Blastogenic response of peripheral blood lymphocytes to mitogens in cynomolgus monkeys (Macaca fascicularis).код для вставкиСкачать
American Journal of Primatology 3:111-119 (1982) Blastogenic Response of Peripheral Blood Lymphocytes to Mitogens in Cynomolgus Monkeys ( Macaca fascicularis ) MASASHI TATSUMI', TOORU FUJIWARA', AND MASANORI HAYAMP 'Department of Veterinary Science and 'Department of Measles Virus, National Institute of Health, Japan Optimal conditions for mitogen-induced transformation of cynomolgus mon key peripheral blood lymphocytes were determined. Maximal stimulation estimated by the incorporation of tritiated thymidine ('H-TdR) occurred at the concentrations between 25 and 50 pglml with phytohemagglutinin (PHA)and concanavalin A (ConA), and between 50 and 100 pglml with poke weed mitogen (PWM)in a culture volume of 120 pl containing 2 x los mononuclear cells supplemented with 10% fresh autologous serum. Lipopolysaccharide (LPS),however, could induce no significant response. In most animals examined, a high response of blastogenesis was induced by Con A, moderate by PHA, and low by PWM. Optimal stimulation occurred after incubation for 78 hours followed by labeling with 1 pCi of "-TdR per culture for 18 hours. Although the extent of blastogenic response was variable in individual animals, the general pattern of time-course and dose-response to mitogens was similar in all monkeys. With 40 monkeys tested, age nor sex did not markedly influence the extent of blastogenic response. Key words: cynomolgus monkeys, macaca fascicularis, mitogens, lymphocyte blastogenesis, optimal conditions INTRODUCTION Lymphocyte blastogenesis, induced by a variety of mitogens derived from plants and bacterias, has become an important and widely used procedure for assessing lymphocyte function both in clinical and in experimental immunology. In general, the extent of in vitro lymphocyte blastogenesis is considered to correlate with in vivo cell-mediated immune response, such as delayed hypersensitivity in human and guinea pig systems [Oppenheim, 19681. As to nonhuman primates, the investigations on mitogen-induced blastogenesis have been done with rhesus monkeys [Mackler et al., 1973; Taylor et al., 19781, baboons [Greene et al., 1977; Eichberg et al., 19811, stumptailed monkeys [Reed, 19761,marmosets [Harvey et al., 1974; Kateley et al., 19771,cynomolgus monkeys [Humber & Hetherington, 19811, and so on. Despite the increasing use of cynomolgus monkeys as experimental models for various human disease, few basic studies concerning their cell-mediated immune response have Received February 2, 1982; accepted April 13, 1982. Address reprint requests l o Masashi Tatsumi, Department of Veterinary Science, National Instilute of Health. 4-7-1, Gakuen. Musashi-Murayama, Tokyo 190-12, Japan. 0275-2565/82/0301-04-0111$03.00 0 1982 Alan R. Liss, Inc. 112 Tatsumi, Fujiwara, and Hayami been undertaken. While the in vitro techniques of blastogenesis have been developed for monitoring cellular immunity mainly in humans, the validity of directly applying them to cynomolgus monkeys has yet to be assessed. Therefore, it seems important to establish the optimal conditions to study the function of cynomolgus monkey lymphocytes with regard to such parameters as cell concentration, mitogen concentration, time of incubation, time of labeling with radioactive DNA precursors, and kinds of sera added to the culture. METHODS Animals Fifty cynomolgus monkeys (Macaca fascicularis) were imported from Southeast Asia, quarantined for more than a year, and were found to be apparently healthy as judged by clinical evaluation and fecal examination. Their age was estimated by the inspection of dentition to be from 1 to over 5 years old. Separation of Lymphocytes Ten ml of heparinized blood (50 Ulml) were aseptically collected from the femoral vein of each monkey, mixed with an equal volume of 3% gelatin solution supplemented with 0.7% NaCl and 0.2% CaCl,*2H,O, and allowed to stand at room temperature for 1hour. Then, the leukocyte-rich fraction was collected and centrifuged at 160 g for 10 minutes. The pellet was resuspended in 0.7 ml of Dulbecco’s phosphate buffered saline and layed onto 0.3 ml of Lymphoprep (Sodium metrizoate-Ficoll, Nyegaad, Oslo, Norway) in a Fisher tube. After centrifugation at 2000 g for 3 minutes in a Fisher centrifuge (Model 59, Fisher Science Co., NJ), the interface layer containing mononuclear cells was collected with a Pasteur pipette and washed twice with RPMI 1640 medium (Nissui Seiyaku Go., Tokyo, Japan). The washed cells were resuspended in culture medium to give an appropriate cell concentration. The viability of cell suspension was usually more than 95% as determined by trypan blue dye exclusion test. The density gradient method employed for human and other species of nonhuman primates was inadequate for cynomolgus lymphocytes because of contamination of erythrocytes which may affect lymphocyte blastogenesis. The exlent of this contamination varied in individual monkeys, usually being more than 1001 for erythrocyte: mononuclear cell ratio. Pretreatment of heparinized blood with the gelatin solution facilitated to obtain constant yield of pure mononuclear cells from most monkey blood by enhancing erythrocyte sedimentation. Preparation of Mitogens Mitogen preparations used were as follows: Phytohemagglutinin (PHA-Y;Difco Lab., Detroit, MI),Concanavalin A (ConA; Pharmacia Chemical, Uppsala, Sweden),Lipopolysaccharide (LPS derived from 055:B5 E. coli; Difco Lab.), and Poke weed mitogen (PWM;GIBCO, Grand Island, NY).The mitogens were dissolved in culture medium and stored in small aliquots at -80°C until used. Lymphocyte Culture Separated lymphocytes were cultured in RPMI 1640 medium supplemented with 25 mM HEPES buffer, 100 pg/ml kanamycin, and 10% serum listed below: fresh autologous serum, inactivated autologous serum, fresh allogenic serum, inactivated allogenic serum, inactivated male monkey pooled serum, and inactivated fetal calf serum (FCS) (Flow Lab., McLean, VA). The monkey sera used were prepared from another blood sample collected at the same time. A 100 pl of lymphocyte suspension at the cell concentrations, indicated in Results, were dispensed into triplicate wells of 96 flat-bottomed microculture plates (Micro-testplate I1 # 3040, Falcon Plastics, Oxnard, CA). To each Cynomolgus Lymphocyte Rlastogenesis 113 well was then added 20 pl of mitogens at the final concentrations in the final reaction volume of 120 p1 indicated in the text. Lymphocyte cultures were incubated at 37°C for 24 to 120 hours in the humidified atmosphere of 5% CO, and 95% air. At a selected time between 2 and 36 hours before the termination of culture incubation, each well received 1 pCi of 3H-TdR(specific activity 20 CilmM, Amersham Co., Buckinghamshire, England) in 10 p1 medium. At the end of the incubation period, lymphocytes were harvested onto glass fiber filters with the aid of a semiautomatic multiple sample harvestor (Labo Mash, Lab0 Science Co.).The filters were washed with distilled water, dried, and placed in scintillation vials with 5 ml scintillator. The amount of radioactivity incorporated by lymphocytes was counted in a liquid scintillation counter (Aloka Co., Tokyo, Japan). Counted values were expressed as mean + standard deviation (cpm).Experiments were performed with at least more than five monkeys to determine the optimal conditions for each parameter and representative data were presented RESULTS Separation of Lymphocytes By the method described, 20 to 50 million mononuclear cells were consistently obtained without detectable contamination of erythrocytes from 10 ml of initial venous blood from each monkey. An average of 74% (range 65-85%) of the mononuclear cells present in the initial blood sample was recovered. The isolated mononuclear cells were morphologically or cytochemically classified into three cell types; lymphocytes (average 87%; range 79-92%), monocytes (10%; 6-14%) andpolymorphonuclear cells (2%;0-4%). Supplemented Sera The effect of added serum on lymphocyte blastogenesis was studied under the condition in which cultures containing 2 x los cells were incubated with 50 pgiml of each mitogen for 78 hours followed by labeling with l pCi of "-TdR for 18 hours. As shown in Fig. 1, the highest reactivity was obtained with 10% fresh autologous serum, whereas relatively poor reactivity was obtained with FCS. Intermediate reactivity was induced with other kinds of sera. Therefore, subsequent experiments were performed in the presence of 10% fresh autologous serum. Cell Concentration As shown in Fig. 2, there was considerable effect of cell concentration on blastogenesis within the range of 5 x lo3to 5 x lo5cells per culture, incubated with 50 pgiml of either PHA, Con A or PWM for 96 hours. In the case of all mitogens except LPS, 3H-TdRincorporation increased with cell concentration per culture, but started to decline after getting to maximum at 2 x lo5 cells per culture. Therefore, subsequent experiment was performed with 2 x lo5cells per culture. Mitogen Concentration Optimal mitogen concentrations to obtain maximal stimulation of lymphocytes were determined under the condition in which cultures containing 2 x lo5 cells were incubated for 78 hours in the presence of 10% fresh autologous serum followed by labeling with 1pCi of 3H-TdRfor 18 hours. Representative data on blastogenesis of lymphocytes from two monkeys differing in the extent of response (high and low responders) are presented in Fig. 3. Although there were considerable variations in the extent of blastogenic response of lymphocytes from individual animals (Fig. 6), the patterns of dose-response curves were in general similar in high and low responder monkeys (Fig. 3). The optimal concentration for the stimulation of lymphocytes ranged between 25 to 50 pgiml of PHA and Con A, and between 50 to 100 pglml of PWM. On the other hand, LPS did not induce a significant response within the range between 0.01 to 500 pg/ml. I t was also 114 Tatsumi, Fujiwara, and Hayami ALL0 FRESH 5 0 15 10 INCORPORATION OF 3H-TdR / 20 CULTURE 25 ( cpm X 30 ) Fig. 1. Representative data concerning the effect of serum added to the cultures. Cultures conlaining 2 x lo5 cells were incubated with mitogens for 78 hours followed by labeling with 1 WCi of '€1-TdR for 18 hours. The culture medium was supplemented to 10%with serum indicated. Each column represents lhe mean counts per minutes Icum)of 'H-TdR incoruorated into triulicate cultures of 1vmDhocvtes. The cells of unstimulated cultures in" _ " cor orated less than 800 cim. Auto.: Autoiogous: Allo.:Allogenic: Inact.: Inactivated a t 56°C for 30 minutes. P P w M : PHA; Con A. 40000-20000 - +I I -;1000u 8000- 6000b3 S 4000 - u . = F 2000I . l LL 0 2 1000: 800z 5 600: LL 400- I 200 7 / r 0.5 1 2 5 10 NUMBER OF CELLS/CULTUHt ( 20 51) X 10 * ) Cynomolgus Lymphocyte Blastogenesis 30 1 115 1 A - n 2 20- X W n +I E s 10- v $ 2 ' 6 O-"' ' 01025 5 0 100 200 LPS 0 J 1 0 2 5 50 100 2 00 1 0 1025 50 0 1025 50 M I T O G E N CONCENTRATlON 100 ° 100 I 200L 200 ( Pg/ml Fig. 3. The determination of optimal mitogen concentration. Cultures containing 2 x lo5cells from two monkeys ( 0 high responder; C>low responder)were incubat,ed with mitogens for 78 hours followed by laheling with 1 pCi of IH-TdR per culture for 18hours. The culture medium was Supplemented with 10% fresh autologous serum. Each point represents the mean counls per minute (cpm)and bar standard deviation (sd)of 'H-TdR incorporated into triplicate cultures of lymphocytes. A: Con A; B: PHA; C PW'M; L): LPS. demonstrated that Con A induced the highest response, PWM the lowest, and PHA intermediate. Incubation Time The effect of incubation time on blastogenesis was studied under the condition in which cultures containing 2 x lo5cells were incubated with mitogens in the presence of 10% fresh autologous serum for various periods, followed by labeling for the subsequent 18 hours. Representative results are shown in Fig. 4. Maximal incorporation of 3H-TdR occurred after incubation for 78 hours with all mitogens except LPS. In very few animals, 3H-TdRincorporation was observed to increase up to 102 hours of incubation with PWM. In the majority of animals, the incorporation was observed to decrease after incubation for more than 102 hours. Labeling Time The effect of labeling time on the uptake of 3H-TdRwas studied after cultures containing 2 x lo5cells were incubated with mitogens for 78 hours. As shown in Fig. 5, maximal incorporation occurred after a labeling time of 18 to 24 hours with all mitogens except Fig. 2. The effect of cell concentration on mitogen-induced hlastogenesis. 1,ymphocytes of each cell concentration per culture from one cynomolgus monkey were incubated with or without (control)mitogens for 78 hours followed by labeling with 1WCi of 'H-TdR per culture for 18 hours. The culture medium was supplemented with 10% fresh autologous serum. Each point represents lhe mean counts per minutes Icpm) and bar standard deviation (sd) of 'H-TdR incorporated into triplicate cultures of lymphocytes. 0 Con A; C)PHA-P; PWM; 0 control. 116 Tatsumi, Fujiwara, and Hayami _- 6ULOO I 4 4uouu ;zoo00 - w, +i ooooy -?'8000 1 + 6000 - 2 4000- \ d P c; I 2000- U 0 rg 1000: - 800- H- 400- 600: c 100' ' 24 1 48 I 72 lNCUBATlON TIME 96 ( 120 hr ) Fig. 4. The determination of optimal incubation time. Culture containing 2 x 10' cells from onc cynomolgus monkey were incubated with or withouL (control)mitogens for indicated periods followed by labeling wiLh 1&i of 'H-TdR per culture for the last 18 hours. The culture medium was supplemented with 1070fresh autologous s t ~ rum. Each point represents the mean count per minute (cpm)and bar standard deviation (sd)of "H-TdRincorporated into triplicate cultures of lymphocytes. 0 Con A; 0 PIIA-P; PWM; Ccontrol. 10- - 1 2 4 6 1 2 18 24 TIME AFTER 'H-TDR ADDlTlON 36 ( hr 1 Cynomolgus Lymphocyte Blastogenesis 70 ,-. I 113457 I I? I I I 260 X I I E I " 50 I 10 137731 353 I I 1 I 1 I I n v 1 e l I I I I I W I e 10 40 I _I I 3 V I \ 30 (r Q I I c I I I m I 20 z l I I I I I I I I I 1 0 0 I U O 117 1 I 2 c a a g 10 0 U z I 0 SE: AGI Fig. 6 . The effect of age or sex on mitogen-induced hlastogenesis. Under the optimal assay conditions determined, cultilres containing 2 x 10' cells from 40 cynomolgus monkeys were incubated with mitogens for 78 hours followed by labeling with 1pCi of 3H-TdRper culture for 18 hours. The culture medium was supplemented with 1070fresh autologous serum. Each point represents the mean counts per minute (cpm)of 'H-TdR incorporated into triplicate cultures of lymphocytes from individual monkey. F: female: M: male: 0 Con A (male); 0 Con A (female);'2PHA (male);WPHA (female). LPS. Longer labeling time led not only to the decrease of specific incorporation of "H-TdR, but also to the increase of background incorporation. Age or Sex Difference The effect of age or sex of donor animals on blastogenesis was studied with 20 male and 20 female monkeys under the optimal assay conditions described above; i.e., cultures containing 2 x lo5cells were incubated for 78 hours with optimal dose of each mitogen in the presence of 10% fresh autologous serum and were labeled for 18 hours with 1pCi of "-TdR. Although the extent of blastogenic response varied from one animal to the other, it did not seem to be correlated significantly with age nor sex of animals. Fig. 5 , The effect of labeling time on mitogen-induced blastogenesis. Cultures containing 2 x los cells from one cynomolgus monkey were incubated with or without (control)rnitogens for 78 hours followed by labeling with 1 &i of 'H-'!.'dR per culture for indicated periods. The culture medium was supplemented with 10% fresh autologous serum. Each point represents the mean counts per minute (cpm)and bar standard deviation Isd)of 'H-TdR incorporated into triplicate cultures of lymphocytes. 0 Con A; 0 PHA-P; W PWM; U control. 118 Tatsumi, Fujiwara, and Hayami DISCUSSION Because of the increasing interest in the use of the lymphocyte transformation test in clinical and experimental immunology, it is necessary to establish a reliable and reproducible culture system. However, there have been only limited numbers of reports on standardization of cynomolgus lymphocyte culture system [Humber & Hetherington, 19811. In the present study, we have established the optimal assay conditions to obtain maximal stimulation of cynomolgus lymphocytes with mitogens using separated lymphocytes in a microculture system. A microculture system has several advantages over macro and semimacroculture systems previously reported in the smaller amount of blood and other reagents, in addition to the ease of setting up larger number of replicate cultures [Kateley et al., 1977; Mackler et al., 1973; Reed, 19761. Recently, Humber and Hetherington 119811reported a micro method of blastogenesis in cynomolgus monkey lymphocytes using a whole blood culture system. However, for the purpose of studying the effect of cellular interactions and serum factors on blastogenic response, the use of lymphocytes separated from other blood elements as responding cells to mitogens may be more suitable than that of whole blood culture. Nonspecific influence of erythrocytes on blastogenic response should also be considered in a whole blood culture system [Luquetli & Janossy, 1976;Mackler et al., 19721. Although the widely used method of separating blood cells by density gradient as described by Boyum is suitable for separating mononuclear cells from peripheral blood of human and other species of nonhuman primates such as rhesus monkeys [Taylor et al., 19781, baboons [Greene et al., 1977; Eichberg et al., 19811, and marmosets [Kateley et al., 19771it is unsuitable for separating cynomolgus lymphocytes because of considerable contamination of erythrocytes. Application of gelatin solution made it possible to avoid this contamination resulting in constant and adequate yield of mononuclear cells from cynomolgus peripheral blood. The present study has demonstrated that cynomolgus lymphocytes were stimulated by Con A, PHA, and PWM, but not by LPS in a similar manner to that reported in other primates including humans. In cynomolgus, as well as rhesus monkeys [Taylor et al., 19781,the most effective stimulant for peripheral blood lymphocytes was Con A which is reported to affect predominantly T cells in mice [Greaves & Janossy, 19721. PHA and PWM also stimulated cynomolgus lymphocytes but to a lesser extent than Con A. However, in the case of humans, PHA has been shown to be the most effective mitogenic stimulant [Douglass et al., 19691. In humans, PHA and Con A are predominantly T cell inducers and PWM may stimulate some U and some T cells. The cell populations stimulated by these mitogens has not been clearly defined in Macaca species. Lack of blastogenic response to LPS which stimulates mouse B cells to differentiate into immunoglobulin-secreting cells was reported also in humans [Chess et al., 19741 and nonhuman primates, such as rhesus monkeys [Taylor et al., 19781and marmosets [Kateley et al., 19771. The optimal dose of PHA or Con A obtained here for blastogenic response in cynomolg u s lymphocytes was similar to that in baboons [Greene et al., 19771, but higher than that reported for rhesus monkeys [Taylor et al., 19781 and cynomolgus monkeys [Humber & Hetherington, 19811. The optimal dose of PHA, Con A, and PWM for maximal stimulation of cynomolgus lymphocytes was much higher than that of humans [Douglass et al., 19691. We also confirmed that the optimal dose was approximately tenfold higher for cynomolgus lymphocytes than for humans (data not shown). Kinetics of blastogenic response of cynomolgus lymphocytes was similar pattern in human and other species of nonhuman primates reported by other investigators. SOfar as a limited number of animals were examined in the present study, there seemed to be no significant relationship between the extent of blastogenic response and age or sex of animals. We are currently investigating whether or not the blastogenic response of individual monkey reflects its immunological response to particular antigens. Cynomolgus Lymphocyte Blastogenesis 119 CONCLUSIONS 1.Optimal a s s a y conditions for mitogen-inducedblastogenesis of cynomolgus monkey lymphocytes were determined; i.e., cultures containing 2 x lo5cells were incubated w i t h mitogens for 7 8 hours in the presence of 10% fresh autologous serum followed b y labeli n g with 1 pCi of "-TdR for 18 hours. 2. O p t i m a l mitogen dose for cynomolgus lymphocytes ranged between 2 5 and 50 pglml w i t h PHA and Con A, and between 50 and 100 pglml w i t h PWM. LPS did not induce a significant response in cynomolgus lymphocytes. 3. Age or sex of donor animals did not markedly influence the e x t e n t of blastogenic response. 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