CHAPTER 81 COMPOSITION AND BIOCHEMISTRY OF LYMPHOCYTES AND PLASMA CELLS
CHAPTER 81 COMPOSITION AND BIOCHEMISTRY OF LYMPHOCYTES AND PLASMA CELLS
THOMAS J. KIPPS
DENNIS A. CARSON
Isolation of Lymphocytes
Lymphocyte Surface Antigens
Composition of Lymphocytes
Ion and Water Content
Fatty Acid and Lipid Synthesis
Protein Synthesis and Amino Acid Metabolism
Nucleic Acid Synthesis and Repair
Hormones and Vitamins
Mature lymphocytes can be divided into several functional types and subtypes. The major classes of lymphocytes are the T cells, B cells, and natural killer (NK) cells. T lymphocytes are derived from the thymus (see Chap. 5 and Chap. 82) and are responsible for cell-mediated cytotoxic reactions and for delayed hypersensitivity responses (see Chap. 84). They also produce the cytokines that regulate immune responses and provide helper activity for B cells. The B lymphocytes concentrate and present antigens to T cells and are the precursors of immunoglobulin-secreting plasma cells (see Chap. 83). NK cells account for innate immunity against infectious agents and transformed cells that have altered expression of transplantation antigens (see Chap. 85). This chapter describes methods for isolating lymphocytes and discusses their physical and biochemical properties. In addition, this chapter also provides insight into mechanisms that may account for the activity of purine deoxynucleoside analogs, ionizing radiation, or glucocorticoids against resting normal or neoplastic lymphocytes.
Acronyms and abbreviations that appear in this chapter include: ADP, adenosine 5′-diphosphate; ATP, adenosine 5′-triphosphate; apaf-1, apoptosis activating factor 1; Btk, Bruton tyrosine kinase; cAMP, adenosine 3′,5′-cyclic phosphate; DHEA, dehydroepiandrosterone; DPPI, dipeptidyl peptidase I; FACS, fluorescence activated cell sorter; GPI, glycerol phosphatidylinositol; lck, leukocyte tyrosine kinase; NAD, nicotinamide adenine dinucleotide; NK, natural killer; TACE, tumor necrosis factor alpha converting enzyme.
ISOLATION OF LYMPHOCYTES
Lymphocytes can be isolated from the whole blood using density gradient centrifugation. Most commonly, this is performed using a step gradient composed of a mixture of the carbohydrate polymer Ficoll and the dense iodine-containing compound sodium metrizoate.1 This technique takes advantage of the low density of lymphocytes (1.07 gm/ml) relative to that of erythrocytes (1.09–1.10 gm/ml), granulocytes (1.08–1.09 gm/ml), or monocytes (1.08 gm/ml).
A Ficoll solution adjusted to a density of 1.077 gm/ml is ideal for isolating human lymphocytes. Whole blood is layered onto a cushion of Ficoll-sodium metrizoate prior to centrifugation at 400 × g for 30 min. The denser red blood cells and granulocytes will sediment to the bottom of the tube, and the monocytes will enter into the Ficoll cushion. The lymphocytes can be collected from the interface formed between the Ficoll-sodium metrizoate cushion and the plasma above, which contains the lighter-density platelets (1.04–1.06 gm/ml). This layer contains lymphocytes along with some monocytes that can be removed by plating the cells in culture flasks and harvesting the lymphocytes that are not adherent to plastic.
LYMPHOCYTE SURFACE ANTIGENS
Lymphocyte subsets generally cannot be distinguished from one another by morphology. Most resting lymphocytes appear as small round cells with a dense nucleus and little cytoplasm (see Chap. 80). However, this homogeneous appearance is deceptive, as these cells comprise many functionally distinct subpopulations.
These subsets can be distinguished through the differential expression of cell-surface proteins. The advent of monoclonal antibody technology has allowed for the generation of virtually unlimited quantities of antibodies, each specific for a particular surface protein or molecule. Coupled with the biochemical analyses of the surface molecules that are recognized by these each of these antibodies, many lymphocyte surface antigens have been defined (see Chap. 13).
Typically, it is necessary to monitor for co-expression of two or more cell-surface proteins to define a functional subset of lymphocytes. The same cell-surface protein is often expressed by more than one cell subset. For example, both helper and cytotoxic T cells express CD3, the proteins associated with the T-cell receptor for antigen (see Chap. 84). Expression of both CD3 and CD4 helps to distinguish mature helper T cells from cytotoxic T cells that express CD3 and CD8, and from other cells, such as dendritic cells, that express CD4 but lack expression of CD3.2 As such, it is the expression of a characteristic constellation of surface molecules, rather than any one particular surface marker, that generally helps to distinguish one subset of lymphocytes from another (see Chap. 13).
The flow cytometer is a highly effective tool for defining these lymphocyte subsets.3 This instrument is based on the principle of fluorescence, or the emission of light resulting from the release of energy gained through the absorption of light at a different wavelength. Monoclonal antibodies specific for desired cell-surface proteins can each be coupled to a fluorescent dye, called a fluorochrome, that will fluoresce with a defined spectrum of light when excited by light at a certain wavelength.4 The flow cytometer can detect cells labeled with such fluorochrome-conjugated antibodies as they pass in a liquid stream through a beam of laser light of defined wavelength. As each cell passes through the laser beam, the laser light is scattered and excites any dye molecules bound to the cell, causing it to fluoresce. Sensitive photomultiplier tubes can detect the scattered light and the fluorescence emissions, respectively providing information on each cell’s granularity and extent to which it bound a given fluorescence dye. This is the most common means used for distinguishing the lymphocyte subsets from one another.
The flow cytometer also can be used to isolate lymphocytes that express selected surface antigens. This requires a fluorescence activated cell sorter, or FACS. With this instrument, the fluorescence signals of cells passing through the laser light are passed back to a computer. This in turn triggers an electric charge that passes from the nozzle through the liquid stream at the precise time the stream is breaking up into droplets containing the desired cell.5 Such droplets therefore will have a positive or negative charge, allowing for their deflection from the main stream of droplets as they pass between plates of opposite charge. In this way, two different subsets of cells can be isolated from each other and from the unsorted cells in nondeflected droplets.
OTHER SEPARATION TECHNIQUES
An effective way of isolating lymphocyte subpopulations is to expose them to paramagnetic beads coated with a monoclonal antibody specific for the distinguishing surface molecule.6 The tube of cells then is placed in a strong magnetic field, thereby attracting the cells that are attached to the beads. The cells attached to the beads are retained, allowing for decanting of the cells that lack the desired surface molecule. The decanted cells lacking the surface molecule are designated as being isolated via negative selection. Bead-bound cells can be harvested and released from the magnetic beads by adding an antibody that reacts with the antibody attached to the magnetic beads, thereby displacing the cells that are bound to the magnetic-bound antibody. The released cells are said to have been isolated via positive selection.
Lymphocyte subsets also can be isolated by binding the cells to plates that are coated with antibodies to a selected surface antigen, a technique known as panning. Alternatively, cells binding a specific complement-fixing antibody can be lysed with complement, leaving behind those cells that lack expression of the targeted surface antigen. All these techniques can be used to enrich for a selected cell subset or to deplete an undesired subset, prior to sorting using the fluorescence-activated cell sorter.
COMPOSITION OF LYMPHOCYTES
Unfortunately, few studies of the composition and biochemistry of lymphocytes have used purified lymphocyte subpopulations. Since mature helper T cells are the predominant blood lymphocyte of normal adults, many reported biochemical parameters are most relevant to this population.
ION AND WATER CONTENT
The resting blood lymphocyte has a mean cell volume of 200 µm3 and contains 71 ± 1.2 percent by weight of water.7 The total lymphocyte cation content is 35 fentamole per cell, of which 22 to 28 fentamole per cell is potassium, and 7.9 ± 3.2 fentamole per cell is sodium.8 Lymphocyte membranes have both voltage-gated and calcium-activated potassium channels that regulate cell volume. Pharmacological inhibition of these channels blocks T-cell activation. The calcium content of resting lymphocytes has been estimated at 580 to 800 pmol/106 cells.9 Cytosolic free calcium concentrations are relatively low in resting lymphocytes (approximately 10–7 M) but increase several-fold after activation.10
The lymphocyte plasma membrane is composed of equal parts of weight of protein and lipid and 6 percent by weight of carbohydrate.11 The molar ratio of cholesterol to phospholipid is approximately 0.5.12,13 Phosphatidylcholine is the predominant phospholipid in the lymphocyte plasma membrane, but phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin are also present. Approximately half the membrane fatty acids are saturated. The membrane proteins are usually glycosylated.
EXTRACELLULAR MEMBRANE-ASSOCIATED ENZYMES
Exposed on the exterior surface of lymphocytes are several enzymes (Table 81-1). Generally, the number of surface enzyme molecules is low compared with that of other surface molecules, such as those involved in lymphocyte adhesion (see Chap. 13). This probably reflects the fact that these molecules are catalytic and have a higher functional specific activity than do molecules involved in adhesion events, where multiple interactions over large surface areas are required. As such, it is possible that many more enzymes are present than the ones currently recognized because they are expressed at levels that are not detectable by conventional methods using monoclonal antibodies and flow cytometry.
TABLE 81-1 SURFACE MOLECULES AND THEIR ENZYMATIC ACTIVITIES
Some of the surface enzymes are involved in nucleotide metabolism (Table 81-1). For example, CD73 is an ecto-5′-nucleotidase that catalyzes the 5′ dephosphorylation of purine and pyrimidine ribo- and deoxyribonucleoside monophosphates to nucleosides that can be taken up by transport systems. This ecto-5′-nucleotidase is attached to the plasma membrane by a glycerol phosphatidylinositol (GPI) anchor (see Chap. 13). In addition, lymphocytes express a membrane-associated adenosine deaminase, the levels of which are increased after activation.14 The shedding of adenosine deaminase by stimulated cells may explain why plasma levels of this enzyme are increased in early HIV infection and in other diseases associated with immune activation.15
The ectoenzymes of nucleotide metabolism may regulate lymphocyte and granulocyte function at sites of inflammation. Activated T lymphocytes can release ATP, which in turn can bind to specific plasma membrane ATP receptors.15 In addition, CD38 can catalyze the transient formation of cyclic ADP-ribose, a new second messenger molecule directly involved in the control of calcium homeostasis by means of receptor-mediated release of calcium from ryanodine-sensitive intracellular stores.16 The consequent increase in calcium mobilization and phospholipid breakdown can provoke activation or death, depending on the target cell. Subsequently, the dephosphorylation of ATP generates adenosine, which can interact with A2 receptors on the plasma membranes of neutrophils, monocytes, and lymphocytes. The engagement of A2 receptors elevates cAMP levels, counteracting the effects of ATP on cell activation.17 The deamination of adenosine permits the cycle to begin anew.
The ectodomains of several other surface antigens can possess proteolytic activity. For example, CD10 (or CALLA) also has neutral endopeptidase activity, and CD26 has dipeptidyl peptidase IV activity.18 These enzymes may play a role in modulating the binding of lymphocytes to other cells and to the extracellular matrix. In addition, inhibition of the catalytic activity of CD26 can provoke many cellular effects, including induction of tyrosine phosphorylation and p38 MAP kinase activation, as well as suppression of DNA synthesis and reduced production of various cytokines. As such, these ectoenzymes may play an important role in lymphocyte activation.
Some membrane-bound proteases have a disintegrin and a metalloprotease domain, termed ADAMs.19,20 One such member of this family of proteins is the tumor necrosis factor alpha converting enzyme, or TACE, otherwise known as ADAM17. These enzymes cleave other surface molecules, such as tumor necrosis factor, thereby releasing the soluble active cytokine.21 In addition, they may play an important role in modifying the activity of cytokines or other cell-surface molecules that are present in the vicinity of the plasma membrane.
INTRACELLULAR MEMBRANE-ASSOCIATED ENZYMES
Transmembrane proteins that have cytoplasmic regions with kinase or phosphatase activities are common in biology although relatively few of these are restricted to lymphocytes. Nevertheless, many cytoplasmic domains of transmembrane proteins interact directly with enzymes that are restricted or preferentially expressed by lymphocytes or lymphocyte subsets (see Chap. 15). B lymphocytes, for example, selectively express Lyn, Syk, and Bruton tyrosine kinase, commonly termed Btk, receptor-protein tyrosine kinases that associate with B-cell-receptor–associated proteins and play a critical role in signal transduction.22 Moreover, mutations that disrupt the function of these kinases can impair B-cell development, leading to dysregulated B-cell function or immune deficiency.23,24 T-cell development and function, on the other hand, rely heavily on receptor-protein tyrosine kinases such as ZAP-70 or leukocyte tyrosine kinase, and lck.25,26 ZAP-70 interacts with the T-cell receptor for antigen, whereas the latter enzyme, lck, is a Src-family tyrosine kinase that interacts with cytoplasmic domains of CD2,27 CD4,28 CD8,28 CD44,29 CD50,30 and CD137.31 Through such interactions, these receptor-protein-tyrosine kinases play important roles in signal transduction following immune recognition and/or cognate intercellular immune interactions.
In addition, lymphocytes possess an important class of intracellular molecules known collectively as adapter proteins that have no intrinsic enzymatic activity.32,33 and 34 Instead, these molecules couple proximal biochemical events initiated by surface-receptor ligation with more distal signaling pathways by recruiting other cytosolic proteins (see Chap. 15).
Beneath the lymphocyte’s plasma membrane is a fully developed cytomatrix with several different structural and mechanical proteins, including tubulin, actin, myosin, tropomyosin, a-actinin, filamin, and a spectrinlike molecule. These are arranged into typical microfilaments, microtubules, and intermediate filaments.35 Lymphocyte activation by antigens or mitogens can lead to changes in the interaction of membrane components with the cytoskeleton, allowing for antigen processing, immunoglobulin secretion, or cell-mediated cytotoxic reactions.
In large part the composition and metabolism of long-lived blood T lymphocytes reflects their resting state. The T cells have a high nuclear-to-cytoplasmic ratio, few ribosomes or mitochondria, and scant endoplasmic reticulum. Glycogen stores are meager. The DNA content of the resting small lymphocyte, 8 pg per cell, is the same amount in other diploid cells. In contrast, the RNA content averages 2.5 pg per cell, yielding an RNA/DNA ratio of approximately 0.32.36 This value is less than in most other human cells, due to the small amount of ribosomal RNA in lymphocytes.
In contrast to most lymphocytes, however, plasma cells have a high RNA/DNA ratio. These cells are the end products of B-cell differentiation and are committed to the synthesis, assembly, and secretion of immunoglobulin. Accordingly, these cells have a well-developed rough endoplasmic reticulum and Golgi apparatus but lack many of the surface receptors found on lymphocytes. Mature plasma cells are probably terminally differentiated and have a low rate of DNA synthesis and abundant RNA, reflecting the plasma cell’s high-level synthesis of immunoglobulin protein.
The few lysosomes in blood lymphocytes contain several different acid hydrolases including acid phosphatase, b glucuronidase, b galactosidase, b hexosaminidase, a arabinosidase, a galactosidase, a mannosidase, a glucosidase, and b glucosidase.37 Acid hydrolase activities are generally higher in T cells than in non-T lymphocytes. Lysosomal acid esterase, assayed histochemically with a-naphthyl acetate as substrate, has a characteristic punctate appearance in mature T lymphocytes.38
In contrast to other lymphocytes, cytotoxic T lymphocytes and natural killer cells possess abundant cytoplasmic granules. These contain a pore-forming proteolytic enzyme, termed perforin, and a series of serine proteinases with specific proapoptotic activity, called granzymes.39 To protect against possible autolysis by granule contents, cytotoxic lymphocytes possess serine-proteinase inhibitors, termed serpins.40 As an additional safeguard, the granzymes of resting lymphocytes are stored as inactive proenzymes.
Cytotoxic lymphocytes rely primarily on the perforin/granzyme system to kill their targets.41,42 Upon contact with its target cell, the cytotoxic lymphocyte converts the granzymes into active forms by a lysosomal cysteine protease called dipeptidyl peptidase I (DPPI).43 Then perforin introduces a pore in the membrane, allowing the activated granzymes and other granule contents to pass into the cytoplasm and then the nucleus of the cell targeted for destruction.44,45 In vitro studies indicate that granzyme nuclear import is independent of ATP, cannot be inhibited by nonhydrolysable GTP analogues, and involves binding within the nucleus, unlike conventional signal-dependent nuclear protein import. The perforin-dependent nuclear entry of granzymes precedes the nuclear events of apoptosis, such as DNA fragmentation and breakdown of the nuclear envelope (see Chap. 11).
FATTY ACID AND LIPID SYNTHESIS
Normal lymphocytes synthesize phospholipids from acetate. The cells contain phospholipases A1, A2, C, and D, and the enzymes of the inositol phosphate metabolic cycle.46
In contrast to monocytes, small lymphocytes probably do not synthesize prostaglandins or leukotrienes; however, small lymphocytes may contain prostaglandin receptors. Prostaglandins synthesized by macrophages inhibit lymphocyte function and may be partially responsible for the impaired immunity associated with chronic inflammatory states such as in Hodgkin’s disease or systemic fungal infections.47 Certain natural fatty acid precursors of prostaglandins, such as gammalinoleic acid, suppress immune function, and may be useful for the treatment of autoimmune disorders.48 However, some prostaglandins may facilitate immunoglobulin class switching and synthesis of selected cytokines or cytokine receptors.49
Quiescent blood lymphocytes have few or no insulin receptors, although these appear following activation. The rate of glucose metabolism is limited by the rate of entry of glucose into the cells by facilitated diffusion. Lymphocytes contain all the enzymes of the glycolytic pathway and the tricarboxylic acid cycle. Although resting lymphocytes consume only small amounts of oxygen in vitro, their mitochondria have typically coupled electron transport chains.
The resting lymphocyte requires energy to maintain its ionic milieu, to replace degraded proteins and lipids, and for active locomotion.50,51 The recirculation of long-lived lymphocytes through the vascular space to the interstitial tissues and back from the lymphatic drainage system requires directed cell movement and utilizes considerable amounts of ATP. Lymphocytes treated with nonlethal concentrations of drugs that specifically inhibit mitochondrial respiration, but not with agents that inhibit glycolysis, recirculate sluggishly. This suggests that the energy for lymphocyte locomotion is derived largely from oxidative phosphorylation.51
The enzymes of the pentose-phosphate pathway account for only a small fraction of energy production in resting lymphocytes.52 As in other cell types, the pathway provides lymphocytes with phosphorylated ribose derivatives necessary for purine and pyrimidine synthesis and with a source of reducing energy in the form of NADPH.
PROTEIN SYNTHESIS AND AMINO ACID METABOLISM
Human blood lymphocytes actively incorporate radioactive amino acids into protein. The protein synthesis is necessary for survival, and inhibition with cycloheximide or puromycin leads to the rapid death of lymphocytes.
The metabolic pathways for the synthesis of two normally nonessential amino acids, L-cysteine and L-asparagine, are inadequate in thymic lymphocytes, and probably in blood T cells.53,54 A similar L-asparagine requirement among certain null and T-cell leukemias is responsible for the L-asparaginase sensitivity of these neoplasms.
NUCLEIC ACID SYNTHESIS AND REPAIR
Blood lymphocytes incorporate radioactive uridine into RNA at a slow but measurable rate. The cells contain the heterogeneous ribonucleoprotein particles that are important for RNA transport and splicing. In B cells, different species of RNA direct the synthesis of immunoglobulin light and heavy chains that are either inserted into the plasma membrane or secreted.55 It is the former that predominate in nonstimulated B cells. These RNA species undergo extensive processing in the cytoplasm prior to translation, including the generation of 5′-terminal cap structures, internal methylations, and the selective removal of intervening sequences.55
The enzymes for the early pathways of de novo purine and pyrimidine synthesis have very low activity in blood lymphocytes, consistent with the small nucleotide requirements of these nondividing cells. The lymphocytes also have minimal ribonucleotide reductase activity and a concomitantly low rate of deoxyribonucleotide synthesis. In contrast, enzymes for purine and pyrimidine intraconversion are easily detectable, with the exception of xanthine oxidase and guanase, which are absent in lymphocytes. The lymphocytes have the capacity to utilize preformed purines and pyrimidines in the plasma, when these are available. However patients with genetic deficiencies of the purine salvage enzymes hypoxanthine-guanine phosphoribosyltransferase (the Lesch-Nyhan syndrome) and adenine phosphoribosyltransferase have normal numbers of lymphocytes and adequate immune function. Hence, the purine salvage pathways are not absolutely necessary for lymphocyte survival.
Genetic deficiencies in two enzymes of purine metabolism, adenosine deaminase and purine nucleoside phosphorylase, are associated with a specific impairment of the development and function of the lymphoid system.56 The primary function of these enzymes is the catabolism of the potentially toxic nucleosides deoxyadenosine and deoxyguanosine. In adenosine deaminase- and purine nucleoside phosphorylase-deficient patients, phosphorylated derivatives of deoxyadenosine and deoxyguanosine may accumulate in lymphocytes. When compared with other cell types, the lymphocytes have high levels of deoxycytidine kinase, for which the purine deoxyribonucleosides are alternative substrates, and low levels of cytoplasmic deoxynucleotidase.
Appreciation of the pathways involved in lymphocyte metabolism of nucleosides prompted development of three antilymphocyte agents, 2-chlorodeoxyadenosine (cladribine), 2-fluoroadenine arabinoside 5′-monophosphate (fludarabine), and 2′-deoxycoformycin (pentostatin) (Fig. 81-1). The two former agents are substrate analogs of 2′-deoxyadenosine that are resistant to adenosine deaminase.57,58 They accumulate selectively in lymphocytes and inhibit both DNA replication and repair. In addition, 2′-deoxycoformycin is a tight-binding inhibitor of adenosine deaminase that prevents degradation of endogenously generated deoxyadenosine.
FIGURE 81-1 Structures of 2′-deoxycoformycin (left), 2-chlorodeoxyadenosine (middle), and fludarabine (right).
2-Chlorodeoxyadenosine and 2′-deoxycoformycin induce sustained remissions in the majority of patients with hairy cell leukemia59 (see Chap. 99). Fludarabine and 2-chlorodeoxyadenosine also exert beneficial effects in chronic lymphocytic leukemia patients (see Chap. 98). The ratio of deoxycytidine kinase to 5′-nucleotidase in chronic lymphocyte leukemia cells has an apparent relationship to the clinical responsiveness of patients to 2-chlorodeoxyadenosine.60 Leukemia lymphocytes with a high ratio respond best, probably because they selectively accumulate the toxic 5′-triphosphate metabolite.
Although the 5′-triphosphate metabolites can be incorporated into the DNA to interfere with its synthesis, these metabolites probably exert other effects to kill normal and leukemic lymphocytes. Other nucleoside analogs that are incorporated into DNA and cause chain termination, such as gemcitabine or cytosine arabinoside, do not kill blood lymphocytes.61 Also, it is unlikely that these metabolites kill lymphocytes through adenine nucleotide depletion, since intracellular ATP levels generally are far higher than the Km values for most vital ATP-dependent enzymes.
Instead, the deoxyadenosine analogs probably exert direct effects on the machinery governing programmed cell death, or apoptosis (see Chap. 11). Various stimuli of apoptosis lead to the activation in the cytoplasm of cysteine proteases with specificity for aspartic acid residues, referred to as caspases.62,63 The activated caspases can cleave structural proteins and enzymes necessary for the survival of both proliferating and resting cells.64 In addition, caspases have been shown to activate the endonuclease responsible for the internucleosomal cleavage of genomic DNA, a hallmark of apoptosis. Activation of the caspase cascade in a cell-free HeLa system, depleted of endogenous low-molecular-weight compounds, requires dATP.65 The dATP interacts with a homologue of the C. elegans death protein ced4, which was designated apoptosis activating factor 1, or apaf-1. In the presence of dATP, apaf-1 forms multimers that combine both with cytochrome c, released from “damaged” mitochondria, and procaspase-9.66,67 This complex induces procaspase-9 processing to generate an active protease that cleaves the “executioner” caspase-3 and perhaps other caspases, with resultant activation of several enzymes that mediate cell death.68 Inactivation of apaf-1 or caspase-9 can substitute for loss of p53 in promoting the oncogenic transformation of cells that overexpress the MYC oncogene.69
As such, adenine nucleotides may play an important role in the modulation of apoptotic and necrotic cell death signals, although different experimental models have yielded conflicting results.70 In the cellfree system, ADP was a good inhibitor of apaf-1-dependent caspase activation, with a Ki of 133 µM. However, to understand the role of ATP and ADP in the regulation of apoptosis, one must measure changes in the concentrations of both nucleotides.
Normal lymphocytes and chronic lymphocytic leukemia cells have been reported to have average cell volumes of 160 to 200 Fl and ADP contents of 1000 to 1200 pmols/107 cells, respectively.71,72 These values yield an estimated ADP concentration of about 400 µM, threefold higher than the Ki of ADP as an inhibitor of caspase activation. Taken together, the data indicate that ADP may work as a physiological intracellular inhibitor of the cytochrome c and apaf-1–mediated caspase pathway.
The positive relationship between the clinical efficacy of the purine deoxynucleosides and the capacities of their corresponding 5-triphosphate derivatives to activate the caspase pathway underscores the relevance of these effects in the therapy of indolent lymphoproliferative diseases. However, the potency of nucleotides as activators of apaf-1 does not fully explain their diverse activities against chronic lymphocytic leukemia and hairy cell leukemia cells. Cladribine is more toxic than fludarabine when tested in purified chronic lymphocytic leukemia cells, and the in vivo dosage of fludarabine is approximately five times higher than cladribine. In contrast, 5′-triphosphate metabolite of fludarabine, F-Ara-ATP, is more effective than that of cladribine in activating caspases. Therefore, there must be additional mechanisms involved in the nucleoside cytotoxicity toward chronic lymphocytic leukemia cells.
One contributing parameter may be DNA strand break formation, which triggers the consumption of adenine nucleotide pools for poly(ADP-ribose) synthesis and reduces ADP constraints on caspase activation. In addition, various adenine deoxynucleotide analogs may interfere with mitochondrial function, perhaps fostering the release of cytochrome c. Thus, purine deoxynucleotides may be able to modulate three different components of the intrinsic apoptosis pathway: (1) DNA damage (2) mitochondrial function, and (3) apaf-1 activation, thus accounting for their activity in inducing apoptosis of resting normal or neoplastic lymphocytes.
Nonreplicating blood T lymphocytes are capable of DNA excision/repair and contain exonucleases, endonucleases, DNA polymerases, and DNA ligase(s). These enzymes play critical roles in the rearrangement and expression of lymphocyte antigen receptors. Mutations in the DNA ligase I gene are a rare cause of an immunodeficiency with a phenotype similar to that of Bloom’s syndrome.73,74 Mice with mutations in the gene encoding the Ku antigen have a defect in repair of double-stranded DNA breaks.75 These animals are not able to effectively repair the double-stranded DNA breaks that occur during rearrangement of immunoglobulin variable region genes and T-cell receptor genes, resulting in impaired lymphocyte development and a severe combined immunodeficiency disease (see Chap. 88). Cortical thymocytes and normal marrow B-lymphocyte precursors contain DNA polymerase g, a DNA-template independent terminal deoxynucleotidyl transferase.76 The enzyme adds new purine and pyrimidine bases at sites of immunoglobulin and T-cell receptor gene rearrangements, during early lymphocyte development.77 As a developmentally restricted enzyme, DNA polymerase g is a useful marker for the classification of acute leukemias.78
The variable, diversity, and joining genes segments for immunoglobulin heavy and light chains, and for the T-cell receptors for antigen, are assembled by lymphocyte-specific recombinases, designated RAG-1 and RAG-2 (see Chap. 83). Mutations that disrupt either of these genes impair lymphocyte development in mice79 and humans.80 Mutations that impair but do not completely abolish the function of RAG-1 and RAG-2 in humans can result in Omenn syndrome, a combined immune deficiency characterized by oligoclonal, activated T lymphocytes with a skewed Th2 profile.81
Despite their resting state, interphase lymphocytes are among the most sensitive cells in the body to the cytotoxic effects of ionizing radiation and ultraviolet light.82 The reasons for their hypersensitivity are not entirely clear. Contributing factors may include the minute pools of deoxynucleotide triphosphates that limit the rate of DNA repair and the presence of abundant endonucleases that degrade DNA at sites of single- or double-strand breaks in the double helix. In addition, the activation of the nuclear enzyme poly(ADP-ribose) polymerase by DNA strand breaks may exhaust the lymphocyte’s minimal NAD stores, leading to a block in oxidation-reduction reactions.83
HORMONES AND VITAMINS
Lymphocytes have receptors for several biologically active peptides, including ACTH, corticotrophin-releasing hormone, calcitonin, calcitonin-gene–related peptide, melatonin, endorphins, enkephalins, vasopressin, oxytoxin, thyrotropin, the tachykinins, bombesin, prolactin, growth hormone, prolactin, somatostatin, vasoactive intestinal peptide, and chemokines.84,85,86 and 87 The various neuropeptides can deliver both positive and negative activation signals to lymphocytes. For example, the tachykinin substance P, which is released by peripheral nerves at sites of injury or inflammation, enhances lymphocyte activation by monocytes.88 Immune function is inhibited by the dopamine D2 receptor-agonist bromocriptine, which causes hypoprolactinemia. Antibodies against prolactin block lymphocyte mitogenesis.85 In general, the receptor density for peptide hormones on lymphocytes increases markedly following activation of the cells.
Glucocorticoids in pharmacological concentrations have a unique lympholytic effect that is not dependent upon cell division, and they are potent immunosuppressive agents.89 Among normal lymphocyte subsets, immature T cells in the thymus are most sensitive. Lymphocytes contain high-affinity receptors for glucocorticoids that may direct and enhance immune functions when they interact with glucocorticoids at physiologic concentrations.90 The glucocorticoid-receptor complexes bind to specific DNA sequences and induce mRNA for proteins that inhibit glucose transport and phospholipid hydrolysis.91 Exposure of lymphocytes to high concentrations of glucocorticoids causes endonuclease activation and DNA fragmentation.92,93,94 and 95 Glucocorticoids also profoundly inhibit the synthesis of interleukin 2 by activated T cells, and of interleukin 1 by monocytes.96 The latter two effects offer an attractive explanation for the immunosuppressive effects of the hormones.
Lymphocytes presumably have receptors for androgens and estrogens, since the sex hormones can modulate immune function.97 The incidence of many autoimmune diseases is higher in females than in males. Androgen therapy may benefit some women with systemic lupus erythematosus but frequently causes unacceptable masculinizing side effects. The androgens may inhibit the formation of proinflammatory cytokines by lymphocytes and monocytes, either directly or through the release of transforming growth factor b1.
The natural adrenal steroid dehydroepiandrosterone (DHEA) stimulates lymphocyte function in old mice, perhaps by interaction with a DHEA receptor complex on T cells.98 Plasma levels of DHEA in people decline with age.99 Whether DHEA supplementation can enhance immune responses in aged humans is still not known.
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Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn