EXPERIMENTAL ANIMALS AND. HUMAN SUBJECTS. Peripheral lymphocyte (PBL), E, EAC and Null cells were determined for 12 adult men and women prior ...
Bovine Lymphocytes: Erythrocyte Rosettes in Normal, Lymphomatous and Corticosteroidtreated Cattle B. N. Wilkie, F. Caoili and R. Jacobs* ABSTRACT
qui provoquent la formation spontanee de rosettes d'erythrocytes, ainsi que de ceux qui
Spontaneous erythrocyte rosettes, antibodycomplement rosettes and nonrosetting cells were enumerated for peripheral blood lymphocytes of normal adult, lymphomatous adult and immature cattle as well as for peripheral blood lymphocytes of adult cows both before and after injection of corticosteroids. Calf thymic lymphocytes were also examined for rosette formation. Results indicate significant reduction in peripheral blood lymphocyte-erythrocyte rosettes and nonrosetting cells in tumour-bearing cows with a simultaneous elevation in percent antibody-complement rosettes. Calf thymus had a significantly greater percent erythrocyte rosettes than did peripheral blood lymphocytes from the same individuals. Corticosteroid injection reduced peripheral blood lymphocytes without altering proportion of cells as erythrocyte rosettes, antibody-complement rosettes or nonrosetting cells.
n'en forment pas, chez les vaches atteintes de lymphosarcome; ces dernieres presentaient par ailleurs une augmentation du nombre des lymphocytes provoquant la formation de rosettes: e'rythrocytes-anticorps-complement. Les lymphocytes thymiques du veau provoquerent la formation de beaucoup plus de rosettes d'erythrocytes que ceux de son sang peripherique. L'injection de corticosteroides reduisit le nombre de lymphocytes du sang peripherique, sans alt6rer la proportion de ceux qui forment des rosettes d'erythrocytes, des rosettes: erythrocytes-anticorps-complement, ou de ceux qui n'en forment pas.
RESUMA Cette experience visait a denombrer les
lymphocytes du sang peripherique qui provoquent la formation spontanee de rosettes d'ery-
throcytes ou de rosettes :erythrocytes-anticorpscomplement, ainsi que ceux qui n'en forment pas, chez des bovins normaux, jeunes et adultes, et chez des vaches adultes, avant et apres l'injection de corticosteroides. Elle visait egalement a verifier la formation de rosettes par les lymphocytes thymiques du veau. Les resultats de cette experience revelerent une reduction appreciable du nombre de lymphocytes *Department of Veterinary Microbiology and Immunology (Wilkie and Caoili) and Department of Pathology (Jacobs), Ontario Veterinary College, University of Guelph, Ontario NlG 2W1. Submitted February 16, 1978.
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INTRODUCTION Identification of subpopulations of lymphocytes, particularly B, T, "Null" and "K" cells on the basis of their membrane and functional attributes has recently found widespread application in the characterization of several diseases. The literature pertinent to lymphocyte identification and enumeration has recently been comprehensively reviewed by Dwyer (4). Published reports on the spontaneous erythrocyte rosette technique (E rosettes) in application to bovine T lymphocyte enumeration are few (9, 10, 11, 19, 21) and evaluation of bovine B lymphocytes by the erythrocyte-antibody-complement (EAC rosettes) has, to our knowledge, been reported only once (8). The study reported here was undertaken to evaluate E and EAC rosetting lymphocytes in normal adult cattle and to compare the values with those of calves, lymphomabearing adult cattle, corticosteroid-treated cattld and normal humans. The results show significant alteration in numbers of E and EAC rosettes with tumour status, age and
Can. J. comp. Med.
steroid therapy and demonstrate the utility of these techniques for investigation of bovine disease.
MATERIALS AND METHODS EXPERIMENTAL ANIMALS AND HUMAN SUBJECTS
Peripheral lymphocyte (PBL), E, EAC and Null cells were determined for 12 adult men and women prior to conducting similar experiments with 15 normal mature female Holstein cattle. Thymic lymphocytes and peripheral lymphocytes were also evaluated from five Guernsey calves at two to four months of age. A series of ten mature Holstein female cattle, each initially suspected of having a lymphoid tumour, were also examined. Seven of these were subsequently confirmed to be lymphoma positive by haematological and pathological examination, while three remained "suspicious". In general, lymphomasarcomatous cattle were in late stages of the disease. SEPARATION OF PERIPHERAL BLOOD LYMPHOCYTES (PBL)
All steps were performed in siliconized glassware. Twenty ml of EDTA blood were centrifuged at 400 g for ten minutes and the supernatant buffy coat discarded. To remove phagocytic cells the lower layer was added to 5 ml of a 5% saline solution of Dextran T-701 containing 400 mg of carbonyl iron powder2 and following incubation in a 37°C water bath for 30 minutes a magnet was added and the nonadherent cell suspension removed and centrifuged at 1000 g for five minutes. The resulting buffy coat was suspended in 15 ml of Seligman's balanced salt solution (SBSS) (12) and centrifuged for five to ten minutes at 1000 g on 6 ml of Ficoll/Hypaque gradient in a 30 ml Corex' tube (6). The Ficoll/ Hypaque gradient consisted of 24 'Pharmacia, Montreal, Quebec.
Lot 469, GAF Cor2Carbonyl iron, SF Specification, poration, New York, New York. 3Corning Glass Ltd., Corning, New York.
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parts of aqueous 9% w/v Ficoll4 and 15 parts of 25% sodium metrizoate3 (18). A lymphocyte rich interface was collected from the gradient, added to an equal volume of SBSS and centrifuged for ten minutes at 400 g to obtain a platelet-rich supernatant which was discarded. Residual erythrocytes were lysed by resuspending the pellet in 2.5 ml of Tris-NH4Cl buffer (17) and incubating at 370C for three to five minutes. Following incubation a further 2.5 ml of SBSS was added and the cells obtained by centrifugation at 400 g for five minutes were twice washed in SBSS before resuspension in 2 ml of medium RPMI1640.0 Cells were counted, viability determined by Trypan blue exclusion and cell concentration adjusted to 4 x 10' viable cells/ml of RPMI-1640 containing 5% fetal calf serum,7 15% normal guinea pig serum (both heat inactivated) and antibiotics (100 Iu penicillin, 50 ,.tg streptomycin/ml). THYMUS CELL SUSPENSIONS
Guernsey calves were killed with barbiturates and thymus cell suspension prepared by forcing the tissue through a stainless steel mesh.8 The cell suspension was treated as above to obtain purified lymphocytes. A sample of whole blood from the same animal was obtained and processed simultaneously with the thymus cells for separation of PBL. TREATMENT OF ERYTHROCYTES WITH NEURAMINIDASE
Sheep erythrocytes (SRBC) were determined in preliminary trials to be superior to erythrocytes of rabbit, guinea pig, horse, chicken, human, mouse, rat or pig in formation of spontaneous (E) rosettes with bovine lymphocytes. Neuraminidase treatment of the SRBC further improved their efficacy as has been previously described 4Pharmacia, Montreal, Quebec. 5Hypaque, Winthrop Laboratories, New York, New York. 6Grand Island Biological Company, Grand Island, New York.
7FCS, Grand Island Biological company, Grand Island, New .York. 840 mesh/cm, W.S. Tyler, St. Catharines, Ontario.
for human (7) and bovine (9) D rosettes. Sheep erythrocytes were obtained weekly and held in Alsever's solution at 4°C. Onetenth milliliter of packed SRBC was added to 0.5 ml SBSS containing 100 units/ml Vibrio cholera neuraminidase,9 mixed and incubated for one hour at 370C. Treated SRBC were washed twice in SBSS and resuspended to 5% v/v.
Rosettes were determined for PBL of normal adult cattle before and 24 hours following intramuscular injection of 25 mg dexamethazone sulphate.'°
ERYTHROCYTE SENSITIZATION FOR EAC ROSETTES
Differences between mean values were examined for significance by two-tailed Student's t test.
Five percent erythrocyte suspension and a subhemolytic dilution of rabbit antiSRBC were mixed in equal volumes, incubated for 30 minutes at 37°C, washed twice with SBSS and resuspended at 5% v/v. Equal volumes of the SRBC suspension and a SRBC absorbed 1/10 dilution of mouse complement were mixed, incubated for 30 minutes at 37°C and resuspended to 5% (AC-SRBC). Mouse complement was used because of its limited ability to mediate hemolysis. E ROSETTES (ER)
One-half milliliter of lymphocyte suspension was mixed with 0.5 ml of 0.5 % neuraminidase-treated SRBC, incubated for five minutes at 37°C, centrifuged at 200 g for five minutes at 4°C and placed in an ice bath for one to two hours. After reducing the volume of supernatant by 50%, the cells were gently resuspended and placed in a haemocytometer chamber. Lymphocytes with more than three adherent SRBC (E rosettes) were counted in a total of 200 lymphocytes.
CORTICOSTEROID TREATMENT OF CATTLE
STATISTICAL EVALUATION
RESULTS ER, EAC-R AND "NULL" CELLS IN ADULT CATTLE AND HUMANS The percentage rosettes obtained for humans and cattle are given in Fig. 1. Values obtained for man were consistent with those reported in the literature (2, 11) and were as follows: ER 64.25%, SD 7.27; EAC 16.21%, SD 8.37; Null 19.54%, SD 6.33. Bovine lymphocytes formed significant numbers of E rosettes only when neuraminidase-treated SRBC were employed. Values for PBL-ER, EAC and Null cells were respectively 26.0% SD 11.82; 22.93% SD 10.30 and 51.37% SD 12.47.
CALF THYMUS AND PBL ROSETTES
(EAC-R)
Calf thymus lymphocytes formed significantly more ER (39.6, SD 14.5, p . 0.01) than did PBL (14.7, SD 4.92) from the same animals (Fig. 2). Erythrocyte-antibody-complement rosettes were sufficiently few with thymic lymphocytes that subtraction of the percent ER resulted in a
One-half milliliter of lymphocyte suspension was mixed with 0.5 ml of a 0.5% suspension of AC-SRBC and rosettes were formed and counted as for E rosettes. The final percent EAC rosettes was taken to be the observed number minus the observed percent of ER obtained for the same lymphocyte suspension.
negative value for EAC rosettes (-12.3, SD 6.48). The calf ER were generally fewer than adults, the respective values being 14.70, SD 4.92 and 25.33%, SD 11.71. The difference was significant with p
