J'HESIF‘ , LIBRARY This is to certify that the thesis entitled HUMAN LYMPHOCYTE IMMUNOREACTIVITY ASSESSED BY EARLY INCORPORATION OF 3H-URIDINE presented by Robert Glenn Ulrich has been accepted towards fulfillment of the requirements for M.S. degreein Biological Sciences fair Pan—”4 Major professor f Date /’7/ NJWA/fi’a 0-7639 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to renom charge from circulation recon HUMAN LYMPHOCYTE IMMUNOREACTI ITY ASSESSED BY EARLY INCORPORATION OF -URIDINE By Robert Glenn Ulrich A THESIS Submitted to iichigan State University in partial fulfillment of the requirements for the degree of aASTER OF SCIENCE Department of Biological Sciences 1980 ABSTRACT HUMAN LYMPHOCYTE IMMUNOREACTIVITY ASSESSED BY EARLY INCORPORATION OF 3H-URIDINE BY ROBERT GLENN ULRICH The detection of an early increase in the rate of RNA synthesis by phytohemagglutinin stimulated human per- ipheral blood lymphocytes has been investigated as a rapid in_zitgg_means of assessing cellular immunoreact- ivity. 3H-uridine uptake and incorporation into RNA precedes the increase of S phase thymidine incorporation providing for quantitation of lymphocyte stimulation occur- ing before DNA synthesis. This assay offers advantages over the commonly used thymidine incorporation method primarily in conveniance and potential accuracy of results. By measuring events occurring during the first stages of blastogenesis this technique may also circumvent the effects of helper and suppressor functions present in culture. Heparinized blood samples were obtained from healthy adult volunteers by venipuncture. The culture conditions that were Optimum for producing the highest increments of stimulatiOn were determined. 1 X 105 lymphocytes isolated by Ficoll-Hypaque density centri- fugation and a phytohemagglutinin concentration of 50 ug per ml led to a level of 3H-uridine incorporation at 20 hours of culture that was over 5 times the rate for unstim- ulated controls. At 12 hours of culture significant up- take of label was obtained, and by 36 hours over 10 times greater accumulation of 3H-uridine had occurred in stim- ulated cells above controls. Beyond 36 hours the stim- ulation index declined mainly because of a plateau of label uptake in stimulated cells accompanied by a rise in uptake by unstimulated cells. The enhanced 3H-uridine incorp- oration in response to phytohemagglutinin was inhibited by actinomycin D and was precipitable by trichloroacetic acid and thus occurs as a result of stimulated early RNA synthesis. Significant RNA synthesis stimulation was observed when lymphocytes were cultured in medium without serum thus providing a chemically defined system for as- sessing immunoreactivity. T and B cell fractions enriched by rosetting T cells with aminoethylisothiouronium bromide sheep red blood cells and characterized by surface markers were incubated with phytohemagglutinin. RNA synthesis was stimulated only for T cells in response to this T cell mitogen. Significant early RNA synthesis was always de- tected but the magnitude of stimulation for the individual's lymphocytes varied between days of observation. 3H-uridine incorportion was exhibited by a specifically stimulated lymphocyte subpopulation, occurred at an early stage of the cell cycle. and thus provides a practical assay for evaluating cellular immune competence. AC 1’. N O‘u‘J LED GIVE NT S I wish to acknowledge the guidance and friendship of Dr. Marek M. Pienkowski. Henry Ford Hospital, while acting as my thesis advisor. ii TABLE OF CONTENTS Page INTRODUCTION 1 REVIEW OF THE LITERATURE u I. Lymphocyte Membrane-Ligand Interactions 4 II. Intracellular Mediators of Lymphocyte 8 Stimulation III. Membrane Transport 10 IV. Other Biochemical and Metabolic Events 11 of Stimulation V. Ribonucleic Acid Synthesis 14 VI. Lymphocyte Cell Cycle 17 VII. Cell-Cell Interactions 20 VIII. Effector Cells 22 MATERIALS AND METHODS 2# Blood donors 2Ll Cell preparation 24 Preparation of lymphocyte T and B cell 24 subp0pulation Cell culture preparation 2? Reagents 28 Statistical analysis 28 EXPERIMENTS AND RESULTS 30 Establishment of Optimum culture conditions 30 Time-dependent incorporation of 3H-uridine 32 iii Lymphocyte stimulation in a single indiv- 36 idual over an extended time of observation Cellular specificity of 3H-uridine incorp- 39 oration Association of 3H-uridine incorporation 40 with RNA synthesis DISCUSSION #1 SUMMARY 52 BIBLIOGRAPHY 53 iv LIST OF TABLES Table 1. Effect of varying cell densities and concentrations of PHA-P in a medium containing serum on (3H) uridine incorpora— tion by human lymphocyte cell cultures. Table 2. Effect of varying cell densities and concentrat'ons of PHA-P in a medium without serum on ( H) uridine incorporation by human lymphocyte cell cultures. Table 3. 3H-uridine incorporation by lymphocytes of a single normal individual sampled over an extended time period. Table A. Cell surface markers analysis of lymphocyte subpopulations. Table 5. 3H-uridine incorporation by lympho- cyte pOpulations exposed to PHA-P in vitro. Page 29 31 35 37 38 LIST OF FIGURES Page Figure 1. Lymphocyte isolation 23 Figure 2. T and B cell preparation 25 Figure 3. T and B cell characterization 26 Figure eu. Incorporation of 3H-uridine by PHA 34 stimulated lymphocytes with time of culture Figure 5. Lymphocyte stimulation #2 vi INTRODUCTION A number of techniques have been established for an- alysis of the immune history and potential immune respon- siveness of the individual. Detection of antigen-spec- ific antibodies is widely relied on for diagnosis of in- fectious disease states. Cell-mediated immune events are generally assessed clinically by cutaneous delayed hypersensitivity reactions which correlate with prior ex- posure to specific antigens. In_xitgg methods have also been developed to assess cellular antigen-specific respon- siveness. referred to as immunoreactivity, most commonly by lymphocyte stimulation (119. 1&0). Positive lymphocyte immunoreactivity has been found in individuals that do not respond to cutaneous hypersensitivity tests but were vac— cinated (145. in?) or were in an active disease state (72. 8“, 133). Similarly lymphocyte immunoreactivity has been found in cases without humoral immunity present (203). 12.21222 techniques are particularly useful in testing for specific hypersensitivity in cases of contact dermatitis. sensitivity tests for multiple antigens, and for patients anergic by cutaneous methods. Specific antigen stimulation of lymphocytes in xitgg has been studied in a variety of systems including challenge with antigens of virus (143, 1A4, 155), bacteria (146, 1A7), fungi (148), protozoa (1h9-151), helminths (152). mycoplasma (15A), allergens (141-1AZ), and tumor antigens (153). The typical blastogenic response of lymphocytes cultured with specific anamnestic antigens or nonspecific mitogens is generally quantitated by radio-labeled thymidine incorp- oration. Using thymidine incorporation as an index of immunoreactivity, however, imposes a few major restrictions on interpreting results and method performance. The ex- tended culture time necessary (at least 72 hours) is in- convenient and introduces nonspecific proliferation from serum factors. and the possibility of in yitgg sensitiza- tion of lymphocytes (178-179). In addition measuring stimulation only in terms of the relatively late event of DNA synthesis complicates the interpretation by intro- ducing the effects of helper and suppressor factors present in culture which influence the G1 to S phase shift in the cell cycle of stimulated lymphocytes (122-125). Thus an analysis of early events in the cell cycle may prove more useful as an assay for cellular immunoreactivity. This study developed a semimicro technique adapted for human peripheral blood lymphocytes which is based on quantitation of RNA synthesis stimulated by the plant lectin phytohemagglutinin (FHA-P), (187). RNA synthesis as determined by incorporation of 3H-uridine precedes DNA synthesis of stimulated lymphocytes. Significant stimulation indices are routinely obtained by 12 hours of culture. with assay sensitivity increasing to 36 hours. The incorporation of uridine by lymphocytes cultured with the T cell mitogen FHA is enhanced exclusively for T cells. mediators of secondary cellular immune responses. The Optimum culture conditions have been established and consistant results were obtained both in the presence and absence of serum supplements. REVIEW OF THE LITERATURE I. Lymphocyte Membrane-Ligand Interactions A complex cascade of physical-biochemical events is initiated in lymphocytes stimulated in zitrg. Agents that induce stimulation or transformation of lymphocytes may be loosely categorized as mitogenic when they produce nonspecific stimulation or antigenic when immunological priming of the organism is required and specific memory cells respond. Allogeneic cells are a special category of antigens that stimulate other lymphocytes based on dif- ferences in histocompatability antigens between individuals. Most biological mitogens are also potentially antigenic as they readily produce antisera in immunized animals (1). The most widely studied class of biological mitogens are the plant lectins which are composed of glycoprotein or protein and possess varying capacities to bind saccharides. The plant lectins conconavalin A (Con A) and phyto— hemagglutinin (PHA) are frequently used in deveIOping mod- els of immunological lymphocyte stimulation. When present ’in soluble form both Con A and PHA are polyclonal T cell activators. The addition ofci-Demethyl mannoside com- petitively inhibits the binding of the Con A molecule to membrane receptors (3). A certain threshold time of incubation with mitogen Lt is necessary for activation to occur (27) and an Optimal cell and mitogen concentration are also observed (4, 12). Lectin mitogenicity is dependent on Ca**concentration, temperature and pH (12, 13). Stimulation progressing to blastogenesis as measured by radioactive thymidine uptake is maximal when 16-25% of the Con A binding sites are filled and 3% of the PHA sites (6). Although only T cells are activated by Con A and PHA the lectins bind equally to B, and T cells (199)- PHA association with the lymphocyte membrane has a t; of of 3 to 5 minutes at 22° C reaching equilibrium at 30 min- utes (12). It is generally agreed that an effective lectin must be at least bivalent for mitogenicity (7) although a re- quired tetravalency (8) and mitogenic monovalent anti- bodies (9) have been reported. The dimeric succinyl- ated form of Con A does not produce a falling limb on the dose response curve while the native tetramer does (93) when the proliferative response is quantitated by thym- idine incorporation. At higher doses of mitogen there is a decline in stimulation resulting from competition for binding sites that reduces the amount of a presumed necessary cross-linking of surface receptors or a toxic effect produced at higher lectin concentrations (198). At lectin concentrations greater than the Optimal dose when lymphocyte viability decreases,lymphokine production, which is DNA synthesis independent,may actually increase (9). The nature of antigen interaction with T cell mem- brane receptors has remained obscure. The surface mem~ brane immunoglobulins IgM and IgD of B cells are clearly implicated in antigenic stimulus of B cells (16). March- alonis has presented evidence (10) in support of an immuno- globulin-like T cell receptor that is an integral membrane constituent like IgD of B cells that is not freely secret- ed into serum. Ig-like molecules have been reported as- sociated with T cells. Szenberg (19) identified molecules isolated from murine thymocytes and thymoma lines with k chain specific fowl anti-IgG (Fab)2 antisera that upon reduction showed k light chain electrOphoretic mobility and a unique heavy chain mobility. Rosenthal (11) using bovine insulin as an antigen model found that guinea pig T cells recognize only a minor portion of the antigen primary structure ( a single amino acid residue) while B cells are able to respond to many different conformational determinants. The "carrier effect” pre- sents another argument against an immunoglubulin T cell receptor. This phenomenon is observed when an animal is immunized with a hapten (chemical modification) conjugated carrier antigen molecule and challenged with a booster of hapten-carrier conjugate or hapten conjugated to a new carrier. Maximum anti-hapten antibody production is obtained only when the original carrier-hapten is used to challenge. Carrier recognition is T-dependent (“0). When an antigen is reacted with antisera from an immun- ized animal and used to challenge another immune animal only delayed type hypersensitivity. which is T cell depen- dent. is exhibited (41). Stimulation of lymphocytes to mitosis requires the continual presence of both antigen and mitogen (1h, 15). Macrophages are required accessory cells for antigenic and most mitogenic stimulation (9u-96). Additionally the macrOphage and lymphocyte must be histocompatability matched for maximum stimulation to occur (17, 18). Pul- sing macrOphages with antigen before lymphocyte addition produces the same results as continuous presence of an- tigen. Rosenthal has suggested a differential route for antigen presentation as opposed to degradation by macro— phages (11). The vast majority of antigen is internal- ized and either fuses with golgi apparatus membrane and is recycled to the cell surface (inhibited by cytochalasin) or fuses with lysosomes and is degraded (ablated by meta- bolic inhibitors). When the fate of ferritin coupled PHA is followed in human blood lymphocytes,within a few min- utes at 37° C the majority of label is found intracell- ularly and after several hours appears to coalesce and condense within the cytOplaSm (25). In “0% of blast cells the labeled PHA was exocytosed to the cell surface. The majority of mitogen is found intact in-T cell blasts (26). Robinson et al (20) found that murine lymphocytes which ordinarily possess few receptors for the lectin wheat germ agglutinin (WGA) were markedly labeled by fluorescent WGA conjugates when cultured in the presence of B or T cell mitogens. Increased WGA binding was also observed with lymph node cells of infected mice (21). Petris and Raff (22) using immuno-ferritin electron micrOSOOpy found the mouse lymphocyte membrane antigens theta, TL, H-2 allo- antigens and Con A receptor sites randomly distributed unless cross-linked by multivalent antibodies or ligands. There is a random distribution of Con A receptors and immunoglobulins when Con A is present in excess (23) but a non-random patch and cap formation whean-D-methyl mannosidecu'the microtubule assembly inhibitors colchi- cine, colcemid, vinblastine and vincristine are added. When the native tetrameric Con A is succinylated to a dimeric form capping and patching of receptors do not occur but mitogenic stimulation still takes place. Glu- cocorticoid receptors increase during mitogen stimul- ation, and there is an increase in membrane fluidity detectable within the first 30 minutes of lectin asso- ciation by spin labeling techniques (2A). II. Intracellular Mediators of Lymphocyte Stimulation Quastel and Kaplan (28) first reported the ouabain sensitivity of human lymphocytes activated by PHA. ' The ouabain inhibition of synthesis of RNA, DNA. and protein as well as blast and mitotic progression could be revers- ed by adding potassium to cultures. K+ is taken up by PHA stimulated cultures (29) thus suggesting a role for Na*. KI-ATPase in stimulation events (30). By medium depletion studies or through addition of cationic sequesterants Ca*+was shown to be necessary for mitogenic stimulation. intracellular accumulation occuring within the first hour Of PHA stimulation (31). Ca**uptake is independent of ATP and membrane depolar- ization, modulated by cyclic nucleotides,and inhibited by mitochondrial uncouplers. colchicine and vinblastine. (32). Ca**accumulation appears to be biphasic with a rapid uptake occurring within 5-30 minutes of mitogen addition, a stable intracellular concentration for 4-6 hours and a gradual efflux over the next 12-20 hours. Although PHA responsiveness is depressed by cation chel- ation using EGTA a significant response still occurs (33). Measurable intracellular calcium accumulation seems to result only at supraOptimal mitogen doses (34). Cal- cium ion fluxes have been associated with secretion in other mammalian cells e.g. insulin secretion by pancreatic islet cells (167) and the possibility remains that Ca** accumulation is required for lymphocyte mediator secre- tion but not as an obligation for progression through the cell cycle. Whitney and Sutherland (35) were able to modulate lymphocyte activation in a mixed lymphocyte culture by varying the concentration of and time of exposure to cAMP (cyclic adenosine 3', 5'-monOphosphate). cAMP functions antagonistically to cGMP (cyclic guanosine 3', 5'-mono- phosphate) in its effect upon mitogen stimulation. Elev- ated levels of cGMP appear to be associated with enhance- ment of cell proliferation while increase in cAMP depress- es proliferation (35, 36). cAMP phosphodiesterase activ- ity is enhanced in Con A stimulation or by addition of 10 cGMP (38). Although significant type I and II cAMP dep- endent protein kinase levels are present in resting human peripheral blood lymphocytes Con A stimulation activates only type I (39). Dibutyryl cAMP at concentrations that block RNA and DNA synthesis results in activation of both enzymes. Their regulation within the lymphocyte may be a consequence of CAMP/cGMP ratios. III. Membrane Transport A rapid increase in membrane transport of many met- abolites after mitogenic stimulation of lymphocytes occurs. 5 fold increase above controls in transport of the non- utilizable glucose analog 3-O-methyl-glucose is Obtained (43) reaching a maximum 30 minutes after PHA addition. There was no change in Km for the glucose analog transport and the bidirectional transport probably was mediated by facilitated diffusion. There is increased transport for the amino acid analog«u-aminoisobutyric acid (AIB) with an increased Vmax and constant Km (200), after a lag of 30 minutes from addition of PHA. AIB transport enhance- ment is inhibited by metabolic and protein synthesis in- hibitors. is independent of RNA or DNA synthesis (45, 46) but parallels the increase in DNA synthesis (44). The relative mitogenicity of different lectins as determined by thymidine uptake by lymphocytes correlates with stim- ulation of proline transport (42). Hanrahan et al (47) prOpose four phases of thymidine transport that are independent of the stimulant dose or nature. First there is a period equal to control uptake; 11 a rapid rise in uptake; exponential increase of uptake; and a declining phase. Uridine transport is increased within minutes and thymidine by 24 hours both as a result of higher respective Vmax but stable Km (48). Uridine and thymidine transport are both believed to operate by facilitated diffusion, IV. Other Biochemical and Metabolic Events of Stimulation In an eloquent series of studies (49-51) Loos and Roos examined carbohydrate metabolism in PHA and antigenic- ally stimulated human peripheral blood lymphocytes. With mitogenic stimulation changes in metabolism were noted within a few hours of culture but antigenic (tuberculin purified protein derivative=PPD) and allogeneic cell stim- ulation required 2-3 days for detection and the magni- tude of change was less. They calculated that 15% of the cellular ATP originated from glycolysis, while 85% formed from oxidative phosphorylation. It was concluded that either glycolysis or oxphos could support the early stage of lymphocyte stimulation but that both were re— quired for extended maintenance of stimulation. Heat evolution in antigen stimulated horse lympho- cyte cultures parallels thymidine incorporation, in- creasing in 48 hours of culture and peaking at 5-6 days. With Con A heat liberation begins at 6 hours while inhibition of RNA and protein synthesis blocked generation of heat for both mitogen and antigen cul- tures, but DNA synthesis inhibition had no effect (52). PHA induces a 10 fold increase of selective incorporation 12 Of inorganic phosphate (Pi) into phosphatidyl inositol of the plasma membrane within 10 minutes of culture and enhances Pi turnover (53, 54). Most mitogens accelerate Pi incorp- oration specifically into phosphatidyl inositol within 30 minutes with the exception of pokeweed mitogen which requires hours of incubation (55). Pi is also incorpor- ated into phosphatidyl choline and phosphatidyl ethanol- amine by tetanus toxoid mitogenic stimulation (56). Label- ed acetate is rapidly found in long chain fatty acids (57). sterols and phospholipids (58). PHA induces a doubling in the rate of glucosamine incorporation into UDP-N-acetylglucosamine (59). T cell blasts take up GDP-mannose into cell surface molecules possibly linked to glycoprotein formation (60). Changes in nucleoproteins also accompany immuno- logical stimulation of lymphocytes. Protein synthe— sis-independent histone acetylation is observed within the first minutes of PHA interaction (63, 168). Increas- ed rate of phosphate turnover occurs during the initial period of RNA synthesis but there is no net increase in histone phosphate content (169). NucleOprotein methyl- ations have also been reported (170). Protein synthesis rates are sensitive to the effects Of mitogens. PHA activation leads to a rapid recovery of labeled amino acids in cell proteins (61- 63). Actino- mycin D which intercalates between the dG-dC base paired dinucleotide sequence of double stranded DNA (64, 190) has been a useful probe for separating DNA-dependent from 13 DNA independent phenomena. By varying the concentra- tion different RNA species may be inhibited from syn- thesis to proportionate degrees. When rRNA synthesis is inhibited by 0.01 micrograms/ml of actinomycin D the characteristic 18 S and 28 S peaks on RNA sedimentation profiles are diminished (65). The FHA enhanced amino acid label incorporation still occurs but the rate of uptake ceases to rise after 24 hours of culture. sug- gesting that the primary increase in protein synthesis rate is independent of transcription. Using RNA-DNA hybridization with RNA from stimulated human lympho- cytes and DNA from resting lymphocytes. and polyacryl- amide gel electrOphoresis of proteins,results of Neiman and MacDonnell (66) suggest that protein synthesis of early stimulation is performed with preexisting RNA tem- plates and the synthesized proteins are amplifications of proteins expressed in resting cells. PHA stimulated lymphocytes after about 72 hours of culture contain 60-70% Polysomes compared to about 25% for controls (67). The polysomal profile and amount of ribosomes sedimenting at 80 S seem to correspond quant- itatively with protein synthesis (68). The majority of ribosomes in resting lymphocytes exist in an inactive 80 S complex (69). Within the first 20 hours of stimula- tion most of the ribosomal units are polysomal or dissoci- ated into subunits. but after 48 hours there again is an increase in the inactive 80 S ribosomes. It would appear that the rate of dissociation of the inactive 80 S to the 14 60 S and 40 S subunits of the ribosome prior to initia- tion of translation limits protein synthesis activity. Kay et a1 (68) concluded that control of the increase in protein synthesis lies in control of the formation Of the protein synthesis-active 80 S complex by initia- tion factors. Cytoplasmic factors support an increased capacity of ribosomes from PHA stimulated lymphocytes to synthesize proteins in a cell-free system (67). When lymphocytes are stimulated with FHA and con- currently inhibited by actinomycin D there is an immed- iate decrease in leucine incorporation along with the de- creased uridine uptake (71). A brief lag period before the decline in leucine uptake when sufficient quantity of actinomycin D is present would be expected if the RNA synthesis was merely a non-limiting corequirement for protein synthesis. There is actually a decrease in the amount of mRNA bound to ribosomes during the first few hours of PHA stimulation (70). V. Ribonucleic Acid Synthesis Precursors for RNA synthesis in mammalian cells are derived from de novo synthesis or from salvage path- ways for degraded nucleic acids. The parent pyrimidine molecule uridine 5'-monOphosphate (UMP) originates from the starting compounds aspartic acid and carbamoyl phos- phate (64). The intermediate orotic acid is joined by a ribose-5-phosphate group derived from 5'-phosphorib- osyl-1-perphosphate (PRPP) and decarboxylated to form UMP. UMP is phosphorylated to uridine-5'-triphosphate 15 through the action of kinases. Cytosine is synthe- sized from UTP through a specific synthetase. The triphosphate pyrimidine derivative is the form incorp- orated into the RNA polymer. The parent purine ribonucleotide inosine-5'-mono- phosphate (IMP) originates from an amino group transfer from glutamine to PRPP. Subsequent steps involve com- bining with glycine, two formylations involving tetra- hydro folate intermediates, another amino group trans- fer from glutamine, carboxylation and aspartate addition. Amination of IMP produces adenosine-5'-monophosphate (AMP) and amination of xanthosine-5'-monOphosphate gives GMP. Mammalian cells also have the capacity to convert free bases via PRPP and nucleosides derived from nucleic acid catabolism to nucleotides for reuse in synthesis of RNA and DNA. The relative importance of the salvage vs de novo pathways in lymphocyte stimulatiaihas been eluc- idated by investigations of certain purine metabolism disorders in humans. Patients with Lesch-Nyhan syndrome have normal blood lymphocyte counts and their lympho- cytes respond well to PHA. pokeweed mitogen, and allo- geneic cell stimulation (73) even though they are defi- cient in the major salvage pathway enzyme hypoxanthine- guanine phosphoribosyltransferase (HGPRT). Analogs of de novo purine synthesis precursors such as azaserine cause complete inhibition of PHA response in these lymphocytes while the purine analog 6-mercaptopurine has no effect. In normal PHA stimulated lymphocytes azaserine has only 16 a slight effect at the same concentration while 6-mer- captopurine strongly inhibits the response. Deficient- cies in adenosine deaminase (74) which deaminates adeno- sine to inosine and purine nucleoside phosphorylase (75) for conversion of inosine to hypoxanthine have been as- sociated with immune disorders. Human lymphocytes incorporate significant amounts of glysine into purine nucleotides within two hours of exposure to PHA and have an increased capacity to take up adenine and adenosine into adenine nucleotides (76). Levels of PRPP are also enhanced in PHA stimulated cells so that both pathways of nucleotide synthesis appear to be functioning in normal lymphocytes. Pyrimidine sal- vage and de novo synthesis are also stimulated by PHA (77). although uracil does not seem to be utilized (1). Adenosine has been shown to inhibit lymphocyte blast cell growth by blocking de novo pyrimidine synthesis (78, 79). Uridine addition to PHA stimulated human lymphocytes shifts the cell from de novo synthesis to the salvage pathways for direct incorporation into RNA (80). The plasma membrane-bound enzyme 5'—nucleotidase that catalyzes dephosphorylation of nucleoside-5'-monophos- phates has increased activity on mitogen stimulated murine lymphocytes (81) and appears to be higher in con- centration on T cells (83). Net RNA increases over the course of stimulation (82) with short lived 4 S and polydisperse species appearing first in the sedimentation profile. Next the ribosomal 17 18 S, 28 S. and 45 S peaks appear. PHA activation reverses the rapid degradation of 28 S and 18 S species that normally occurs in the nucleus of the resting lympho- cyte (85), although there is no apparent change in cyto- plasmic ribonuclease activity (86). Extractable levels of RNA polymerase I. II. and III increase upon cell act- ivation (87). The activity of soluble deoxyribonucleo- protein-bound RNA polymerase increased after a detectable rise in uridine uptake (88). Heterogenous nuclear RNA (hnRNA) is specifically lab— eled by short pulses of radioactive nucleotides in the presence of quantities of actinomycin D sufficient to block rRNA synthesis (89). Polyadenylation may be re- lated to stability and processing of mRNA (89). In the human lymphocyte PHA stimulation appears to increase rates of RNA maturation and transport from nucleus to cytOplasm (90). Processed tRNA seems to appear in cytoplasm be- fore rRNA is detected. Polyadenylation of RNA and the appearance Of polyadenylated RNA in the cytoplasm does not occur until several hours of lymphocyte exposure to the lectin (91. 92)- VI. Lymphocyte Cell Cycle The human lymphocyte is fastidious in culture with a steady loss of viable cells within 48 hours (99). Media"conditioned" by stimulated cells (5) or lectin, antigen stimulation is necessary to maintain a cont- inuously growing culture. Generally less than one per- cent of the cells respond to antigens (97, 98). and 18 approximately 50% respond to mitogens (99). In the normal individual 90% of the peripheral blood lymphocytes are in a resting state possessing few visible organelles, generally scattered ribosomes, large mito- chondria. and undeveloped Golgi apparatus (171). Large blast cells appear in culture at least by 48 hours of stim- ulation and 50% or more of the cells are blasts at 72 hours of lectin activation (100). These changes are accomp- anied by increasing nuclear volume (101) and cytOplas- mic basophilia (102). Nucleoli undergo morphological transformations that follow the phase of the cell cycle (103)- Understanding Of the lymphocyte cell cycle has in- creased from data of flow cytometry studies. The in- strumentation has become available (at considerable cost) allowing rapid 50,000 cell/minute multiple parameter anal- ysis of cell pOpulations. The flow microfluorometer measures the fluorescent intensities of tagged cells pass- ing rapidly single-file through a beam Of laser light which activates the fluorescent labels. Data is collated by computer interfacing. Using the stoichiometric DNA stain mithramycin, Cassidy et. al. (104) were able to detect blastogenic responses of human lymphocytes to PHA, PPD and viral antigens. DNA-specific prOpidium iodide (105) and the metachromatic stain acridine orange (106) have also been used. Stacking of acridine orange to RNA and intercalation in DNA produces different fluorescence peaks (107, 108). Cell transition from cell cycle phase Go to 19 G1 reflected by an increase in DNA fluorescence and decrease in RNA peak occurring in absence of DNA syn- thesis is observable after 6 hours in cultures stim- ulated by PHA or allogeneic cells (201, 202). After two days of culture the number of cells in transition decreases andthe population of cells in Go and G1 be- comes distinct. There was observed a heterogenicity in duration of the S phase, with cells having the highest RNA concentration completing S phase at the fastest rate. A shift in the average length of G1 in cul- tured cells is usually the way a change in cell gen- eration time is mediated (112). The quiescent lymphocyte exists in Go phase until antigenic or mitogenic stimulus pushes the responsive cell into G1 growth phase. The majority Of blast cells appear to be derived from repeated mitoses of the same cell pOpulation (97). For some mammalian cells G1 may be bypassed during a period of rapid cell cycling (113. 114). DNA synthesis occurs in S phase up to the boundary of G2, the tetraploid stage before mitosis (115). The S phase is the point frequently assessed by thymidine incorporation. The activity of thymidine kinase is increased 200 fold (116) and thymidine catabolizing en- zymes have a much decreased activity during S phase (117) of lymphocytes. Low molecular weight DNA is excreted into the culture medium (118). Cells restimulated after a decline in DNA synthesis require less time for onset of 20 blastogenesis and DNA synthesis is more rapid and greater in activity than unprimed cells (120. 131). VII. Cell-Cell Interactions The term lymphokine (109) was first suggested to characterize biological activities expressed by cell- free soluble factors of lymphocyte origin. "Migration inhibition factor" was the first reported (110, 111) as a factor obtained from culture media of stimulated lympho- cytes that inhibited the random migration of macrOphages. Currently there is an extensive list of lymphokines that affect many functions of lymphocytes (2. 121) and any as- say of cell mediated immunity must take into account their actions. There is evidence that PHA induces only a shift to the G1 activated state in responsive lymphocytes and lympho- cyte activating factor (LAF or Interleukin I) produced by macrOphages is necessary for induction of S phase (2, 174). cGMP mimicks the affects of LAF when present during G1. Another factor produces a rise in cAMP in late G1 and in- hibits DNA synthesis and mitosis in T, B cells and fibro- blasts. Pretreatment of lymphocytes with Con A suppress- es subsequent response of thymidine incorporation to anti- genic or mitogenic stimulants (122). With allergic rhinitis patients desensitized to ragweed antigen E anti- gen-specific suppressor cells could be detected in vitro when generated by incubation with antigen (123). Both "suppressor" or "helper" functions have been described as defective in certain immunological disorders (124, 125). 21 A defect in helper activity causing immunoglobulin def- iciency is suspected when cO-cultured X-irradiated normal T cells augment in vitro immunoglobulin synthesis or blast— ogenesis of patient's lymphocytes. Suppressor function is assessed in a similar manner. 1-2 KR of radiation ablates suppressor activity, while leaving helper intact. A cycloheximide sensitive, actinomycin D and colchicine insensitive B cell suppressor of-T cell blastogenesis has been reported (126). There are also T helper cells that induce another T cell subset to exert suppressor activity (134, 135). The macrophage plays another role besides antigen processing and presentation. Addition of normal per- itoneal or splenic macrophages to murine splenic T lympho- cytes at a level beyond 5-10% suppresses rather than en- hances T cell proliferation (127). "Activated" macro- phages exhibit an even greater suppressor activity (128). Lymphokines such as macrOphage activating factor (129) activate the macrOphage. and suppression may be mediated by prostaglandins and hydrogen peroxide (128). A soluble factor produced by polymorphonuclear leuko- cytes has been found to enhance thymidine incorporation in stimulated lymphocytes (130). Parasitic infections have been linked to sup- pression of the cellular and humoral immune response mounted against the parasite (132). A recent study (133) found that patients with patent microfilaremia were un- responsive to in vitro lymphocyte stimulation by filiarial 22 antigens although cell-mediated responses to other non- parasitic antigens such as PPD were normal. VIII. Effector Cells Immunological stimulation of specific T cells that recognize foreign antigens in association with self anti- gens or mitogenic stimulation leads to maturation of a variety of cells of the effector limb of the immune re- sponse. In some cases a maturation of the effector occurs without leading to mitosis (136). Allogeneic cell stim- ulation produces "natural killer" cells that specific- ally kill non-self cells (137). In the murine system the inducing T cell has the Ly 1*23' cell surface pheno- type and the killer cell has the Ly 23+Ly1' antigens (138). The natural killer cells slowly disappear in culture but stimulation of the lymphocytes produces an "activated lymphocyte killing" of targets unrelated to the stim- ulating antigen (162). This new cytotoxic potential may be induced by interferon. occurs prOportionally with blast formation and proliferation but does not require cell division (139). 23 Lymphocyte Isolation -—hlood A U --flcoll-hypoque Nd, lymphocytes / ol lnlerfoce\ and! l l REG! 5 FUN! «a _'""" In cell pellel CS” \ washed reeuspend. fl U lymphocytes count Figure 1 MATERIALS AND METHODS Blood donors: 40 healthy volunteers of both sex, 22 to 35 years of age, donated blood (20 to 50 ml) for these experiments. Cell preparation: Diluted 1:1 (v/v) with RPMI 1640 (Gibco, Grand Island, NY), heparinized (20 ug/ml, Sigma, St. Louis. MO) venous blood samples were carefully layer- ed on the top of Ficoll-Hypaque density medium (p=1.078 ug/ml, Ficoll, MW 40,000,Sigma; Hypaque, sodium diatri- zoate, Sigma) in sterile 15 ml polystyrene centrifuge tubes (5 ml of the density medium and 10 m. of diluted blood) and the tubes were centrifuged applying 400 G at the in- terface for 20 min. (Figure 1). Cells which appeared at the interface were pipetted out, washed twice with the medium by centrifugation (300 G for 10 min.), cell con- centration counted with a hemocytometer and adjusted apprOpriately. Preparation of lymphocyte T and§_cell subpopulations: T and B cells were separated from the original lympho- cyte preparation using sheep red blood cell (SRBC) ros- ette formation. SRBC pretreated with aminoethylisothio- uronium bromide (AET, Sigma) and adjusted to a 10% concen- tration in phosphate buffered saline (PBS) free of Mg++ and Ca*+containing 5% dextran (avg. MW 80,700. Sigma) 24 25 T and B Cell Preparation lymphocytes AET-SRBCs slow centrifuge at 4°C t 2 eye es ¢ entl-lg heads rosettes resuspend e incubate at 4°C a l T rosettes LLllLLls.’ tlcolt-hypoque B a null cells density centrltuootion .05.... M11 ”Q'a" rosettes e SRBCs Figure 2 26 T and B cell characterization peHet . interface T cells B 8 null cells hypotonic lyflna ot SRBCs V wash———)CU|NYO wosh——-) culture odd anti-la beads odd anti-lg beads V 00 no bead rosettes 00% Q bead rosettes 0000 8 null cells Figure 3 27 were mixed with an equal volume of lymphocytes at 5 x 106per ml in the same medium and immediately centrifuged (50 G for 10 min. at 4°C),resuspended and incubated one hour on ice (Figure 2). After another 10 min. centri- fugation (at 50 G) the SRBC + lymphocyte mixture was gently brought back into suspension and layered on the Ficoll-Hypaque density medum and centrifugated 20 min. at 400 G. The pellet was composed of rosette forming cells (T), while non-rosetted forming cells (B + null) remained at the interface of the density medium. The pellet was treated with hypotonic NHACI EDTA solution and the isolated lymphocyte fractions were washed twice with RPMI 1640. The purity of the subp0pulation iso- lates (Figure 3) was confirmed using polyacrylamide beads coated with rabbit anti-human heavy and light chain imm- unoglobulins (Immunobeads, Bio-Rad Laboratories. Richmond, California), which form rosettes (3 or more beads or SRBCs bound per cell) with B cells but not with T cells. Monocytes phagocytize the beads and may thus be distin- guished from other mononuclear cells. The initial heter- ogenous lymphocyte preparation was also assessed for B and T cells by double rosetting with polyacrylamide immuno- beads and SRBCs. Cell culture preparation: All manipulations were per- formed under sterile conditions using a vertical laminar flow hood. Lymphocytes were cultured in microculture plates, 0.35 ml capacity wells (Linbro, New Haven, Conn.) with RPMI 1640 supplemented with 3.2 mM L-glutamine and 28 containing penicillin (100 IU/ml) and streptomycin (100 ug/ml: both Gibco). 5% heat inactivated fetal calf serum (FCS: Gibco) was added to the medium in some experiments. the cultures were composed of 100 ul of cell suspension per well, 10 ul of 3H-uridine (5.6-3H, 40.8 uCi/mM: New England Nuclear, Boston, Mass.) at 10 uCi/ml and 10 ul of PHA—P solution at appropriate concentration with 3 or more wells used per each experimental variant. The cul- tures were incubated at 37°C in a C02 incubator (5% 002- 95% air. humid atmosphere). Cell cultures were collected on fiber glass filters (93H, Reeve-Angel, Whatman. NJ) with a semiautomatic multiple sample harvester (Otto Hiller 00., Madison, WI) using 5% TCA or 0.9% NaCl in distilled water for wash and precipitation. Dried filters with cell precipitate were placed in a toluene scintillation cock- tail supplemented with Triton X-100 (New England Nuclear). A Searle Model 6890 Delta 300 Liquid Scintillation Counter was used for detection of cell precipitate radioactivity. Reagents: Phytohemagglutinin (PHA-P) was purchased from Difco, (Detroit, MI). Dilutions were made in RPMI 1640 medium. sterilized by passage through a 0.22 um mem- brane filter and used within 10 days. Actinomycin D (Calbiochem, La Jolla, CA) was diluted in serum-free media and filter sterilized just before use. Statistical analysis: Statistical tests performed were standard error. Student's t-test, coefficient of var- iation, and analysis of variance. .Houuu Unopnmum a afiE\muaaoo :mmzn .Houum vumccmum H xmccfl cowumHnfiwum cmozm mm H :flE\mu:soo 29 Hso.~n~oo.vm mvmnmfls.mu Hanemm.m Hewnsmm.e nmsnmmm.a massage Houucoo mH.onH.v om.ono.m sm.onv.m Hm.onm.v we.ono.m ooH o~.ons.¢ mH.on~.m m~.onoumd Hv.onm.m sm.onv.e om ~m.onm.e om.ono.m -.onm.m mm.ono.e mm.onm.m mm sm.onv.v ma.one.v o~.ono.v om.ono.¢ mm.onm.~ m.~H mm.onm.m mfl.onm.m ea.onm.m m~.onm.m ma.ono.~ m.e Hm.onm.m ma.onm.~ ofl.onv.~ H~.ono.~ mo.ons.a H.m om0© «H.0Ho.H mo.onv.H mo.onm.H mo.onm.H «so.on~.a flexes m.H «mm won x a sea x m sea x H moa x m mom x m.~ assuage HE\mHHmo mo Hmnfiaz .mun om How mononus Ammv HE\fiU: 0H mo mocmmoum mnOSCHucoo on» an poumnsocfl mama Essen mama Hmumw mm suw3 voucmamammsm finance cw HE H.o mo mmuduaso mmmDBADU AAMU MENUOIQZNA 2&293 Nm ZOHfidmOAMOUZH mZHQHmD Ammv 20 ZDme GZHZH¢BZOU ZDHQmZ fl 2H mldmm ho mZOH94MBZMUZOU Qz¢.mMHBHmzma AAQU UZHMM<> m0 BUMhhm H mHHm¢ds EXPERIMENTS AND RESULTS Eatabliahment of Optimum culture conditions The Optimum culture conditions were first established by manipulation of the cell concentration in the fixed 100 microliters/well volume and by varying the PHA dose. The stimulation index (SI) was determined by dividing the counts per minute of radioactivity (cpm) of PHA stimulated cultures by the nonstimulated control which provides a reference of uridine incorporation. 20 hours of culture was chosen as a convenient harvest time from preliminary studies. Labeled uridine was present continuously during cul- ture. Radiotoxicity is not a severe problem when culture times as short as these are used and label pulses of less duration had insignificant effect on SI. A final concen- tration of 3H uridine'at 10 microcuries/ml was used to give optimum incorporation by stimulated cells while decreasing the variability of background label incorporation by con- trols. This concentration of 3H-uridine also produces a higher level of radioactive labeling which decreases the amount of time needed for liquid scintillation counting. The initial cultures contained 5% serum in medium. The PHA concentrations were made by serial dilutions over the range of 1.5 to 100 micrograms/ml of final con- centration. Within this range the SI exhibited a dose effect (Table 1). Significant stimulation (at p<0.01) was 30 31 .Houum pumpcoun H GflE\mucsoo cmmzn .uouuo pumuaoum H xm©2fi coflumHsEHum amaze mm H :HE\mu::oo Hem.mnmso.av mmo.MHSHm.m~ mmo.anmmm.ou nommnm.am~.m agendas Houucoo mfl.onsm.m s~.onmm.m ma.onma.m mm.onHm.m ooH sfl.on0m.m mo.onmo.m mfl.onem.m mm.onms.~ om eo.onmo.m Ha.onmm.~ o~.onve.m mv.ons~.m mm HH.onme.~ sa.onmm.~ mm.onmm.~ ev.onam.m m.NH ma.on~s.fl mH.onso.H AH.on m.H H~.onsm.a ~.o mo.onmm.H NH.onsm.H No.0nmo.H ma.onms.a H.m anon mo.ono~.a No.0nam.a ma.0nea.a mmo.onos.a Hs\ma m.H «mm mos x v sea x m 00H x H moH x m assuage HE\mHHmU mo Hmnfisz .mun on How ocwvwws Ammv HE\MU: 0H mo mocmmoum mzoscflucoo any cw moumnsocw mum3 endows GM as H.o mo mounuaau mmmDBADU AAMU mBNUOmmzwq ZflZDm Mm ZOHB m0 Bomhhm N mfiHm¢du 32 found at all concentrations of PHA for 1 x106 cells/ml. The control cpm exhibited a linear increase propor- tional to the increase in cell concentration over the range of 2.5 to 40 x 105 cells/ml. The highest SI value 5.6 was obtained at 1 x 106 cells/ml and 50 micrograms/ml of PHA. although there was no statistical difference (p>0.4) be- tween SI for the upper and lower limits of the cell dil- utions for this PHA concentration. With increasing cell numbers per culture well there is decreasing variation with- in the pooled data. The coefficient of variation (CV) varies from 24% for 2.5 x 105 cells/ml to 12% for 4 x 106 cells/ml at 50 micrograms/ml of PHA. Table 2 shows the data for the same set of experiments when the lymphocytes were cultured in a medium without FCS. The overall SI are still Obtained at significant levels while being lower values than that obtained with serum present. Similar trends are observed within the data when serum is present or omitted from the medium. The highest variation of SI between wells is found at the lowest cell concentration. 1 x 106 cells/ml again presents the high- est stimulation at a dose of PHA that is however 50% the Optimal amount when serum is present. Time-dependent incorporation of 3H-uridine A series Of experiments were performed to study the relationship of uridine incorporation by PHA stimulated lymphocytes with time of culture. 5 x 105 cells/ml were incubated with 100 micrograms/ml of PHA and 10 microcuries/ ml of 3H-uridine. Incorporation of radioactivity 33 Figure 4. Incorporation of 3H-uridine by PHA stimulated lymphocytes with time of culture. Lymphocyges were cultured at a concentration of 0.5 x 10 /ml with 100 ug/ml of PHA in RPMI 1640 containing serum. 10 uCi/ml of 3H-uridine was added at the beginning of culture. The stimula- tion index is the ratio of cpm for PHA stimulated cultures to cpm for nonstimulated (control) cul- tures. Vertical bars indicate the standard error of the mean. cpm for + PHA. --—~ Cpm-PHA. —u——stimulation index. 34 .I I I. xmoz. zo. P4432. Pm mil— nuw 8 6 4 2 - . . . . . . \.\\\\ \HA. 3\ .\\ . n.o w: 1.. 4 \ ,. x w \ .. \ \ \ 6 .Isflu . 1.3 ./ / e I I O x .1. l/ . 5 O v11 .0 r gum / my w LII. 0 .r 1m x < w .V. P 9.6 III e w I. I ' P12 / . .l Tbvnl 4 4.5 / . / e / _ x m p - p . p b p h V O O 0 0 w m w m 4 3 2 .. <1§t¥l.l<1a¢$lsz.o.¢3-Inv m¢3h430\mo. x Eco TIME OF INOUBATION (HOURS) Figure 4 Mm a mosam> cmozm a Hw.onw.m a mmfimasms.mfi 35 m.m Hoo.s oofi ans o.m mso.mfi oofi mmfi m.m mmfi.s om RNH 3.: OH:.0H om HNH 0.: mmm.m om ma o.m mfia.fim HE\ms mm o Roccfi mthPHSO muo QMHm2HQZH A¢Emoz mqwsz < mo mmewoozmswa Mm ZOHB we once; esp new mucoswsoaxo m Ease mzpm> mmmgm>< a .uoc as couosssucw mmgwacmg mumwnoczssw can ommm so mm: msooceapaswm museumn seepagmumu no: meson co armouxuommcsm Amptm vo.m A mup vo.~ no 2 mcv~wpzuommsa mummnocasev cup: Ammtosvo.pm Ammumpvm.m~ an 2 mcwuwuzuoumsq mcmpummosuco co: ozH ”as Am.-opvm.F~ scapummog-co Ampum vo.o— Apmtwmvm.¢s Aopumvo.m mcwpummog unmnocsssH Asm-Fmvm.Na o slow-mavm.ma m=_ssmmoa ummm cowumgmawgq corumcmamza cowumgmamnn _Pmu-p r, swppmu-=m= seasona2s4 .suswcs massagsmcoees m._wu gowns—saoa Ppmu mo :o_usoaoga mzo-<42aoam2m mmp>uozaz>4 no mHm>4.mW mt¢zm OB ammomxm mZOHB