Veterinary hnmunologT and Immunopathology, 28 ( 1991 ) 29-35
29
Elsevier Science Publishers B.V., Amsterdam
Numbers and percent of T lymphocytes in bovine peripheral blood during the periparturient period J.A. Harpa, M.E. Kehrli JrL, D.J. Hurleyb, R.A. Wilsonb and T.C. Boone c aUnited St~:tesDepartment of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, 1,4 50010, USA bPennsylvania State University, University Park, PA 16802, USA CAMGEN, Inc., Thousand Oaks, CA 91320, USA (Accepted 23 April 1990)
ABSTRACT Harp, J.A., Kehrli, Jr., M.E., Hurley, D.J., Wilson, R.A. and Boone, T.C., 1991. Numbers and percent of T lymphocytes in bovine peripheral blood during the periparturient period. Vet. ImmunoL lmmunopathol., 28: 29-35. To determine if periparturient immunosuppression in dairy caule might be due to an alteration in total numbers or percent of T lymphocytes, we examined the numbers and percent of T lymphocyte subsets in peripheral blood from periparturient dairy cows, some of which received recombinant bovine granulocyte colony stimulating factor (rbG-CSF) during the study. Beginning 2 weeks preparturn through 4 weeks postpartum, peripheral blood mononuclear cells (PBMC) were collected and labeled with monoclonal antibodies to BoCD5, BoCD4, and BoCDS, and the percent of cells positive for each marker measured by flow cytometry. The percent of PBMC expressing BoCD5 (total T cells), and BoCD8 (T suppressor/cytotoxic cells) was not significantly different between the groups, or at different times before and after calving. The percent of PBMC expressing BoCD4 (T helper cells) was not significantly different between the groups, however, within both groups there was a higher percent of BoCD4 + cells after calving than during the prepartum period. In cows receiving rbG-CSF, total numbers of PBMC were significantly increased compared to controls during the postpartum treatment period.
INTRODUCTION
Previous studies with dairy cattle have shown that some in vitro lymphocyte and neutrophil functions are impaired during the periparturient period (Kehrli and Goff, 1989; Kehrli et al., 1989a, 1989b). This in vitro impairment corresponds to an increased susceptibility to infections, notably mastitis, in the weeks before and immediately after calving (Malinowski et al., 1983; Smith et al., 1985 ). In the present study, we wished to determine whether there were changes in the total numbers or relative percent ofT lymphocyte subsets in peripheral
30
J.A. HARP ET AL.
blood of periparturient dairy cows that might explain the in vitro and in vivo immunosuppression seen during this period. We examined these parameters in periparturient dairy cows, some of which received recombinant bovine granulocyte colony stimulating factor (rbG-CSF) during the study. MATERIALS AND METHODS
Experimental design Eight multiparous Holstein cows were used in the study. Four cows received daily subcutaneous injections of rbG-CSF (AMGEN Inc., Thousand Oaks, CA) at a dose of 5/zg/kg body weight beginning 14 days prior to the projected calving date and then for 10 days postpartum (recipient cows ). The other four cows received carrier alone following the same schedule (control cows). Blood samples were collected weekly from all cows by jugular venipuncture beginning 5 weeks prepartum until 4 weeks postpartum. The sample collected within the 7 days before calving is designated week - 1 and the sample collected within the 7 days following calving is designated week + 1; thus, there is no time point labeled 0.
Enumeration of peripheral blood mononuclear cells Total leukocyte counts were determined using an electronic cell counter (CellTrack, Angel Engineering Corp., Trumbull, CT) as previously described (Kehrli et al., 1989a). Slides for differential cell counting were prepared by cytocentrifugation (CytoSpin, Shandon, Sewicldey, PA), stained with a combination Wrights/Giemsa stain (StatStain, Volu-Sol, Henderson, NV), and > 200 cells were counted and identified as neutrophils, eosinophils, or mononuclear cells. The total numbers of mononuclear cells per mm 3 of blood were calculated from these data.
Lymphocyte preparation Lymphocytes were isolated using a modification of previously described procedures (Kehfli et al., 1989b). Briefly, blood was collected into 250 ml bottles containing anticoagulant (acid-citrate dextrose) and centrifuged for 20 min at 1171 g. Five ml ofthe buffy coat layer was harvested with a Pasteur pipet and mixed with 20 ml of phosphate-buffered saline (PBS). Twelve ml of this mixture was placed in a 50 ml conical centrifuge tube and underlaid with 10 ml of Percoll (Pharmacia AB, Uppsala, Sweden) adjusted to a specific gravity of 1.084. Tubes were centrifuged for 40 min at 400 g. The interface layer, containing lymphocytes, was harvested with a Fasteur pipet and rinsed by centrifugation to remove excess Percoll. Contaminating erythrocytes were removed by hypotonic lysis, and the lymphocytes were rinsed again by centrifugation. After this final rinse, lymphocytes were resuspended at a
T. LYMPHOCYTES IN CATTLE DURING THE PERIPARTURIENT PERIOD
31
concentration of 2× 106 cells/ml in RPMI 1640 (Gibco, Grand Island, NY) and prepared for flow cytometric analysis.
Antibodies Monoclonal antibodies used in the study were IAH CCI 7 (Howard et al., 1988) which recognizes BoCD5, a membrane glycoprotein found on virtually all bovine peripheral blood T lymphocytes, IAH CC8 (Howard et al., 1989) which recognizes the BoCD4 molecule delineating the T helper cell subset, and IAH CC63 (C. Howard, personal communication, 1989) which recognizes the BoCD8 molecule delineating the T suppressor/cytotoxic cell subset. These antibodies were obtained from Dr. Chris Howard, Agricultural and Food Research Council, Institute for Animal Health, Compton, Great Britain. All the monoelonal antibodies were used at 1:1000 final dilution, which was 2 to 4 times minimum saturating concentration.
Flow cytometry Fifty/d of cell suspensions (2 × 10 6 cells/ml) were stained in 96-well roundbottom tissue culture plates. Cells were washed with PBS containing 2% fetal bovine serum (FBS), and incubated with 50/d of antibody for 20 min at 4°C. After two rinses with PBS-2% FBS, the cells were incubated with fluorescein isothioeyanate-labeled anti-mouse IgG (US Biochemical, Cleveland, OH) for 20 min. The cells were washed twice more, then resuspended in PBS-2% FBS. Cells were diluted with Isoton (Coulter Diagnostics, Hialeah, FL), filtered through 44 micron nylon mesh (Small Parts Inc. Miami, FL) to remove clumps and analyzed using a Coulter 753 flow cytometer.
Statistical analysis Data for total numbers of PBMC were divided into four time periods each for recipient or control cows. The pretreatment period included counts of PBMC during weeks - 5 through - 3. The prepartum treatment period (prepartum Tx) included weeks - 2 and - 1. The postpartum treatment period (postpartum Tx) included weeks + 1 and + 2. The posttreatment period ineluded weeks + 3 and + 4. The mean _+S.E.M. was taken of counts obtained from all blood collections from recipient or control cows within each time period. The data for percent positive BoCD5, BoCD4, and BoCD8 cells were similarly analyzed, with the exception that these parameters were not measured during the pretreatment period. Data between groups were compared using Student's t-test. RESULTS
Fifty-one to 60% of PBMC expressed the BoCD5 (T lymphocyte) marker during the study. This percent was not significantly different (P> 0.05 ) be-
32
J.A. HARP ET AL.
tween recipient and control cows or between time periods. Twenty-nine to 42% of PBMC expressed BoCD4 (T helper cells). There was no significant difference (P>0.05) between the percent of BoCD4+ cells from recipient compared with control cows at any time. Within the control group, there was a significantly higher ( P < 0 . 0 5 ) percent of BoCD4+ cells during the posttreatment time period compared to the prepartum treatment time period (Fig. l ). Thirteen to 22% of PBMC expressed BoCD8 (T suppressor/cytotoxic cells). This percent was not significantly different ( P > 0.05 ) between recipient and control cows or between time periods. The ratio of helper to suppressor cells in control cows was 2.2 during the prepartum treatment period, 2.0. during the postpartum treatment period, and 2. l during the posttreatment period. These ratios in recipient cows were 1.8, 2.3, and 1.9, respectively. Total numbers of PBMC in blood of recipient cows were significantly increased (P , D
(0 O
T
4 0 ~-
20
Prepartum T x
Poatpartum T x
Posttreatment
Period relative to calving and treatment
Fig. i. Percent of BoCD4+ lymphocytesin peripheral blood from cows receiving (hatched bars) or not receiving(solid bars) rbG-CSF. Data are presented as mean+ S.E.M. of all samplingsfrom cowsduringthe indicatedtime periods. See text for details.
33
T. LYMPHOCYTES IN CATTLE DURING THE PERIPARTURIENT PERIOD
20-
"0
o
_o .O
E E_ 10 L-
T
L
0
Pretrestment
Propartum T x
Postpartum
Tx
Posttraatmont
Period relative to calving and t r e a t m e n t Fig. 2. Total numbers o£ mononuclear cells per cubic mm of peripheral blood from cows receiving (hatched bars) or not receiving (solid bars ) rbG-CSF. Data are presented as mean + S.E.M. of all samplings from cows during the indicated time periods. See text for details.
the numbers of PBMC in the blood of these same cows during the pretreatment and posttreatment periods (Fig. 2 ). DISCUSSION
Periparturient cows have an increased incidence of disease, which may be related to the in vitro immunosuppression observed during this period. We wished to determine if this immunosuppression was reflected in vivo by change3 in the numbers or percent of peripheral blood T lymphocyte subsets. Decreases in both the total number and relative percent of helper T cells have been used as an indicator of compromised immune function. For example, human patients with acquired immunodeficiency syndrome (AIDS) commonly have decreased numbers of T cells and an inversion of the CD4/CD8 (helper/suppressor) ratio in peripheral blood (Fahey et al., 1984; Kalish and Schlossman, 1985). In immunocompetent humans, this ratio is normally about 2.0, while in AIDS patients, it is often 0.5 or less~ In the present study, there was no evidence that periparturient immunosuppression in cows is caused by an inversion of the ratio of helper to suppressor T cells. This ratio remained near 2.0 for all cows throughout the study.
34
J.A. HARP ET AL.
We did observe a lower percent ofT helper cells in both groups of cows during the prepartum period compared to the postpartum periods (in the control cows this difference was statistically significant), however, more study is needed to determine the biological significance of this observation. Granulocyte colony-stimulating factor (G-CSF) has been shown to be essential for the proliferation and differentiation of neutrophils in mice and humans (Nicola et al., 1983; Metcalf, 1985; Souza et al., 1986). In addition, G-CSF may be important in activation of neutrophils for killing by antibodydependent cellular cytotoxicity (Souza et al., 1986). Some of the cows in the present study received rbG-CSF to determine its effect on numbers and function of bovine neutrophils (Kehrli et al., submitted to J. Dairy Sci. ). During the time cows received rbG-CSF, the numbers of both granulocytic and monocytic cells in peripheral blood increased significantly. This may indicate an effect of rbG-CSF on bone marrow precursors of the lymphocytic, as well as the granulocytic lineage, either directly or through the induction of lymphopoietic factors. Alternatively, increased numbers of granulocytes may result in greater availability of lymphokines such as IL-1 (Lindemann et al., 1988; Canning and Neill, 1989), which along with other factors might lead to activation and cell division of peripheral lymphocytes. The doses of rbG~CSF used in this study appeared to have no undesirable side effects on the recipients. Additional work is needed to determine if larger doses of rbG-CSF would further increase the numbers of lymphocytic and granulocytic cells and if this would have any beneficial effect on disease resistance of cows during the periparturient period. ArKNOWLEDGMENTS
The authors thank Bruce Pesch and Sally Crandell for collection and processing of lymphocytes.
REFERENCES Canning, P.C. and Neill, J.D., 1989. Isolation and characterization ofinterleukin-I from bovine polymorphonuclearleukocytes. J. Leuk. Biol., 45:21-28. Fahey, J.L., Prince, H., Weaver, M., Groopman, J., Visscher, B., Schwartz, K. and Detels, R., 1984. Quantitative changes in T helper or T suppressor/cytotoxic lymphocyte subsets that distinguish acquired immune deficiency syndrome from other immune subset disorders. Am. J. Med., 76: 95-100. Howard, C.J., Parsons, K.R., Jones, B.V., Sopp, P. and Pocock, D.H., 1988. Two monoclonal antibodies (CCI7, CC29) recognizing an antigen (Bo5) on bovine T lymphocytes, analogous to human CDS. Vet. lmmunol, immunopathol., 19:127- ! 39. Howard, CJ., Sopp, P., Parsons, K.R. and Finch, J., 1989. In vivo depletion of BoT4 (CD4) and of non-T4/T8 lymphocyte subsets in cattle with monoclonal antibodies. Eur. J. Immunol., 19: 757-764.
T. LYMPHOCYTES1NCATTLEDURINGTHEPERIPARTURIENTPERIOD
35
Kalish, R.S. and Schlossman, S.F. 1985. The T4 lymphocyte in AIDS. New Eng. J. Med., 313: ll2-113. Kehrli, M.E. and Goff, J.P., 1989. Periparturient hypocalcemia in cows: Effects on peripheral blood neutrophil and lymphocyte function. J. Dairy Sci., 72:1188-l 196. Kehrli, M.E., Nonnecke, B.J. and Roth, J.A., 1989a. Alterations in bovine neutrophil function during the periparturient period. Am. J. Vet. Res., 50:207-214. Kehrli, M.E., Nonnecke, B.J. and Roth, J.A., 1989b. Alterations in bovine lymphocyte function during the periparturient period. Am. J. Vet. Res., 50:215-220. Lindemann, A., Riedel, D., Oster, W., Meuer, S.C., Blohm, D., Merteismann, R.H. and Herrmann, F., 1988. Granulocyte/macrophage colony-stimulating factor induces interleukin l production by human polymorphonuclear neutrophils. J. Immunol., 140:837-839. Malinowski, E., Krzyzanowski, J., Wawron, W., Slawomirski, J. and Gluszak, J., 1983. Analysis of cases ofEscherichia coli mastitis in cows. Med. Weter., 39:608-610 (in Polish, with English abstract). Metcalf, D., 1985. The granulocyte-macrophage colony-stimulating factors. Science, 229: 1622. Nicola, N.A., Metcalf, D., Matsumoto, M. and Johnson, G.R., 1983. Purification of a factor inducing differentiation in murine myelomonocyticleukemia cells. J. Biol. Chem., 258:90179023. Smith, K.L., Todhunter, D.A. and Schoenberger, P.S., 1985. Environmental pathogens and intra-mammary infection during the dry period. J. Dairy Sci., 68:402-417. Souza, L.M., Boone, T.C., Gabrilove, J., Lai, P.H., Zsebo, K.M., Murdock, D.C., Chazin, V.R., Bruszewski, J., Lu, H., Chen, K.K., Barendt, J., Platzer, E., Moore, M.A.S., Mertelsmann, R., and Welte, K., 1986. Recombinant human granulocyte colony-stimulatingfactor: Effects on normal and leukemic myeloid cells. Science, 232:61-65.
29
Elsevier Science Publishers B.V., Amsterdam
Numbers and percent of T lymphocytes in bovine peripheral blood during the periparturient period J.A. Harpa, M.E. Kehrli JrL, D.J. Hurleyb, R.A. Wilsonb and T.C. Boone c aUnited St~:tesDepartment of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, 1,4 50010, USA bPennsylvania State University, University Park, PA 16802, USA CAMGEN, Inc., Thousand Oaks, CA 91320, USA (Accepted 23 April 1990)
ABSTRACT Harp, J.A., Kehrli, Jr., M.E., Hurley, D.J., Wilson, R.A. and Boone, T.C., 1991. Numbers and percent of T lymphocytes in bovine peripheral blood during the periparturient period. Vet. ImmunoL lmmunopathol., 28: 29-35. To determine if periparturient immunosuppression in dairy caule might be due to an alteration in total numbers or percent of T lymphocytes, we examined the numbers and percent of T lymphocyte subsets in peripheral blood from periparturient dairy cows, some of which received recombinant bovine granulocyte colony stimulating factor (rbG-CSF) during the study. Beginning 2 weeks preparturn through 4 weeks postpartum, peripheral blood mononuclear cells (PBMC) were collected and labeled with monoclonal antibodies to BoCD5, BoCD4, and BoCDS, and the percent of cells positive for each marker measured by flow cytometry. The percent of PBMC expressing BoCD5 (total T cells), and BoCD8 (T suppressor/cytotoxic cells) was not significantly different between the groups, or at different times before and after calving. The percent of PBMC expressing BoCD4 (T helper cells) was not significantly different between the groups, however, within both groups there was a higher percent of BoCD4 + cells after calving than during the prepartum period. In cows receiving rbG-CSF, total numbers of PBMC were significantly increased compared to controls during the postpartum treatment period.
INTRODUCTION
Previous studies with dairy cattle have shown that some in vitro lymphocyte and neutrophil functions are impaired during the periparturient period (Kehrli and Goff, 1989; Kehrli et al., 1989a, 1989b). This in vitro impairment corresponds to an increased susceptibility to infections, notably mastitis, in the weeks before and immediately after calving (Malinowski et al., 1983; Smith et al., 1985 ). In the present study, we wished to determine whether there were changes in the total numbers or relative percent ofT lymphocyte subsets in peripheral
30
J.A. HARP ET AL.
blood of periparturient dairy cows that might explain the in vitro and in vivo immunosuppression seen during this period. We examined these parameters in periparturient dairy cows, some of which received recombinant bovine granulocyte colony stimulating factor (rbG-CSF) during the study. MATERIALS AND METHODS
Experimental design Eight multiparous Holstein cows were used in the study. Four cows received daily subcutaneous injections of rbG-CSF (AMGEN Inc., Thousand Oaks, CA) at a dose of 5/zg/kg body weight beginning 14 days prior to the projected calving date and then for 10 days postpartum (recipient cows ). The other four cows received carrier alone following the same schedule (control cows). Blood samples were collected weekly from all cows by jugular venipuncture beginning 5 weeks prepartum until 4 weeks postpartum. The sample collected within the 7 days before calving is designated week - 1 and the sample collected within the 7 days following calving is designated week + 1; thus, there is no time point labeled 0.
Enumeration of peripheral blood mononuclear cells Total leukocyte counts were determined using an electronic cell counter (CellTrack, Angel Engineering Corp., Trumbull, CT) as previously described (Kehrli et al., 1989a). Slides for differential cell counting were prepared by cytocentrifugation (CytoSpin, Shandon, Sewicldey, PA), stained with a combination Wrights/Giemsa stain (StatStain, Volu-Sol, Henderson, NV), and > 200 cells were counted and identified as neutrophils, eosinophils, or mononuclear cells. The total numbers of mononuclear cells per mm 3 of blood were calculated from these data.
Lymphocyte preparation Lymphocytes were isolated using a modification of previously described procedures (Kehfli et al., 1989b). Briefly, blood was collected into 250 ml bottles containing anticoagulant (acid-citrate dextrose) and centrifuged for 20 min at 1171 g. Five ml ofthe buffy coat layer was harvested with a Pasteur pipet and mixed with 20 ml of phosphate-buffered saline (PBS). Twelve ml of this mixture was placed in a 50 ml conical centrifuge tube and underlaid with 10 ml of Percoll (Pharmacia AB, Uppsala, Sweden) adjusted to a specific gravity of 1.084. Tubes were centrifuged for 40 min at 400 g. The interface layer, containing lymphocytes, was harvested with a Fasteur pipet and rinsed by centrifugation to remove excess Percoll. Contaminating erythrocytes were removed by hypotonic lysis, and the lymphocytes were rinsed again by centrifugation. After this final rinse, lymphocytes were resuspended at a
T. LYMPHOCYTES IN CATTLE DURING THE PERIPARTURIENT PERIOD
31
concentration of 2× 106 cells/ml in RPMI 1640 (Gibco, Grand Island, NY) and prepared for flow cytometric analysis.
Antibodies Monoclonal antibodies used in the study were IAH CCI 7 (Howard et al., 1988) which recognizes BoCD5, a membrane glycoprotein found on virtually all bovine peripheral blood T lymphocytes, IAH CC8 (Howard et al., 1989) which recognizes the BoCD4 molecule delineating the T helper cell subset, and IAH CC63 (C. Howard, personal communication, 1989) which recognizes the BoCD8 molecule delineating the T suppressor/cytotoxic cell subset. These antibodies were obtained from Dr. Chris Howard, Agricultural and Food Research Council, Institute for Animal Health, Compton, Great Britain. All the monoelonal antibodies were used at 1:1000 final dilution, which was 2 to 4 times minimum saturating concentration.
Flow cytometry Fifty/d of cell suspensions (2 × 10 6 cells/ml) were stained in 96-well roundbottom tissue culture plates. Cells were washed with PBS containing 2% fetal bovine serum (FBS), and incubated with 50/d of antibody for 20 min at 4°C. After two rinses with PBS-2% FBS, the cells were incubated with fluorescein isothioeyanate-labeled anti-mouse IgG (US Biochemical, Cleveland, OH) for 20 min. The cells were washed twice more, then resuspended in PBS-2% FBS. Cells were diluted with Isoton (Coulter Diagnostics, Hialeah, FL), filtered through 44 micron nylon mesh (Small Parts Inc. Miami, FL) to remove clumps and analyzed using a Coulter 753 flow cytometer.
Statistical analysis Data for total numbers of PBMC were divided into four time periods each for recipient or control cows. The pretreatment period included counts of PBMC during weeks - 5 through - 3. The prepartum treatment period (prepartum Tx) included weeks - 2 and - 1. The postpartum treatment period (postpartum Tx) included weeks + 1 and + 2. The posttreatment period ineluded weeks + 3 and + 4. The mean _+S.E.M. was taken of counts obtained from all blood collections from recipient or control cows within each time period. The data for percent positive BoCD5, BoCD4, and BoCD8 cells were similarly analyzed, with the exception that these parameters were not measured during the pretreatment period. Data between groups were compared using Student's t-test. RESULTS
Fifty-one to 60% of PBMC expressed the BoCD5 (T lymphocyte) marker during the study. This percent was not significantly different (P> 0.05 ) be-
32
J.A. HARP ET AL.
tween recipient and control cows or between time periods. Twenty-nine to 42% of PBMC expressed BoCD4 (T helper cells). There was no significant difference (P>0.05) between the percent of BoCD4+ cells from recipient compared with control cows at any time. Within the control group, there was a significantly higher ( P < 0 . 0 5 ) percent of BoCD4+ cells during the posttreatment time period compared to the prepartum treatment time period (Fig. l ). Thirteen to 22% of PBMC expressed BoCD8 (T suppressor/cytotoxic cells). This percent was not significantly different ( P > 0.05 ) between recipient and control cows or between time periods. The ratio of helper to suppressor cells in control cows was 2.2 during the prepartum treatment period, 2.0. during the postpartum treatment period, and 2. l during the posttreatment period. These ratios in recipient cows were 1.8, 2.3, and 1.9, respectively. Total numbers of PBMC in blood of recipient cows were significantly increased (P , D
(0 O
T
4 0 ~-
20
Prepartum T x
Poatpartum T x
Posttreatment
Period relative to calving and treatment
Fig. i. Percent of BoCD4+ lymphocytesin peripheral blood from cows receiving (hatched bars) or not receiving(solid bars) rbG-CSF. Data are presented as mean+ S.E.M. of all samplingsfrom cowsduringthe indicatedtime periods. See text for details.
33
T. LYMPHOCYTES IN CATTLE DURING THE PERIPARTURIENT PERIOD
20-
"0
o
_o .O
E E_ 10 L-
T
L
0
Pretrestment
Propartum T x
Postpartum
Tx
Posttraatmont
Period relative to calving and t r e a t m e n t Fig. 2. Total numbers o£ mononuclear cells per cubic mm of peripheral blood from cows receiving (hatched bars) or not receiving (solid bars ) rbG-CSF. Data are presented as mean + S.E.M. of all samplings from cows during the indicated time periods. See text for details.
the numbers of PBMC in the blood of these same cows during the pretreatment and posttreatment periods (Fig. 2 ). DISCUSSION
Periparturient cows have an increased incidence of disease, which may be related to the in vitro immunosuppression observed during this period. We wished to determine if this immunosuppression was reflected in vivo by change3 in the numbers or percent of peripheral blood T lymphocyte subsets. Decreases in both the total number and relative percent of helper T cells have been used as an indicator of compromised immune function. For example, human patients with acquired immunodeficiency syndrome (AIDS) commonly have decreased numbers of T cells and an inversion of the CD4/CD8 (helper/suppressor) ratio in peripheral blood (Fahey et al., 1984; Kalish and Schlossman, 1985). In immunocompetent humans, this ratio is normally about 2.0, while in AIDS patients, it is often 0.5 or less~ In the present study, there was no evidence that periparturient immunosuppression in cows is caused by an inversion of the ratio of helper to suppressor T cells. This ratio remained near 2.0 for all cows throughout the study.
34
J.A. HARP ET AL.
We did observe a lower percent ofT helper cells in both groups of cows during the prepartum period compared to the postpartum periods (in the control cows this difference was statistically significant), however, more study is needed to determine the biological significance of this observation. Granulocyte colony-stimulating factor (G-CSF) has been shown to be essential for the proliferation and differentiation of neutrophils in mice and humans (Nicola et al., 1983; Metcalf, 1985; Souza et al., 1986). In addition, G-CSF may be important in activation of neutrophils for killing by antibodydependent cellular cytotoxicity (Souza et al., 1986). Some of the cows in the present study received rbG-CSF to determine its effect on numbers and function of bovine neutrophils (Kehrli et al., submitted to J. Dairy Sci. ). During the time cows received rbG-CSF, the numbers of both granulocytic and monocytic cells in peripheral blood increased significantly. This may indicate an effect of rbG-CSF on bone marrow precursors of the lymphocytic, as well as the granulocytic lineage, either directly or through the induction of lymphopoietic factors. Alternatively, increased numbers of granulocytes may result in greater availability of lymphokines such as IL-1 (Lindemann et al., 1988; Canning and Neill, 1989), which along with other factors might lead to activation and cell division of peripheral lymphocytes. The doses of rbG~CSF used in this study appeared to have no undesirable side effects on the recipients. Additional work is needed to determine if larger doses of rbG-CSF would further increase the numbers of lymphocytic and granulocytic cells and if this would have any beneficial effect on disease resistance of cows during the periparturient period. ArKNOWLEDGMENTS
The authors thank Bruce Pesch and Sally Crandell for collection and processing of lymphocytes.
REFERENCES Canning, P.C. and Neill, J.D., 1989. Isolation and characterization ofinterleukin-I from bovine polymorphonuclearleukocytes. J. Leuk. Biol., 45:21-28. Fahey, J.L., Prince, H., Weaver, M., Groopman, J., Visscher, B., Schwartz, K. and Detels, R., 1984. Quantitative changes in T helper or T suppressor/cytotoxic lymphocyte subsets that distinguish acquired immune deficiency syndrome from other immune subset disorders. Am. J. Med., 76: 95-100. Howard, C.J., Parsons, K.R., Jones, B.V., Sopp, P. and Pocock, D.H., 1988. Two monoclonal antibodies (CCI7, CC29) recognizing an antigen (Bo5) on bovine T lymphocytes, analogous to human CDS. Vet. lmmunol, immunopathol., 19:127- ! 39. Howard, CJ., Sopp, P., Parsons, K.R. and Finch, J., 1989. In vivo depletion of BoT4 (CD4) and of non-T4/T8 lymphocyte subsets in cattle with monoclonal antibodies. Eur. J. Immunol., 19: 757-764.
T. LYMPHOCYTES1NCATTLEDURINGTHEPERIPARTURIENTPERIOD
35
Kalish, R.S. and Schlossman, S.F. 1985. The T4 lymphocyte in AIDS. New Eng. J. Med., 313: ll2-113. Kehrli, M.E. and Goff, J.P., 1989. Periparturient hypocalcemia in cows: Effects on peripheral blood neutrophil and lymphocyte function. J. Dairy Sci., 72:1188-l 196. Kehrli, M.E., Nonnecke, B.J. and Roth, J.A., 1989a. Alterations in bovine neutrophil function during the periparturient period. Am. J. Vet. Res., 50:207-214. Kehrli, M.E., Nonnecke, B.J. and Roth, J.A., 1989b. Alterations in bovine lymphocyte function during the periparturient period. Am. J. Vet. Res., 50:215-220. Lindemann, A., Riedel, D., Oster, W., Meuer, S.C., Blohm, D., Merteismann, R.H. and Herrmann, F., 1988. Granulocyte/macrophage colony-stimulating factor induces interleukin l production by human polymorphonuclear neutrophils. J. Immunol., 140:837-839. Malinowski, E., Krzyzanowski, J., Wawron, W., Slawomirski, J. and Gluszak, J., 1983. Analysis of cases ofEscherichia coli mastitis in cows. Med. Weter., 39:608-610 (in Polish, with English abstract). Metcalf, D., 1985. The granulocyte-macrophage colony-stimulating factors. Science, 229: 1622. Nicola, N.A., Metcalf, D., Matsumoto, M. and Johnson, G.R., 1983. Purification of a factor inducing differentiation in murine myelomonocyticleukemia cells. J. Biol. Chem., 258:90179023. Smith, K.L., Todhunter, D.A. and Schoenberger, P.S., 1985. Environmental pathogens and intra-mammary infection during the dry period. J. Dairy Sci., 68:402-417. Souza, L.M., Boone, T.C., Gabrilove, J., Lai, P.H., Zsebo, K.M., Murdock, D.C., Chazin, V.R., Bruszewski, J., Lu, H., Chen, K.K., Barendt, J., Platzer, E., Moore, M.A.S., Mertelsmann, R., and Welte, K., 1986. Recombinant human granulocyte colony-stimulatingfactor: Effects on normal and leukemic myeloid cells. Science, 232:61-65.
