc., Cambridge, MA), and [3Hlthymidine incorporation was determined by liquid scintillation spectrophotometry. As a measure of oxidative metabolism of mononuclear cells, parallel tissue culture plates were prepared to determine the activity of the mitochondrial enzyme, succinate dehydrogenase, in cultured PBMC as previously described (6). Methylthiamltemmlium (MTT), a yellow tetrazolium salt, is specifically reduced by succinate dehydrogenase into purple formam crystals, and the optical density of this end product is measured at 560 MI. In Vitro Antibody Production. Pokeweed mitogen (PWM)-driven production of polyclonal IgM by cultures of bovine B lymphocytes was determined by harvesting culture Supernatants and assaying IgM by ELISA (30). Tissue culture media (.5 ml of RPMI 1640) was supplemented with the following: heatinactivated fetal bovine serum to a 7.5% final concentration, 50 ~IM2-mercaptoethanol, and antibiotic, antimycotic solution. Bovine PBMC were plated at a density of 4 x Id cells per well and incubated for 12 d at 39'C in a humidified 5% CO2 atmosphere. Cultures were unstimulated or stimulated with .1 pg of P W m l of tissue culture medium. Media in all mononuclear cell cultures were replenished on d 4 of incubation with the same media used for unstimulated cultures. After an additional 8

Serum Complement, Conglutinin, and immunoglobulin Activity

Blood samples were collected by jugular venipuncture, and serum was frozen at -70°C until tested. Complement activity was determined by hemolysis induced by bovine sera in agar gel containing guinea pig erythrocytes opsonized with specific bovine antisera (32). Serum conglutinin activity was determined by two methods as described (33). Serum IgM and IgGl concentrations were determined by ELISA (30). Data Analysis Leukocytes isolated daily from 4 Holstein steers were used as internal laboratory standards to determine the daily mean for each assay. This mean was used to reduce the day to day component of variance typically seen with immune cell function assays by standardizing the daily individual cow results as a percentage of the steers' daily mean (17). Immune cell function data from each cow were then coded relative to actual calving dates. Standardized values for lymphocyte blastogenesis and M'IT reduction as well as raw data values for serum calcium, Ig , hemolytic complement, conglutinin, and in vitro IgM production for individual cows were averaged to obtain a single value for each of four periods. The four periods included the baseline period for all determinations made prior to placebo or G-CSF injections, the prepartum injection period, the postpartum injection period, and the period after the fimal injections for each cow. Significant differences ( P e .05) between the G-CSFtreated cows and controls for any period by infection status were determined by Student's t test. Effects of G-CSF administration, chronic S. aureus IMI,and their interaction were also tested using general linear models with a repeated measures analysis of variance (REPEATED PROFILE analysis) (28). Main and interaction effects were evaluated by three sequential contrasts of the four experimental periods for each variable. Raw data values for serum calcium for individual cows were averaged within control or Journal of Dairy Science Vol. 74, No. 11. 1991

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Figure 1. Effect of recombinant bovine granulocyte colony-stimulatingfactor (G-CSF) administration on blood calcium levels. Time of parturition was assigned a zero time value, and the days represent 24-h periods before or after calving. Each data point represents the mean f SEM of 3 to 6 cows.

G-CSF treatment groups in the immediate periparturient period. Time of parturition was assigned a zero time value, and other time points represent 24-h periods surrounding parturition, ranging from 11 d before to 10 d after calving. RESULTS

Prrp.am 1 x

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Figure 2. Tbe IgM concentrations in culture supemaof unstimulated or pokeweed mitogen 0stimulahxi lymphocytes isolated from peripamuient COWS treated with recombinant bovine granulocyte colonystimulating factor (G-CSF)compared with placeboinjected control cows. Data are the concentration of IgM (nghnl) in culture supernatants as determined by ELISA. Time of parturition was assigned a zero time value, and the periods represent baseline, prepartum injection flx), postpartum injection, and posttreatment periods. Bars represent the mean f SEM of all cows in that group for that time period; *P < .05. tants

Periparturient Changes in Lymphocyte Function

Table 1 contains data from cows receiving G-CSF versus controls across IMI status. Cows receiving G-CSF had higher (P c .05) levels of lymphocyte blastogenic activity in unstimulated cultures than control animals during the postpartum treatment period (Table 1). In addition, a significant (Pc .01) increase in lymphocyte blastogenesis from baseline levels was observed due to G-CSF administration General Observatlons at Parturltion during the penparturient period. In contrast, GThere were no differences in overall health CSF significantly decreased (P c .05) mitogenbetween the animals that received G-CSF or induced blastogenesis in lymphocyte cultures placebo injections. Clinically, the cows did not containing .5 pg/ml of Con A and tended to exhibit any adverse effects of G-CSF therapy decrease blastogenic responses to 1.0 pg/ml of during the experiment. No new IMI were de- Con A (Table 1). This effect, however, was not tected in any quarters of any cows during the apparent for the highest concentration of Con time of G-CSF administration. A (which resulted in the highest incorporation of [3Hlthymidine). The PBMC from cows receiving G-CSF also had greater (P c .05) Plasma Calclum Levels hT"reduction in unstimulated cultures during Blood calcium determinations (Figure 1) in- the treatment period, as well as in cultures dicated that G-CSF administration reduced the receiving the lowest concentrations of Con A severity of postpartum hypocalcemia experi- used (.5 pgJml; Table 1). enced by cows in this study, especially on the Figure 2 shows the effect of G-CSF admin1st d after calving. istration on in vitro IgM production by B

On average, cows were sampled from 64.5 f 6 d prepartum (range = 31 to 115 d) to an average of 50 k 2.7 d postpartum (range = 25 to 77 d). Ten cows received G-CSF for 19.1 f 1.8 d prepartum (range = 10.3 to 37 d) and for 10 d postpartum.

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GRANULOCYTE COLONY-STIMULA'IDlG FACTOR

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Figure 3. Meet of recombinant bovine granulocyte colony-slimdating factor (G-CSF)administration on Ig concentrations in periparturient cows compared with placebo-injected control cows. Data are h e concentration of Ig (&a) in serum as detennined by ELISA. Time of parturition was assigned a zero time value, and the periods represent baseline, prepartum injection, postpartum injection, and pos-tment periods. Bars represent the mean f SEM of all cows in lhat group for h a t time period; *P < .05.

lymphocytes in mixed cultures of lymphocytes and monocytes. In vivo administration of G.05) in CSF prevented the reduction (P PWM-stimulated antibody production by B cells observed in prepartal control animals. Serum Ig, Complement, and Conglutinin Levels

A significant decrease (P c .05) in serum IgM concentration was observed around p a rition in three of the four treatment groups (Figure 3). Serum IgM concentrations remained fairly constant throughout the peripar-

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Fig e 4. Effect of recombinant bovine granulocyte colony-slimdating factor (G-CSP) administration on serum coqlutiain and complement levels in periparhuient cows compared with placebo-injected control cows. Data are the reciprocal log2 titer concentration of serum conglutinin collcentrations as determined by a bacterial conglutination. Tme of parturition was assigned a zero time value, and the periods represent baseline, prepartum injection nx), postpartum injection, and posttreatment periods. Bars represent tbe mean f S E M of all cows in that group for that time period. *Ininfection (MI)different from controls (00 IMI)at P < .05.

turient period in cows without IMI and receiving G-CSF. Serum IgGl also declined at parturition with no significant effects of GCSF administration observed (Figure 3). Across IMI status, administration of G-CSF minimized (P < .OS) a drop from baseline levels in serum complement and attenuated the decline in serum E. coli agglutinin. For serum complement and conglutinin, this effect was most apparent in cows without S. aureus IMI (Figure 4). Journal of Dairy Science Vol. 74, No. 11. 1991

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TABLE 1. Effect of recombinant bovine granuloq3e colony-stimulatingfactor (GCSF) administration to peaipaxturiat Holstein cows on lymphocyte function^.^ BSelinc period DNA Synthesis unstimutated c u l m Controls GCSF .5 pg of Con A Conlmls G-CSP 1.0 pg of Con A Controls G-CSP 5.0 pg of Con A Controls G-CSF M l T Reduction unstimulated cultures Conkols G-CSP .5 pg of Con A Controls GCSP 1.0 pg of Con A Controls GCSP 5.0 ue of Con A Controls GCSP

.-

sz 211 174 228 217 206 197 193 191

SEM 57 26 16 20 15 17 12 15

105 106 125 122 125 123 127 127

5 6 4 8 5 9 7 10

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Postpartam

Post-G-CSP

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treatment paid SEM 158 24

@od X

SEM 112 80 13 17 9 13 6 8 7 12 5 8 5 8 10 8

sz

391 524 213 159 187 160 173 169

167 142 23 14 16 10 13 8

123 155* 126 141* 119 124 112 114

4 8 3 7 3 6 5 6

bnatment

423* 170 134 156 127 162 165

20 14 15 11 19

357 274 165 145 159 147 158 158

136 174, 136 147 126 128 115 117

11 11 6 11 6 10 8 11

135 148 129 130 125 122 135 122

89 17

~~~

'Data are mean percentage of standards f SEM of all placebinjected (n = 11) or GCSPijected cows (n = 10) for each lymphocyte function evaluated. Raw data means f SEM e x p r d in counts per minute for steer lymphocyte DNA synthesis averaged over all sampling days were as follows:aostimulated, 2315 f 266, .5 pg of CollCBaavalin A (Con A)/ 131,112 f 6264; 5.0 pg of Con A/ml, 192,474 f 8715. MTT = ml. 90,736 f 5391; 1.0 pg of Con Methylthiazoltetrazolium. *Indicates mean significantly different (P < .05) from placebo-injected controls for any particular period.

DISCUSSION

An unexpected finding of G-CSF administration to periparhxient dairy cows was a significant increase in circulating mononuclear cells (16). This effect of G-CSF on mononuclear cells uniformly affected the number of circulating CD5+, CD4+, and CD8+ T lymphocytes; IgM+-bearing B lymphocytes; and monocytes (13). For these reasons, it is interesting to note the effects of G-CSF administration on lymphocyte blastogenesis, oxidative metabolism, and Ig secretion, as well as total serum Ig, complement, and conglutinin concentrations. There were no consistent effects of G-CSF on mitogen-induced lymphocyte blastogenesis or oxidative metabolism, which is not surprising, because the primary activity of this cytokine is induction of bone marrow granulopoiesis. However, the higher numbers of circulating CD5+ T lymphocytes, IgM+ B lymphocytes in vivo, and the prevention of reduced IgM produced during in vitro culture Journal of Dairy Science Vol. 74, No. 11, 1991

suggest that G-CSF does affect lymphocyte function. Whether this is a direct or indirect effect of G-CSF cannot be determined from our data. It is known that GM-CSF treatment of neutrophils in vitro induces secretion of IL1 (19). If in vivo administration of G-CSF to cows increased the recently recognized production and secretion of ILl by bovine neutrophils (3), it may explain the increased lymphocyte activities reported here. Another explanation may be the increased numbers of monocytes and B lymphocytes observed with G-CSF treatment. Treatment of human bone marrow cultures with G-CSF increased cellular proliferation with a selective expansion of total granulocytes and monocytes (5). Monocytes are a primary source of IL-1, which in turn may stimulate proliferation and differentiation of B lymphocytes. Therefore, upregulation of IL-1 production by leukocytes induced by GCSF may also be responsible for modulating Ig secretion by B cells, especially in cows receiving G-CSF with no IMI. Administration of G-CSF lessened the degree of decline in serum complement and

GRANULOCYTE COLONY-STIMULATING FACXOR

conglutinin observed during the penparturient period in all treated groups. This efFect on complement and conglutinin activity was most apparent in cows receiving G-CSF but without chronic S. aureus IMI, which might suggest that chronic inflammation may stimulate an endogenous cytokine-mediator cascade similar to that induced by exogenous G-CSF. Lymphokines that activate monocyte complement biosynthesis have been reported (8). Cows receiving G-CSF during the periparturient period demonstrated no adverse reactions, and milk production and constituency were unaffected (data not shown). The lessened severity of hypocalcemia in cows receiving G-CSF is an interesting linding that is of unknown biological significance at this time. Administration of G-CSF increased the number of monocytes, some of which may be precursors to the boneresorbing osteoclast cells (31). It is possible that the number of osteoclasts or their activity might be increased by G-CSF treatment, increasing the bone resorption abilities of the cow, which aid in the maintenance of plasma calcium. Thus, there may be a potential use for G-CSF in the prevention of parturient hypocalcemia. There was little evidence in this study to indicate that establishment of S. aureus IMI prior to G-CSF administration altered its effectiveness in the reduction of periparturient immunosuppression. Indeed, experimental evidence indicates that G-CSF may be a useful therapeutic in the treatment of established infections (38). Bacterial challenge studies in a limited number of cows given G-CSF have demonstrated reductions in the rate of new IMI (nearly a 509i reduction in the rate of new infections) (24). Studies in mice have indicated that prophylactic therapy with human G-CSF may augment the resistance of immunocompromised (cyclophosphamide-induced) mice to infections (22). In addition, no severe bacterial infections were observed in children with se vere congenital neutropenia treated with GCSF (36). 'Zhe immunomodulatory effects reported here (increased basal lymphocyte activity, in vitro IgM production by B lymphocytes and serum complement, and conglutinin levels in periparturient dairy cows) as well as the striking increase in the number of circulating neutrophils available to respond to new infections

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(16) should contribute to increased resistance to new infections. Many of the in vivo effects observed with G-CSF administration may be indirectly mediated by induction of other known and unknown mediators and members of a cytokine cascade. Further experiments to study the resistance of the mammary gland to postpartum challenge exposure to a mastitis pathogen during G-CSF therapy are warranted based on previous bacterial challenge studies and the immunomodulatory data reported here. ACKNOWLEDGMENTS

The authors thank Arlen Anderson, Sally

Crandell, Bruce Pesch, Gary Fry, and Kim Driftmier for excellent technical assistance and Norm Tjelmeland, Creig Caruth, Jack Moore, and Ben Durbey for animal care. REFERENCES 1 Reference deleted in proof. 2Avalos, B. R., J. C. Gasson, C. Hedvat, S. G. Quan, G. C. Baldwin, R. H.Weisbart, R. E. Williams, D. W. Golde, and J. P. Dipasio. 1990. Human granulocyte biologic &ties and ~ol~ny-~hdatin factor: g receptor characterization on hematopoietic cells and small cell lung cancer cell lines. Blood 75:851. 3CanniOg. P. C., and 1. D. Neill. 1989. Isolation and charactaization of intmleukjn-1 from bovine polymorphonuclear lcalocytcs. 1. Leukocyte Biol. 45:21. 4cludra, R K. 1987. Trace elements and immune response. Clin. Nutr. 118:118. 5 Couthho, L. H..A. Will, J. Radford, R. Schir6, N. G. Testa, and T. M. Dexter. 1990. Effects of recombinant humau grauulocyte colony-stimulating factor (CSF), haman grarmlocytc mauvphageKSF, and gibbon intaleukb3 on hematopoiesis in human long-term bone marrow coltare. Blood 722118. 6Deaizot, P., and R. Lang. 1986. Rapid coiorimehric assay for cell growth and survival: modifications to the tetrazolium dye procadwe giving improved sensitivity and reliability. J. Immunol. Methods 89271. 7Dinartll0, C. A. 1987. clinical relevance of intdeukbl and its mdtipk biological activities. BuU. Iust. Pasteur 85:267. 8 Drouet, C., A. Reboul, and M. Colomb. 1989. IdenWication of a human non-mterfemn lymphokine activating mollocyte complement biosynthesis. Biochem. I. 263: 157. Y. 9 h%sawa, M.,Y. Kobayashi, T. Okabe, F.Takak~~, Komatsu, and S. Itoh. 1986. Recombinant human Brarmlocyte wlony-stimulating factor induces granalocytosis in vivo. Jpa J. Can= Res. n:866. 10J.F.T. 1989. Stress and immunity: a anifying concept. Vet. Immunol. Immunopatbol.20263. 11 Gross, R L., and P. M. Newbeme. 1980. Role of nutrition in iaununologic fanctioa Physiol. Rev. 60: 188. 12 Hamblin, A. S. 1988. Lymphokines and interleukins.

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