Springer-Verlag 1998
Cell Tissue Res (1998) 293:525±534
REGULAR ARTICLE
J.R. Moffett ´ K.L. Blinder ´ C.N. Venkateshan M.A.A. Namboodiri
Differential effects of kynurenine and tryptophan treatment on quinolinate immunoreactivity in rat lymphoid and non-lymphoid organs
Received: 10 July 1997 / Accepted: 7 October 1997
Abstract Quinolinate is a tryptophan metabolite and an intermediary in nicotinamide adenine dinucleotide (NAD+) synthesis in hepatocytes. Kynurenine is an upstream metabolite in the same biochemical pathway. Under normal physiological conditions, kynurenine is thought to be produced primarily in the liver as an NAD+ precursor. However, during immune stimulation or inflammation, numerous extrahepatic tissues convert systemic tryptophan to kynurenine, and its concentration subsequently rises dramatically in blood. The fate and role of extrahepatic kynurenine are uncertain. In order to begin addressing this question, the present study was performed to determine which cell types can produce quinolinate from either systemic tryptophan or kynurenine. By using highly specific antibodies to protein-coupled quinolinate, we found that intraperitoneal injections of tryptophan led to increased quinolinate immunoreactivity primarily in hepatocytes, with moderate increases in tissue macrophages and splenic follicles. In contrast, intraperitoneal injections of kynurenine did not result in any significant increase in hepatocyte quinolinate immunoreactivity, but rather led to dramatic increases in immunoreactivity in tissue macrophages, splenic white pulp, and thymic medulla. These findings suggest that hepatocytes do not make significant use of extracellular kynurenine for quinolinate or NAD+ synthesis, and that, instead, extrahepatic kynurenine is preferentially metabolized by immune cells throughout the body. The possible significance of the preferential metabolism of kynurenine by immune cells during an immune response is discussed.
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J.R. Moffett ( ) ´ C.N. Venkateshan1 M.A.A. Namboodiri Department of Biology, Georgetown University, 37th & O Sts. N.W., Washington, DC 20057±1229, USA Tel.: +1±202±687±5997; Fax +1±202±687±5662; E-mail
[email protected] K.L. Blinder Laboratory of Central Nervous System Studies, NINDS, National Institutes of Health, Bethesda, MD 20892, USA
Key words Quinolinic acid ´ Tryptophan dioxygenase ´ Indoleamine 2,3-dioxygenase ´ Nicotinamide adenine dinucleotide metabolism ´ Liver ´ Spleen ´ Rat (Sprague Dawley)
Introduction Quinolinate, a metabolite of the kynurenine pathway of tryptophan metabolism (see Fig. 1), has attracted attention recently as a potential neurotoxin in inflammatory diseases (Heyes et al. 1992). The only known physiological role for quinolinate is as a precursor of nicotinamide adenine dinucleotide (NAD+) in hepatocytes (Bender 1989). However, recent studies (Saito et al. 1993a; Moffett et al. 1994a,b, 1997; Espey et al. 1995) have suggested that extrahepatic quinolinate synthesis may be limited to certain cell types in the immune system. Both hepatocytes and macrophages utilize the kynurenine metabolic pathway (Fig. 1) for the synthesis of quinolinate (reviewed in Bender 1989). However, the initial ratecontrolling enzyme in liver is distinct from that in other cell types. Liver is unique in utilizing tryptophan dioxygenase (TDO, E.C. 1.13.11.11), which is principally regulated by tryptophan concentrations and general regulators of metabolism, such as corticosteroids and insulin. Its activity is not increased in inflammation. Macrophages and most other cells of the body perform the same reaction via the much more ubiquitous enzyme indoleamine dioxygenase (IDO, E.C. 1.13.11.17), which is specifically induced in inflammation. Although only hepatocytes and macrophages have been shown to be able to synthesize quinolinate from tryptophan, many tissues including lung (Takikawa et al. 1986) have the initial part of the kynurenine pathway. Thus, many extra-hepatic cells can convert tryptophan to kynurenine by using IDO to catalyze the first step (Saito et al. 1993c). As a result, the concentration of kynurenine in the blood and many tissues is greatly increased during an immune response (Saito et al. 1992). The purpose of this investigation has been to study the effects of extracellular kynure-
526 Fig. 1 Schematic representation of the kynurenine pathway. a Tryptophan dioxygenase (TDO), E.C. 1.13.11.11, b indoleamine dioxygenase (IDO), E.C. 1.13.11.17, c kynurenine formamidase E.C. 3.5.1.9, d kynurenine aminotransferase, E.C. 2.6.1.7, e kynurenine hydroxylase, E.C. 1.14.13.9, f kynureninase, E.C. 3.7.1.3, g 3-hydroxyanthranilate dioxygenase, E.C. 1.13.11.6, h aminocarboxymuconic acid semialdehyde decarboxylase, E.C. 4.1.1.45, j quinolinate phosphoribosyl transferase (QPRT), E.C. 2.4.2.19, k spontaneous non-enzymatic reaction, m aminomuconic acid semialdehyde decarboxylase
nine and tryptophan on quinolinate immunoreactivity (QUIN-IR) in several tissues that are known to regulate differentially tryptophan degradation through the kynurenine pathway.
Materials and methods Reagents Chemicals were from Sigma (St. Louis, Mo.) unless otherwise noted. Male Sprague-Dawley rats (approximately 200 g) were acquired from Zivic-Miller (Zellenople, Pa.). Horseradish-peroxidase (HRP)coupled goat anti-rabbit antibodies were purchased from Kirkegaard and Perry (Gaithersburg, Md.). Avidin-biotin complex (ABC) kits and normal goat serum (NGS) were from Vector (Burlingame, Calif.). Nickel and cobalt (Ni/Co)-enhanced diaminobenzidene kits were acquired from the Peirce Chemical Company (Rockford, Ill.). Polyclonal rabbit antibodies against protein-coupled and gold-adsorbed quinolinate were generated and purified as previously reported (Moffett et al. 1994a, 1997).
Experimental treatments Three hours prior to sacrifice, rats were injected i.p. with either 300 mg tryptophan per kg body weight (n=3, tryptophan diluted in sterile endotoxin-free 0.9% saline), 300 mg/kg kynurenine (n=3), or with saline only (n=3). Immunohistochemistry Rats were deeply anesthetized with 100 mg/kg pentobarbital i.p. prior to transcardial perfusion with 500 ml of an aqueous solution containing 6% dimethyl sulfoxide, 6% EDAC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride) and 1 mM N-hydroxysuccinimide. Tissues were postfixed overnight in 4% formaldehyde in 100 mM phosphate-buffered saline (PBS), brought to 30% sucrose in PBS over 3 days, embedded in OCT embedding medium (Miles, Elkhart, Ind.), frozen at ±20 C, and cut at a thickness of 20 mm on a cryostat. Endogenous peroxidase was blocked by a 30-min incubation in 1% hydrogen peroxide in 50/50 methanol/water with 10 mM dithiothreitol. Tissue sections were incubated overnight with purified antibodies diluted 1:800 in 2% NGS, washed thoroughly, and then developed by using the ABC-HRP system with
527 Ni/Co-enhanced diaminobenzidene as chromogen. The control experiments employed in this study have been previously described (Moffett et al. 1997).
with elements of the blood-brain barrier were substantially greater in kynurenine-treated rats than in tryptophantreated rats. All QUIN-IR cells observed in brain tissue sections exhibited the morphological characteristics of macrophages or lymphocytes.
Results Brain
Thymus
Quinolinate-immunoreactive (QUIN-IR) cells were not observed in the brain parenchyma of any of the animals examined. However, QUIN-IR cells were observed in the brain vasculature (Fig. 2A,B), meninges, and choroid plexus (Fig. 2C,D) of tryptophan-treated and kynureninetreated animals. The intensity of the immunoreactivity for quinolinate and the density of QUIN-IR cells associated
QUIN-IR was present in the thymus of control rats as previously reported (Moffett et al. 1994a). Numerous pleomorphic cells were observed throughout the medulla, and these ranged from lightly to intensely immunoreactive (Fig. 3A). Scattered QUIN-IR cells were present in the cortex of the thymus. Three hours after administration of tryptophan, the level of QUIN-IR in the thymus had moderately increased (Fig. 3B). The density of QUIN-IR cells in medulla and cortex and the proportion of cells with moderate to intense QUIN-IR in both areas were notably increased under this treatment. The effect of kynurenine-treatment on QUIN-IR in the rat thymus was much more pronounced than that of tryptophan administration (Fig. 3C). Both the density of QUIN-IR cells and the intensity of immunoreactivity were increased to a much greater extent. The response to kynurenine in the thymic medulla was striking, with the great majority of cells there containing moderate to intense QUIN-IR.
Fig. 2A±D QUIN-IR cells in brain vasculature and choroid plexus. Three hours after i.p. administration of 300 mg/kg tryptophan, QUIN-IR cells were occasionally observed in association with structures comprising the blood-brain barrier. A QUIN-IR cell inside a thalamic capillary from a tryptophan-treated rat is shown in A. In the tryptophan-treated animals, moderately stained cells were infrequently observed in the choroid plexus (C). Three hours after kynurenine administration (300 mg/kg), scattered QUIN-IR cells were also seen within the brain vasculature, such as the small QUIN-IR cell seen within a capillary in the supraoptic nucleus of the hypothalamus in B. A notable difference observed between kynurenine- and tryptophan-treated animals was the greater number of QUIN-IR cells observed in the choroid plexus in the rats given kynurenine (D). Bar 12 mm
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certain. In spleens from tryptophan-treated rats, moderate increases in QUIN-IR were observed in follicles (Fig. 4B). Virtually all the cells in the follicles of tryptophan-treated rats exhibited light QUIN-IR, and some follicles contained centrally located cells that had a dendritic morphology and that were moderately to strongly immunoreactive for quinolinate (Fig. 4D). Modest increases in the density of QUIN-IR cells were also noted in the red pulp (Fig. 4F). Staining for quinolinate was relatively unchanged in the PALS after tryptophan administration (Fig. 4H). In contrast, kynurenine administration dramatically increased QUIN-IR in the follicles (Fig. 4C,E) and significantly increased the levels in the red pulp (Fig. 4G) and PALS (Fig. 4I). In the PALS, large QUIN-IR dendritic cells, possibly interdigitating cells, surrounded many central and penicillar arterioles. The number of QUIN-IR cells with a dendritic morphology in the PALS was much greater in animals receiving kynurenine than tryptophan (see Fig. 4H,I). QUIN-IR was very marked in virtually all cells in the follicles of the kynurenine-treated rats. In some splenic nodules, marginal zone lymphocytes were more intensely stained for quinolinate than those within the follicles after kynurenine administration (Fig. 4E). QUIN-IR cells in the red pulp were much more numerous after kynurenine treatment than after tryptophan treatment. Liver QUIN-IR in control liver was restricted to a very small number of probable macrophages and Kupffer cells scattered around the sinusoids and veins. Very light, diffuse QUIN-IR was observed around some hepatic veins (Fig. 5A). Tryptophan administration led to significant increases in QUIN-IR throughout the liver (Fig. 5B). In the animals given tryptophan, QUIN-IR was moderate in all hepatocytes, but strong in a small population of
Fig. 3A±C QUIN-IR in the rat thymus. In the thymus of control rats, most QUIN-IR cells were observed in the medulla, and only scattered immunoreactive cells were seen in the cortex (A). Three hours after tryptophan administration, the number and staining intensity of QUIN-IR cells increased significantly (B). Kynurenine administration had a more pronounced effect on QUIN-IR in the rat thymus, with especially large increases observed in the medulla (C). Bar 250 mm
Spleen QUIN-IR cells in control spleens were seen primarily in the periarteriolar lymphoid sheaths (PALS), but also scattered in the red pulp and subcapsular space (Fig. 4A). QUIN-IR cells in the red pulp had the morphological characteristics of macrophages and lymphocytes. There was also very slight QUIN-IR in follicular lymphocytes. The identity of the QUIN-IR cells in the PALS is still un-
Fig. 4A±I Effect of tryptophan and kynurenine treatment on QUIN- c IR in the rat spleen. Immunoreactivity for quinolinate is shown for spleens from a control animal (A), a tryptophan-treated rat (B), and a rat treated with kynurenine (C). Very slight, diffuse QUIN-IR was observed in many follicles in the untreated rat spleen, and tryptophan administration increased follicular staining moderately. QUIN-IR cells with a dendritic morphology were observed in the center of some follicles in tryptophan-treated animals (e.g., arrow in D). In comparison, kynurenine administration led to much larger increases in follicular QUIN-IR, wherein most cells in the splenic nodules were found to be moderately to strongly immunoreactive (E). QUIN-IR was darker in the marginal zone (MZ) than in the germinal centers in some nodules, and this was often more obvious in kynurenine-treated rats (E). The splenic red pulp of tryptophantreated animals contained more immunoreactive cells than control spleens, but the number was still relatively low (F). Numerous QUIN-IR cells with the appearance of macrophages and lymphocytes were observed throughout the red pulp in kynurenine-treated rats (G). QUIN-IR cells in the PALS of tryptophan animals (H) were similar to controls. In contrast, the PALS regions in kynurenine animals contained numerous QUIN-IR cells with the morphology of dendritic cells (I), which were not observed in control or tryptophan-treated animals. A Arteriole. Bar 250 mm (A±C), 50 mm (D±I)
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Fig. 5A±G Differential effects of tryptophan and kynurenine on QUIN-IR in rat liver. Very little QUIN-IR was observed in control livers (A) compared with the livers from tryptophan-treated animals (B). In the tryptophan-treated animals, all hepatocytes were moderately immunoreactive for quinolinate, and some macrophage-like cells were more strongly stained (D, F). QUIN-IR hepatocytes in tryptophan-treated rats were unique among QUIN-IR cells, because
their nuclei were more immunoreactive than their cytoplasm (D, F). The liver from animals receiving kynurenine, like control animals, did not exhibit QUIN-IR in hepatocytes but did contain strongly stained macrophage-like cells, which were most numerous around hepatic veins (C, E, G). Bar 250 mm (A±C), 50 mm (D±G)
Fig. 6A±H QUIN-IR in rat lung. The number of QUIN-IR alveolar macrophages in the lungs of kynurenine-treated rats (B) was substantially greater than that observed in the lungs of tryptophan-treated rats (A). Similarly, QUIN-IR connective tissue macrophages were more numerous in the lungs from kynurenine-treated animals (D, F), than in the lungs from tryptophan-treated animals (C, E).
Lung lymphoid tissue, or BALT, from tryptophan-treated animals c contained some strongly QUIN-IR cells and many lightly to moderately stained cells (G). However in the BALT from kynureninetreated rats, QUIN-IR was intense in the great majority of the cells (H). Bar 50 mm (A±D, G, H), 12 mm (E, F)
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cells with the appearance of macrophages and Kupffer cells (Fig. 5D,F). Hepatocyte QUIN-IR in the tryptophan-treated rats was unique when compared with leukocyte staining, in that the nuclei of the liver cells were more immunoreactive than the cytoplasm. This observation is congruent with the use of quinolinate for NAD+ synthesis, and the localization of NAD+ synthesis to hepatocyte nuclei. Unlike tryptophan, kynurenine treatment did not significantly increase QUIN-IR in hepatocytes (Fig. 5C). Instead, QUIN-IR was increased in scattered cells with the morphology of tissue macrophages (Fig. 5E,G). These strongly immunoreactive cells were particularly numerous around central veins and portal triad areas. Lung Scattered presumptive alveolar and interstitial macrophages were immunoreactive for quinolinate in control rat lung tissue, whereas QUIN-IR in the bronchiole-associated lymphoid tissue (BALT) was moderate (see Moffett et al. 1994a). In the tryptophan-treated animals, the number of QUIN-IR cells was increased in alveoli, interstitium, and BALT relative to the controls (control data not shown), but the increase was minimal (Fig. 6A,C,E,G). After kynurenine administration, the number, size, and staining intensity of QUIN-IR cells in all lung compartments was increased substantially (Fig. 6B,D,F,H). QUIN-IR cells were scattered in the alveoli (Fig. 6B), and numerous strongly immunoreactive macrophages were observed in lung connective tissue (Fig. 6D,F). QUIN-IR was very intense in the majority of cells in the BALT from kynurenine-treated animals (Fig. 6H).
Discussion Based upon previous immunohistochemical studies that have demonstrated that strong staining for quinolinate is limited to cells of the immune system, we have postulated that quinolinate serves an immune-system-specific function (Moffett et al. 1994a,b, 1997; Espey et al. 1995, 1996; Venkateshan et al. 1996; Namboodiri et al. 1996). The present study has been designed to assess the effect of increased extracellular tryptophan and kynurenine on quinolinate staining in several organs known to contain varying amounts of quinolinate (Saito et al. 1993b). In liver, quinolinate is a metabolic precursor for NAD+ in the kynurenine pathway of tryptophan degradation but has no known function in the many extrahepatic cells in which it is present (Bender 1989). Although the initial part of the kynurenine pathway (from tryptophan to kynurenine) is found in many cell types (Werner-Felmayer et al. 1989; Saito et al. 1993c), only hepatocytes are reported to have the complete pathway. As such, the liver is believed to be the sole site of de novo NAD+ synthesis and of total tryptophan oxidation, but extrahepatic synthesis has not been excluded (Bender 1989). The first enzyme of the kynure-
nine pathway in hepatocytes, TDO, is activated by its substrate, tryptophan, and is induced by corticosteroids. In cells other than hepatocytes, the rate-limiting first step of the kynurenine pathway is catalyzed by a different enzyme, IDO, which is regulated in a distinct manner. IDO is induced by interferon-gamma, lipopolysaccharide, pokeweed mitogen, and other immune system activators (Yoshida et al. 1986). It is also known that immune activators such as pokeweed mitogen reduce the activity of hepatic TDO (Saito et al. 1993b). This differential activation of hepatic vs leukocytic tryptophan degradation pathways suggests that NAD+ metabolism may be altered during an immune response. It is also worth noting that IDO is distinct from the liver enzyme not only in its regulation, but also in the oxidizing agent it utilizes to cleave the pyrole ring of tryptophan. TDO in hepatocytes utilizes O2 to break the ring structure, whereas IDO uses the superoxide anion for this purpose (Sun 1989; Bender 1989); the superoxide anion is produced by phagocytes during chemotactic stimulation or phagocytosis (Johnston Jr and Kitagawa 1985; Baggiolini and Wymann 1990). Hypotheses advanced regarding the function of increased IDO activity during inflammation include tryptophan depletion as a biostatic defense mechanism (Pfefferkorn 1984; Hayaishi 1996) and detoxification of reactive oxygen intermediates (Sun 1989). Either of these roles could be fulfilled by activating the ubiquitous initial part of the kynurenine pathway (see Fig. 1). These possibilities do not address the downstream consequences of the large increases in extrahepatic kynurenine pathway flux during inflammation (Saito et al. 1993b). Kynurenine pathway metabolites that are normally produced transiently in the liver are instead increased in concentration in the blood and extrahepatic tissues. We have postulated that quinolinate may be synthesized and released by activated macrophages and lymphocytes as a modulator of the immune response (Moffett et al. 1994a,b, 1997; Espey et al. 1995). A question that has not been addressed is whether de novo hepatic NAD+ synthesis is compromised by the extrahepatic conversion of tryptophan to kynurenine and quinolinate. It is known that hepatocytes use tryptophan much more efficiently for NAD+ synthesis than niacin or nicotinamide (Bender and Olufunwa 1988), and this may also be true of extracellular kynurenine. It remains to be determined whether diverting tryptophan degradation from the liver to extrahepatic sites, especially lymphoid tissues, during inflammation is associated with a shift to extrahepatic de novo NAD+ synthesis, or to an overall change in the amount of NAD+ synthesis. In the present study, tryptophan and kynurenine administration is intended to provide substrate for the kynurenine metabolic pathway above and below the rate-limiting step (see Fig. 1). Provision of excess tryptophan, the initial metabolite above the rate-limiting step, leads to modest increases in QUIN-IR in lymphoid structures and liver. Because the liver enzyme, TDO, is activated by its substrate, tryptophan, the moderate increases in hepatic QUIN-IR are expected. Because both tryptophan and its downstream metabolite, kynurenine, can partici-
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pate in quinolinate synthesis in the liver, increased systemic kynurenine, like tryptophan, might be expected to increase hepatocellular QUIN-IR. However, this has proved not to be true in the present study. Intraperitoneal kynurenine administration increases QUIN-IR in some hepatic macrophages or Kupffer cells, but not in hepatocytes. In contrast, extracellular kynurenine increases QUIN-IR in many cells in primary and secondary lymphoid organs, and in tissue macrophages in all the tissues studied, except for the brain. These findings suggest that tryptophan is taken up much more efficiently by hepatocytes than is kynurenine. Studies have indicated that some hepatocytotic transport of kynurenine occurs in rat. Incubation of rat liver slices with kynurenine leads to small dose- and time-dependent increases in quinolinate in the medium (Speciale et al. 1988), primarily by a sodium-independent mechanism (Speciale and Schwarcz 1990). However, liver-slice preparations contain numerous Kupffer cells and blood-borne phagocytes; these may have contributed to the observed kynurenine uptake. In contrast to liver, kynurenine is avidly taken up by brain slices by both sodium-dependent and sodium-independent mechanisms (Speciale and Schwarcz 1990). The transport of kynurenine into liver and spleen merits further examination to determine whether differences exist between hepatocytotic and leukocytotic kynurenine uptake mechanisms. During immune system activation, the metabolite in the kynurenine pathway that reaches the highest concentration appears to be kynurenine itself, and tissues such as lung are significant producers of kynurenine (Saito et al. 1993b). If kynurenine is taken up poorly by hepatocytes and avidly by leukocytes, as our results suggest, then one function of the extensive activation of IDO in tissues such as lung may be to shift tryptophan utilization from the liver to the immune system. The complete metabolic fate of tryptophan during an immune response has not been well examined. It is not known which of the three potential metabolic fates, viz., the total oxidative route, the picolinate path, or the quinolinate/NAD+ pathway, is favored during inflammation (see Fig. 1). Research in this area has focused on the quinolinate branch, but picolinate has also been implicated in the immune response (Varesio et al. 1990; Melillo et al. 1994). Because the enzyme responsible for quinolinate catabolism in the liver, quinolinic acid phosphoribosyl transferase (QPRT), has a Km that is 200 times higher than the usual concentration of its substrate, it is thought that, as the concentration of quinolinate increases, the rate of incorporation into NAD+ increases proportionately (Bender 1989). It is possible that quinolinate produced in leukocytes such as macrophages during an immune response (Venkateshan et al. 1996; Namboodiri et al. 1996) is released and then processed further in the blood. The presence of the degradative enzyme for quinolinate, QPRT, in certain blood cells including platelets (Foster and Schwarcz 1995) argues in favor of further quinolinate metabolism in the blood or lymphatic system. Although there is little evidence to support the notion of de novo extrahepatic NAD+ synthesis under normal metabo-
lism, it remains to be determined whether such synthesis occurs through an interaction of kynurenine-producing cells and certain blood cells during an immune response. Further research into the role of enhanced extrahepatic kynurenine pathway metabolism during the immune response to pathogens or tumors is thus warranted.
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