Document jm27jpZ63XmBoNLYKBLLdzKr9

786633. Hhwut f-or'i Afp-;.'V.-.t h,s, PF-tOPFti . i AM Y I i- i[ V>A P, .*t l ! 5^ v*co 35s -7l B&S 102275 Splenic Lymphocyte Transformation in Culture as a Tool for Immunotoxicologic Evaluation of Chemicals R. P. Sharma X 37 CRC-Uniscience Series None TCDO, Vinyl chloride HET 1.5-5-(5) i 2 Ron Davis Legal P. J. Gehring 1803 Midland John Davidsc' Ag Products Hugh Farber Inorganic Chemicals Earl Smith Inorganic Chemicals X V. K. Rowe Toxicology Research T. Torkelson Corporate Medical H&ER April 5, 1978 636-1C89 April 30, 1978 R. J. Kociba j < -,. B. A. Schwetz .Til 7^? P. J. Gehri,hg--:/>. ; S n o COP; "O L\' MIDLAND a4 /l LZZ splenic lymphocyte transformation in culture AS A TOOL FOR IMMUNOTOX1COLOC1C EVALUATION OF CHEMICALS 3) 9P 0) I SeptenEner 22, I97S R. P. Sharna Toy.icol op.y Research Laboratory Health and Environmental Research Dow Chemical ITS.A. Midland, Michigan 4S640 J R&S 102277 ABSTRACT Splenic colls from mouse wore cultured in nicroplatvs and the upi "V-of thymidino determined co evaluate the lymphocytic transt ornutic . in short term (64-6S hrs) cultures. The cells undergo spohtan<uus blast formation as veil ..s selective activation of suhpupalation olymphocytes by addition of phytomitogens in culture media. Thu two phytomitogens used in these studies were phytohemugglutinin (I'HA) .. 1 pokeweek mitogen (1'U'M) , which are known to be selective for thymusdependent (T-lymphocytes) and thymus-independent (B-eolls) lymphocytes from mouse spleen, respectively. The suitability of these cultures for immune com ieologic studies involving various chemicals was evaluated. Tiie blast formation of lymphocytes from mouse spleens decreased consider ably with aye in growing animals. Addition of small amounts of either mouse serum or liver homogenate significantly inhibited the chymidine up take by cells in culLure. Mlcrusomes isolated from liver when added to tlu-se cultures had only a little influence, on cliyr; id i uptake. l-.xposuro of male mice to different chemicals altered cite transformation character istics of their splenic lymphocytes. Addition of chemicals directly to lymphoevte cultures also caused difleruiit effects indicat in;.; that some of the effects can be caused directly on the cells. Based on these studies it is suggested that lymphoc>Le cultures from spleens can be utilised for toxicity evaluation of chemicals on immune functions of tiie organism. Tiie results can be used to predict immunosuppression or R&S 102278 immunostimulaticn potential of different chemicals. Further studios with a lar^e number of chemicals will help ascertain the mechanisms and implications of the effects seen in lymphocyte cultures. R8ls 102279 -3- I NTROIH'CT ION Lymphoid organs and lymphocytes have been known to be intimately in volved with different immune mechanisms in the body. Both cell mediated and humoral type immune responses are mediated through different types of lymphocytes, A general plan by which lymphocytes may acquire their d i f f erent in 1 and selective action is shown in Fig. 1. The; population of peripheral lymphocytes consist of at least two different types of cells, i.e., T-cells t!:at are involved in cell mediated and delayed type of immunity, and B-eells that are implicated in the secretory process of antibodies. All lymphocytes originate in the bone marrow of higher organisms. Some of these cells migrate to the thymus, usually early in life, and become thymus dependent or "T"**lvmpiiocy Les. These cells proliferate into effector cells or memory cells. They are also involved in the pro duction of various factors (i.e. lymphokines) that regulate the activity of other ceils (e.g. ll-eells or macrophages) and also have important biological actions. The outer membrane of T-Lymphocytes possess a small amount of immunoglobul in molecules. 11-eeLls, also originating in the bone narrow, are differentintid elsewhere in the body. In birds, this differentiation takes place in the lymphoid follicles located near the llursa of Fabr icius. In mammals, fetal liver and/or otiier lymphoid organs associated with the digestive tract may he involved in this differentiation. The surface of 15-cells is covered with specific receptors or miorociohulins chat may be specific for the production of different immunoglobul ins. Those cells are the precursors of plasma cells that produce antibodies. (For more information see Hobart and McConnell, 1975). In 1960, Nowell found that plant lectins such as phytohemagglutinin (P1LA, a lectin derived from Phaseo lus vulgar is) stimulates lymphocytes in vitro in a manner that is morphologically and biochemically similar to the responses induced by antigenic stimulation in vivo. Douglas _ct al., (1967) reported that another phvtomitogen from uokeweed (PWM, obtained from Phytolacca amcricana) stimulated a significant number of cells to develop structural characteristics of the plasma cell line in vivo. A number of other chemicals that stimulate the lymphocyte responses include bacterial llpcpolysaccharide (LI'S, from K. Coli), concannva 1 in A (Con A) and ant i lymphocy t ic serum (ALS) (Andersson o_t al., 1971). The fact that the lymphocytes can be selectively stimulated by different mitogens provided a tool for studying the subpopulation of these cells in vitro. Almost simultaneously, several workers reported that P1LA was selective for mammalian T-cells whereas PWM induced mitosis in Ulymphocytes (.Janossy and Greaves, 1971; Stockman et a_l. , 1971). Some of tliese findings were made possible by the techniques that utilized UNA synthesis in cells as a measure of their mitotic activity. In short term cultures, the mitotic activity is exhibited by the formation R&S 102280 R&S 102281 o- o: hi.isc colls (honco chose chemicals arc also referred to as bins tokens) and can bo readily estimated by t h t? incorporation of a 3 labeled DN'A base (e.g. H-thvmidLne). The evaluation of lymphocyte populations in_ vitro was suggested for monitoring the immunologic competence of lymphocytes from patients with various immunologic disorders and those undergoing immunosup pressive therapy (Oppenheim, 1968). It has also been used to describe the hypersensitivity reactions in responsive individuals (Haipern, 1972), and also to evaluate the immunosuppressive potential of en vironmental chemicals (Nalias ej^ al_. , 1974; Vos and Moore, 1974; Caworski and Sharma, 1977). This report describes a method that has been developed on the basis of those mentioned above and can be used as a routine test for immunologic evaluation in lcag or short term toxicity studies. The method can also he used for in vitro addition of chemicals or their metabolites to the cultures if a direct effect on the lymphocytes is to be evaluated. R&S 102282 -6- MKTHODS Colls and Materials. The spleen is a convenient source of lymphocytes for culLuring front animals exposed to a test chemical. If a tost needs to be performed in the interim periods, it can also be performed on whole blood samples (Turk and Good, 1972). The presence of erythrocytes does not interfere with the assay and tlie blood samples can be used directly without any processing. If necessary, blood can be hemolyzed by diluting in a hypotonic saline solution and lymphocytes separated immediately by centrifugation. In the case of spleen, the organ is taken out asepLically from animals (decapitation is satisfactory) and stored in a sterilized physiological saline solution mill the lympho cytes are isolated. It is suggested that tiie spleen not be allowed to sit for more than 2 hours for maximum viability of the cells. The spleen car. be weighed if desired and rinsed in sterilized saline before use. Other materials required for this method are: sterilized physio logical saline solution, a set of sterilized forceps, supply of sterilized culture tubes, culture medium (RI'MI 16-'t0, supplemented just prior to use with 10.7 fetal call' serum, inactivated by heat ing at 5G8C for 1/2 to 1 hour, penicillin 100 units/ml, streptomycin 0.1 mg/ml and I.-glutamine 0.3 mg/ml), phyLoniitogens (see below), mierocul Lure plates and covers (Falcon 30-40), ilppendorff or a similar pipeting system wKh sterilized Lips. R&S 102283 The method described below is the one typically employed in our studies. The ail inula used in the following; studies were male CD-I mice obtained from Charles River Breeding I. iboratories, Inc. (Wilmington, Mass.). Preparation of Lymphocyte Suspensions. The whole spleen of a mouse or part of the spleen of ocher laboratory animals (usually 0.1-0.2 g tissue provides enough lymphocytes) was rinsed in sterilized saline solution and placed in a petri dish containing 3 ml physiological saline. The tissue was mashed with forceps until most of it was disrupted. The cell suspension was mixed well (by flushing through an 18-gauge needle with a syringe) and passed through a series of hypodermic needles (using a syringe and 18, 21 and 26 gauge needles in succession) to separate the unhro'sen tissue and cell aggregates. The suspension was finally trans ferred to a glass culture tube and allowed to stand for nearly 10 minutes to allow the cell aggregates to settle or adhere to the sides. I'sing a disposable transfer pipet, the suspension was transferred to a different culture Lube leaving the bottom portion (ca 0.5 ml) behind. Tl'.e suspension (nearly 400 g) was then centrifuged at room temperature for 15 minutes. The supernatant was removed by suction and 3-4 ml of culture medium was added immediately over the cell pellet. The cells were suspended in the medium using a Vortex mixer. After allowing to stand lor a lew minutes, the cull suspension was transferred to another tube leaving the aggregated clumps behind. It was considered desirable to process only 16-30 samples simul taneously and no norc than 3d samples were processed at any one time so the process could be completed in one-half day. An aliquot of tile cell suspens'on was removed and the concentration of lymphocytes determined using an electronic counter. The red cells were lysed before counting. The viability of tlte cells was checked occasionally by adding a little trypan blue to an aliquot of ceil suspension and the cells counted in a iiemocytometor. The viability in our experience was always belter than 95,3 and was not routinely cheeked. live original suspension was then adjusted to provide approximately S x 1U^ cells/ml. The ceils were counted again after ution and culturing to cheek the final concent ration. Lymphocyte Cultures. Aliquots of the above suspension, 50 al repre senting approximately 400,000 lymphocytes, were placed in each well of a microculture plate. The cultures were conducted either in triplicate or quadruplicate for each animal or addition of a mitogen, and thus S animals were accomodated In each culture plate. An arrangement of > plating scheme is shown in Figure 2, in order to keep a re'ord of culture numbers, a diagram was prepared on paper and labeled iceordingly. Tlie lymphocytes were allowed to settle on the bottom before any other additions. After all samples were plated, 50 pi of culture medium was added to (each well. In cases when in vitro addition of a test chemical or other substance (e.g. liver supernataut) was desired, med icm containing the appropriate chemical was added. L'sually liver homogenate or scrua constituted less than 25,". of the medium (12.5 ill) and the mixture added instead. Subsequently, an additional 50 pi of either medium alone or medium containing Pi LA or PU'M (see below) was added and the plates covered and incubated for 43-50 hrs. at 37C in a humidified 52 CO., in air atmosphere. The mitogens, P1L\ and PU'M (obtained from Grand island Biologicals, Grand Island, N.Y.) were reconstituted to 10 ml and portions diluted in medium to provide 0.5 pi in eaeli culture well. This provided optimum con centrations of lectins (about 2.5 pi/ml in final incubation medium). After Lhe above incubation, the cultures were spiked with 50 pi of 3 medium containing 0.5 qCi of ll(metlivl)-thymidine (New England Nuclear, Boston, Mass.). The plates were reincuhated for an additional 16 hours. The cells were then harvested using a Model M12V Cell Harvester (Braudel, Rockville, Md.) and collected on Reeve Angel 934A11 glass fiber discs. The cells were vigorously washed with saline. Precipitation by trichloroacetic acid (TCA) and repeated washing of cells with 32 TCA had no influence on the thymidine uptake; thus, the cells were washed with saline rather than TCA. The glass fiber discs, nearly dry because of vacuum suction in the harvester, were directly placed in regular scin tillation vials. Scintillation fluid (Aqunsel, New England Nuclear, Boston, Mass.), 10 ml, was added to each vial and the solution was well shaken. These vials were counted in a Mark 111 Scintillation Counter 37 CO O ttoo Co Oj R&S 102286 (Searle Analytics, Inc.) tor one minute each; the counts were coaverted to disintegrations per minute (dpm) using a standard quench curve. The dpm were averaged for replicate samples and adjusted for 10^ cells in culture. The stimulation index for THA or PW>! was determined by dividing the dpm for cultures with respective mitogen by dpm : rom Lite similar culture containing no mitogen. Whenever necessary, values from different animals in a group were aver aged and the results were expressed as mean and its standard error. R&s 102287 -11- KCSULTS AND DISCl'SSION The purpose of this report is to describe and evaluate a method utiliz ing lymphocyte transformation (blast formation) in a culture system. The method used above has been modified primarily from that described by Janossy and Greaves (11)71) and adapted for automated harvesting of cells. Many of the parameters have been evaluated previously and were either used or optimized from the information available. Generally the factors that may be of importance in evaluation of toxicity of chemicals were furLher examined. .3 Determination ot H-Thvnidinu Uptake. The cells were harvested on a mechanical harvester and were vigorously washed using saline as a wash solution. Whether this procedure disrupts cells so that only DMA bound thymidine (and not the soluble pool) is determined was questioned. In one of the experiments the cells were precipitated by addition of 72 Lr icliloroacetic acid (TCA) and were ! lien washed on the harvester using a 3,1 TCA solution. The washing was repeated several times in one case. Results of such an experiment are shown in Table 1. It is apparent that precipitation of cellular material is not necessary and saline wash provides the same values as after the TCA precipitation and washing. Concentration of Cells and 1111ytomitogens in Culture. The effect of cell concentration on lymphocyte transformation using flat-bottom microculture system has been described by Janossy and Greaves (1971). These authors have reported the optimal concentr.it ion of cells to be -12- 4 x lO"5 per culture well, the transformation being proportional to the number of cells to tills point but declining sharply if the number of cells exceeded this limit. Our system generally employed less than or equal to tiiis number of cells. The cell preparation described above generally yielded more than 952 viable lymphocytes. Because of tills constant recovery it i.; possible to use an electronic counter (Coulter, Inc.) after lysing the red cells. The optimal amounts of phvtomitogens, P1L\ and PWM are also described by Janossv and Greaves (1971). Excess of mitogen generally results in inhibition of lymphocyte transformation. The concentrations were optimized to provide the above mentioned values using the available mitogens. It is suggested that the concentrations be optimized for each preparation used. Variability in Replicates and Within Animals. Variability within replicates of the same culture system is inherent in these procedures (Graf and Mather, 1972). In our system a 102 variation between differ ent replicates was usual but occasionally larger variations were observel. Sufficiently large anima1-to-animal variations were often noted. Graf and Mather (1972) have suggested a nonparnmetric analysis of variance using geometric means of replicates. In our experience, use of parametric test methods was sufficient to detect effects of chemical treatments. L'se of a uouparametrie system, although more R&S 102288 13- I sensitive, was not employed since wc do not: know the causes ol vari ability and the related implication. Sensitive statistical tests tor significance may, however, be applied if considered suitable. Influence of Agi- on Lymphocyte Transformation. In natty toxicologic studies, growing animals are used for short eon.: repeated exposures to chemicals. Like certain other effects, the lymphocyte transformation is also altered with age. Adult animals possess considerably fewer lymphocytes tiiat will form either spontaneous blasts or can be stimu lated by phytonitogonic agents. Fig. 3 and Table 2 illustrate the effects of age in different experiments. These experiments were not conducted separately but the data were obtained from control groups used in several other experiments involving evaluation of chemical inmunotoxicit>. It is apparent that there is a relatively rapid decline in spontaneous blast formation of mou:.e splenic 1 ym.phocytes and also of the stimulation indices obtained with l'IL\ and I'WM. In imm.unotoxicologic evaluation, the use of juvenile animaLs may have some advantage, al though the variability is reduced considerably in older and mature animals. At the same time the immunotoxLeo 1ogicaL effect of chemical exposure is also considerably reduced in adult animals (Vos and Moore, 1974; Sharma et al. , 1977b.) . A drastic dec.line with age in one of the experiment:; shown in Fig. 3 nay have b eon due to the stress produced by housing conditions. The animals in tins group were housed in inha LaL ion chamber.; and were R&S 102289 R&S 102290 -14- deprived of food and water during simulated exposure (although these were used as controls for respective exposure groups). Repeated hand ling of cages and other stress factors such as water deprivation for an extended period nay be responsible for such a decline. Stress induced modulation of immune responses has been described previously (Monjun and Collector, 1977) where environmental stress both depressed and enhanced the immune responsiveness. In immunotoxicologic studies, an appropriate control, exposed to similar environmental conditions, ic, therefore, highly desirable. Addition of a Metabolizing System or Other Tissues to the Cultures. In the case where a chemical exposure alters tiie trunsformation of lympho cytes, addition of the chemical to the culture system directly in vitro can assess whether the effect is due to the chemical per se. It is possible tiiuL the chemical exerts its effect v_i vo after being either itself modified chemically (biotruns format ion) or by modifying some tissue components (hapten conjugation). To verify such cases it was considered desirable that the chemical he added to culture simultane ously with an active metabolizing system or tissues like serum or liver from exposed animals. Addition of different amounts of serum and liver homogenate was thus attempted and Lite representative data are given in Table 3. It appeared that even a small amount of these biomnteriuls, as little as 5 1 of serum and 0.03 Ug of livor Lissuo to a 300 ;il culture system R&S 102291 -15- prodticed appreciable inhibition of the lymphocyte trunsform.it ion. A reduction in both spontaneous tmustarrantion as well as in mi Logon induced trans format, ion was observed. Tin.: of foot of scrum was surprising sine; tiic culture medium contained 10.". fetal calf serum (heat-inact ivated). Janossy and Oreaves (1971) have suggested that fetal calf scrum stimu lates lymphocyte Lraasformation. In another report, Vos and Moore (1971) reported that rat serum inhibits cell trunsfuntil ion in cultured thymocytes or splenic lymphocytes. In a subsequent experiment, manipoint ions were made to serum and liver supernatent to determine the nature of inhibitory factors. The results of such a preliminary experiment are shown in Table 1. dentine (ob'CI for 1 hour) and dialyzing (in a dialysis bag, 15,000 dultons cut-off, against 0.01 M phosphate buMcr, pH 7.1, for 18 hours) appeared to reduce the inhibitory effect of mouse serum only partially. Similar treatments were, however, noL effective in tin* case of liver supernatent. When pur i tied mierosum.es (100,0'K) g pallet from a pnsl-r.ilocltoudr in 1 , 9,000 g supernatent) were used in an amount equivalent to the added liver tissue, these were not found to In* inhibitory. At this stage, tiie nature of inhibitory factor(s) in mouse serum and liver against its own splenic lymphocytes is not clear but some other reports also support this contention. In addition to the inhibitory effect of rat serum referred Lo above, serum and liver factors were reported to be inhibi tory for hepatocyte mul t ip 1 ieni ion in culture (Nadai et_ a_l_. , 197G). Suppression of lymphocyte stimulaiion in culture was also reported by uterine and placental extracts (Koullab el al, 197fi). Based on preliminary results, it ma; bo suggested tli.it tin- inhibitory factors in liver are primarily in the soluble fraction and the chemicals can be metabolized by utilizing, a purified microsomal system in lympho cyte cultures it necessary. This hypothesis, however, has not been experimentally tested at this point. lit tout OL Antigen Challenge on Lvnphocvte Transformation. If an immuno suppressive role ot a chemical has to be evaluated, it may be necessary to challenge the exposed animals simultaneously to a known antigen and determine the specific immune responses. In some of our immunotoxicoLogy experiments we attempted such a technique. Exposure to 2,3,7,3-r.e Z racii lorodilienno-p-dLoxin was found to be immunosuppresive (measured by skin reactivity to tuberculin and antibodies to tetanus toxoid in sensitized animals) but exposure to vinyl chloride did not show a similar effect. This correlated well with the mitogen siimulatiea indices in lymphocyte cul Lures fror.i animals exposed to these chemicals. In these experiments, tlie animals were challenged with a mixture of tetanus toxoid and Freund's complete adjuvant and the etleet of this tetanus antigen challenge in control groups of mice is si.own in Table 5. In both cases, antigen challenge increased the spontaneous blast for mation of lymphocytes in culture and also increased the stimulation indices against I'llA and FU'M. The increase in spontaneous C r. ms forma L ion was similar to those observed with exposure to chemicals in some cases and no aildition.il interactions ot the two (ehemicaL exposure and antigen R&S 102292 -17- chal leuge) wore observed. Known inmunosuppressants like 2,3,7,8tet rach lorodibcnzo-p-d io.xin caused a docroase in the stimulation indices even after antigen innoculations. Although these results surest that antigen challenge caused a stimu lation of spontaneous and induced lymphocyte transformat ion in culture, these differ somewhat from some of the earlier reports. In one case where chickens were injected with killed Mycobacteriuni tuberculosis organisms, the peripheral lymphocytes from these animals showed a variable efloe.t to PllA (Visco and Buening, 1977). Only one out of four birds showed enhancement of the stimulation index, while all 4 showed increased stimulation when purified protein derivative (l'l'll) was added to the culture. Challenge of chickens to M.rek disease virus (Mi>V) decreased the stimulation ind Lees by PllA, I'U'M and Con A (l.u and Lapen, 1974). Our results in mice cha]longed to a bacterial antigen suggest that tlie splenic lymphocytes are sensitized to the phytomitogeus. In addition to using an antigenic ciialUnge for immimo-1 o:< ico 1 og i c evalu ation, it is imperative that the health status of animals in such studies should he carefully monitored, since the presence of in fee lion can alter the results. Lvaluat ion of Se 1 ec ted Cliem i cn Is Using, mouse splenic lymphocyte transformation as one of the methods for toxicologic evaluation, a number of chemicals have been tested. The details of these are reported elsewhere (Sharma et_ a_l. , 1977 a, b, e). A summary of these ohservat Lons R&S 102293 -18- 1s listed In Table 0. Sonic of those chemicals end their biotransfor mation products were used tor direct addition to cultures in vitro and these results are presented in Table 7. It is interestin>; that both vinyl chloride monomer and styrene caused enhancement of spontaneous lymphocyte transformation when the animals were exposed to these chemi cals j_n vivo. fn vi_tro, on the other hand, these chemicals were in active, but their metabolites caused a similar stimulation. This il lustrates tlie role of h iot rans format ion in immunologic effects. R&S 102294 R&S 102295 -19- CONCt.l'S IONS Bused on Clio testing of a 1 ini toil number of chemicals, it nay bo said that splenic lymphocyte transformation in vitro can ho considered as a nodoi svston for the evaluation of tlio of foot of chemicals on immune mechanisms. Unless a .rood correlation by other testing methods is estab lished, it should bo used as a supplementary Cost in conjunction with other tests on the immune system, i.e., an effect on the immune responses to ant iconic stimulation. The mouse appears CO be a satisfactory species but a.co of the animal, honsin;.; and care, and adequate controls should he considered in any testinp, protocol. SLn.ce the responsiveness of lymphocytes to t ransformat ion is greater in younc, animals as compared to adults, the animals should bo used helore achieving maturity. Mature animals can, of course, he used for cornpar ison . K.iuet it::p 1 ieat i on of alteration of lymphocyte t runs forma t ion is not known at present. 1'he responsiveness of lymphoey t os to phy tomi colons is said to involve die same derepression phenomenon that is induced by ant ip.cn ic substances _in vivo (I'ppenhcim, 19bS) . In addition, phytomi top.ens cause a selective induction of DMA synthesis and this can be used as a LooL for meusuri in; tin1 respousiveness of difforont populations of lymphocytes (Audi-rssou ct ai_., 19/J: .lano-. ii-y and Urcuvcs, l`>7l: Stockman it a!., l`J7l). Mouse splenic cells have relative selectivity R&S 102296 -:o- for some r.:itokens and in ease of the two used in this report, mouse 11lymphoeytes are stimulated by i'U'M, whereas T-colIs exhibit selectivity to PJiA (Jauossey and Greaves, J471; Stockman et aj.., 1971). FHA is considered as a genera', nonspecific stimulant in other species (Viseo and ilueniny, 1977). Regardless of their selectivity it is desirable to use several mitogens. In a mixed cell system, such ns the one described above, cooperation amony different cells is likely and the activity of one may influence the transformat ion of cells in anoLher subpopulation of lymnlioey tes. Although it is desirable sometimes to use a purified population of iympho-eytes, it is difficult for routine testing of chemicals. Manipu lation ot lymphocytes has been known to alter their transformation properties, e.y. lym.phocyte activation by cell separation procedures lias been reported (U'ardley e_t a_l. , 197u). An alteration ol stimulation indices to phy t om i t o.-.ous by exposure of animals to chemicals may be considered as an effect on immune mechanisms. Depression ui such indices is comp.irnble to immune suppression in the oryunism, as was the case with 11, 1, 7,3- te t rach 1 orod i henco-p-d iox i n. Tinexact in-plication of an increased stimulation index is not yet apparent but it may be considered as an immune enhancement or modulation of immune responses. Such a phenomenon has boon described in the case of levamisole (Keaoux ._t a_l. , 197(>). -:i- influcnce of chemical exposure on spouLuneous blast formation on lympho cyte cultures is difficult to explain. Several chemicals tested hy this protocol exhibited this phenomenon, i.e. increased t runs format ion in culture. Such .m effect, however, should not he considered universal or nonspecific since exposure of nalc nice to toxic he.ivy metals did not produce such effect, although the sL initiation indices to mitogens were altered (C.iworski and Sharmu, 1977). Orgad and Cohen (1974) have iiapiied that increased lymphocyte sensitivity, particular1y that of T-cells is indicative of au lo itr.muuologi e effects. It would not he unlikely that such effect is produced hy hapten-eonj ui;.i I ion mechaui seas caul is similar to in vJ_vo antigenic stimulation. If such is the case, the sens i t i in.; potential of chemicals can be tested using these techniques. A direct evidence of tliis effect can he made if the sens i t i c.ed lymphocytes sltew increased t r.mst ormat ion in tin* presence of chemicals i'' culture, hut often factors such as hiolruns formalion and specificity towards a conjuguting-macromolccule in the organism may make such testing difficult, if not impossible. A reduction in lymphocyte truns forma;ion may he indicative of immune suppression asnl may he mediated cither through damage of 1ymphaiic organs (as appears Lo be the case in high doses of 2, 3 , 7 , S-let racli 1 orodibonxo-p-dioxin) or through steroid suppression medianisms. The latter may he the reason for dcereusod respons i veuess in adult animals or the animals exposed to an cnvLronmontu1 stress. R&S 102297 In some of our studies, the enhancement of spontaneous lymphocyte trans formation by chemical exposure was similar to one caused by antigenic stimulation, and the fact that these two did not have an additive effect, further supports the argument that this may be an autoimmunologic phe nomenon . Written by: R. P. Shnrma / Reviewed by: R&S 102298 -23- R&S 102299 REFKRENCF.S Andersson, J. , Moller, G. , .mJ Sjoberp, 0. (1972). Selective induction of DNA synthesis in T- end B-lynphocytes. Cell. Immunol. 381- 393. Douglas, S. D., Hoffman, 1'. F. , Horjeson, J. and Cites sin, L. N. (1967). Studies on human peripheral hlood lymphocytes in vitro. HI. Fine structure features of lymphocyte transformation by pokeweed mitop.cn. J. Immunol., 9S: 17-30. Cavorski, C. 1.. and Sharna, R. P. (1977). Heavy metals and lymphocytes. A possible site of immunosuppression by chemicals. Toxicol. Appl. Pharmacol. 41 :149-130. Oral, p. A. and Mather, F. (1973). A statistical model for the evaluation of antij;en-sLimula ted hi astopcnic transformation as measured hy scinti11 at ion spectrometry. Cell. Immunol. 4: 143-151. Halpern, H. N'. (1972), Antibodies produced by drops and methods for their detection. In, International Kneycloped 1a of Pharmucology and Therapeutics, Hypersensitivity to Drops. Perpamon Press, Oxford and New York. Vol. 1, Sec. 75. Hobart, M. J. and McConnell, I. (1975). The Immune System: A course on the molecular and cellular basis of immunity. Blackwell Scientific Publications, Oxford. 357p. Janossy, G. and Greaves, M, F. (1971). Lymphocyte activation. Response of T and B lymphocytes to phvtomitogens. Clin. Exp. Tmmunol., 9: 4SJ-498. Kouttab, S'. M., Fowler, A. K. , Strickland, J. K., and Heilman, A. (1976). Suppression of in vitro lymphocyte stimulation in mice by uterine and placental extracts. J. Immunol., 117: 1644-1650. Lu, Y. S., and Lapen, R, F. (1974). Splenic cell mitogenic response in Marek's disease: A comparison between noninfected tumor-bearing and nontumor-boaring infected chickens. Amur. J. Vet. Res., 35: 997-9S0. Monjan, A. A., and Collector, M. I. (1977). Stress induced modulation of the immune response. Science, 196: 307-308. N'adel, C., Lombard, M. N., and Zajdela, F. (1976). Inhibition of rat hepatocyte mu 11iplication by serum and liver factors. Physiological development and experimental induction. Virchows Arch. B. Cell Pathol. 22: 277-285. R&S 102300 R&S 102301 -25- Nahas, C. G., Suciu-Foca, N. , Armand, J. P., and Moristiima, A- (1974). Inhibition of cellular mediated immunity in marihuana smokers. Science, 183: 419-420. Nowell, P. C. (1960). Phytohacr.ugglutinin: An initiator of mitosis in cultures of human leukocytes. Cancer Res., 20, 462-466. Orgud, S., and Cohen, I. R. (1974). Autoimmune encephalomyelitis: Activation of thymus lymphocytes against syngeneic brain antigens in vitro. Science, 183: 1083-1085. Oppenhoim, J. J. (1968). -.elationship of in vitro transformation to delayed hypersensitivity in guinea pig and man. Federation Proc., 27, 21-28. Park, 13. H. and Good, R. A. (1 972). A new micromethod for evaluating lymphocyte response to phytohemagglutin in. Quantitative analysis of function of thymus dependent cells. Proc. Nat. Acad. 8c i., 69, 371-373. Renoux, C., Kenoux, M., Teller, M. N., McMahon, S. A., and Guillaumin, J. M. (1976). Potentiation of T-cell immunity by lovumisol. Clin. Exp. Immunol., 25: 288-296. R&S 102302 -26- Sharma, R. 1'., Ko.iha, K. J. and Gehring, P. J. (i *.) 7 7; i) . Innunotoxicologic studies involving 2,3,7,8-totraehlorodiben?.o-p-dioxin (TCDD) in mice and rabbits. (in manuscript) Sharma, K. P., Ko-iba, R. J. and Gehring, P. .J. (1977b). Reversal of immunologic and toxicologic. effects of a single exposure of 2,3,7,8te t rac.hlorod ibenzo-p-diox in in mice. (in manuscript) Sharma, R. P., Kociba, R. J., Yakel, H. 0., and Gehring, P. ,J. (1977c). Stimulation of lymphocyte transformation in splenic cultures of animals exposed to vinyl chloride. (in manuscript) Stockman, G. D. , Gallagher, M. T. , Heim, I.. R. , South, M. N., and Trentin, d. J. (1971). Differential stimulation of mouse lymphoid cells by piiy tohemagg 1 u t i u i u and pokeweek mitogen. Proc. Soc. Kxp. Biol. Med. 130, 980-982. Yisco, R. J. and Buening, G. M. (1977). Specific in vitro lymphocyte response of chickens injected with killed Mycohacterium tuberculosis. Amur. J. Vet. Res., 38, 395-397. '.`os, J. G. and Moore. .1. A. (1977). Suppression of cellular Immunity in rats and niee by maternal treatment with 2,3,7,8-lelracli 1orodibcucop-dioxin, hit. Arch. Allergy 77, 777-797. Wardley, R. C., Rouse, B. T. and Bahiuk, I,. A. (1970). Lymphocyte activation by cell separation procedures. I; .-.uno 1. Comm. 5, 637- 07 8. -27- Table 1 EFFECT OF WASHINC WITH TRICHLOROACETIC ACID (TCA) ON SPLENIC LYMPHOCYTE CULTUKF.Sa Treatment Wash with Saline Wash with TCA once Wash with Saline Wash with TCA repeatedly DPM/106 cells 25673 29476 30122 31251 St inti la t ion Index PI LA PWM 4.0 3.3 3.5 3.6 6.9 5.2 6.4 5.7 The cells were Created with and washed with a 32 solution of trichloroacotic acid to cause precipitation of nucleic acids and proteins. All cultures run in quadruplicate and averaged. R8tS 102303 -23- Tnble 2 IN'FI.l'ENCE OF ACE OF CROWING MICE ON THE MITOGEN INDUCED STIMULATION INDICES 01' SPLENIC LYMPHOCYTE TRANSFORMATION IN CULTURES Ex per inent Nunhera 1 2 3 Approx inn to Ace,dovs 32 46 60 88 43 57 71 99 38 43 50 64 Stimulation Index PHA PWM 4.6il.S 31.U5. > 2.S10.4 12.4H.2 15.916. 1 17.6i5.0 1.7t0.2 5.110.4 4.611.7 10.Ill .9 2.911.1 4.211.6 2.310.7 2.210.4 1.4;0.2 0.710.2 8.412.2 10.912.4 3.610.8 6.Si 1.0 3.2i0.S 4.510.5 4.2i0.7 4.110.8 The first two experiments ore ns those also indicated in Fij;. 3. MenniSE, 4 aniwals/v;roup, each culture in triplicate. R&S 102304 -29- Table 3 EFFECT OF MOUSE SERUM AND LIVER SUPERNATANT ON LYMPHOCYTE TRANSFORMATION IN VITRO'1 Age nc and amount added, None Serum , 1 Ul Serum , 5 H Serum , 10 yl DPM/106 Cellsb 65311174 8077.-2103 541912052 471011794 None Liver,, 0.25 al (0.05 Lg) Liver,, 2 ul (0.4 ag) Liver, 10 :.l (2 ;.g) 9252139S7 603911316 579411434 761313497 Stimulation I ndex PHA P'.O-t 3.8 4.4 2.3 2.5 3.1 2.1 1.9 5.0 3.4 2.3 3.2 2.4 1.9 2.2 1.5 1.3 The Serum or 9000 g liver supernatant, 207 (post-mitoohondrial fraction) was mixed with appropriate amount of media and added to culture wells. The equivalent amount of liver tissue is shown in parenthesis. MeaniSE of 4 cultures. R&s 102305 R&S 102306 -30- Table 4 EFFECT OF DIFFERENT TREATMENTS ON MOl'SE SERUM AND LIVER SITERNATANT ADDITION TO SPLENIC LYMPHOCYTE TRANSFORMATION IN CULTURES'* Agent and Treatmentt)I'M/IQ6 ColIs None Serum, uncreated Heated Serum Dialyzed Scrum Liver Supernatant, untreated Heated Liver Supernatant Dialyzed Liver Supernate Microsor.es iron Liver 25190 1749 5*51 604 3 3129 134 3 716 19 390 Sc Iruil.it ion f tide i'[L\RUM 4.0 3.0 2.9 2.1 _ , /** 1.4 2.4 1.7 6.6 9.8 5.7 3.6 1.2 2.4 3.4 3.7 The amount of different factors added to the cultures was, serun 10 ;.l, liver 2.5 ,.g, of tissue equivalent, and :u i c rosomes obtained from, liver reconstituted to represent 2.5 of liver tissue. All values are average of 4 cultures. -31- Table 5 EFFECT Of AXTIGEX cnAi.r.xx:i:;: ox sci.extc I.VMI'HOCVTE T RA X S f0 RMA T10 X IX CL'LTl'RES3 Croup Control Antigen Treated Control Antip.cn Treated Dry,/;or' coils 6158i654 107642632 24321799 11 736*3584 St ir.iul.ition Index by HU PUT! 1.7*0.2 6.0:1.6 5.1:0.4 12.7r2.8 1.410.2 S.114.8 0.710.2 6.512.9 The anir.-.a 1 s wore injected subcutaneously with 0.02 nl of an antigen fixture containing equal amounts of tetanus toxoid and Freund's .o::,.[']ete adjuvant, 2 and 4 weeks before sacrifice and culture preparation. Mean and SE of 4 nr. ir;a 1 s/group , all cultures conducted in triplicate. R&S 102307 i -32- Table o EVALUATION' OF LYMPHOCYTE TRANSFORMATlON TECHS'[Ol'K AFTER IN' VIVO FNTOSURK OF MICE TO DIFFERENT CHEMICALS R&S 102308 Chen ion 1 s Kv.i 1 ua ted Spontaneous Transformation Mitogen Induced Transformation TCDI) (2,3,7,8-tetrachloro- Increased, not dibenzo-p-dioxin dose related, more pronounced after short time exposures Decreased stimulation indices with both PHA and HOI, generally at high exposure levels (l gg/kg/week or more) Vinyl Chloride Monomer Increase observed depending or. ex posure level and time, effect declining at later times Increase seen both with PHA and PVT!. The effect persisted throughout an eight week exposure period S tvrene Dose and time independent increase observed. Effect not consistent Little effect seen with both mitogens Thirdiglyeol ic Acid Slight increase, dose related No consistent effect after two weeks of exposure -33- Table 7 EVALUATION OF .YMPHOCYTE t:<au;s format ion technique af EK IN VITRO ADDITION Or CHEMICALS TO MOUSE SPLENIC LYMPHOCYTE CULTURES c. p-diox in Vinyl chloride monomer Thiod i.clveo l ic acid3 N-acetyIhydroxyethyL cysteine3 Styrene S_ tyrene oxidie b Hose rei trod decreets e No effect Stimulation at 10" 3m No effect No effect Stimulation at 1076M and inhibition at 10 M or mo r e No effect No effect Slight in No effect No effect No effect Renortoti metabolites of vinyl chloride monomer (VCM) . Transforn.it ion product of styrene in tile bodv. 33 CO too Co o CO R&S 102310 - K.- LMCMNDS TO FIGtRF.f, Schematic representat inn of origin, differentiation and function of different populations of lymphocytes. Suggested arrangements for arranging lymphocyte cultures in microplates: a, cultures run in triplicate; and b, cultures in quadruplicate. In cadi case a total of 3 snm.( 'us can Lie used. The last two rows in "a" can bo used Lor additional samples continuing in another microplate. Thymidine uptake representing blast formation in 1ymphocyte cultures from mouse spleen as a function of aye in growing animals. The data shown here lias been taken from control groups of two independent experiments. In experiment shown, by solid circles (), the male animals were housed in groups of two each and given a dose of corn oil once a week. In the other one, with erosses (X), the males were housed in groups of four and were kept in inhalation chambers starting at the age of 311 days. Mach point is an average of ' animals, all cultures " ;n in triplicate. Of i(jm Boot; U.iffji'. S:--si CON LYMPHOCYTE DIFFERENTIATION AND FUNCTION Dif Iff L'flll.ltlOfl Pfoltfcr .(tmn ^ FT In tin Ci 11 ,---------/ -------- ------- * I'tts ' t)[ P'HlV I COA COs 'ils M ,, " - [ i .: " * M..... .. C. 9 PI.,SOM C- * l. . ssoi Function Antif|(*tt Rrcrifimtnm, C''M Dostructirm, l yntplioV me Rirli'.i'.c, ( 1 '.in'ilni F.)( (Of, Afj'ifi-ij.itmt), Miloi|(.'m(., Mtcio|jh;i!]L' InltilHlion, Lynt|il ioIuxk; P;u:lors, I nli'f lor (ui, i:tc, i Cc'l Con| Iff .ll Iflfl I in'mji;ol<)f]iC Mt.'luOf y Anti! ii )i ly Bi'Cf i"! if in |||M, l*|fi. I'lA. I-ID. li)E ^.................. . it..... B:, C y' loin mi; Af.l ti in i in S. mm;i/i-iI Cols I i ;;u rc 1 uezoi. ssa R&S 102312 I i- >